WO2018134389A1 - Methods and compositions for treating infections - Google Patents

Abstract

The present invention relates to a method for treating an infection, particularly HCMV infection. To date, there are very few reports on the role of DC-SIGN+ dendritic cells in very early phases of viral infections. By using four anti-human DC-SIGN monoclonal antibodies, the inventors have demonstrated that DC-SIGN might be considered as the most important receptor for both pre- and post-fusion HCMV gB on monocyte-derived dendritic cells (MDDCs). In particular, the invention relates to a method for treating an infection in a subject in need thereof comprising a step of administering to said subject an agent that blocks the interaction between DC-SIGN and an infectious ligand.

Description

METHODS AND COMPOSITIONS FOR TREATING INFECTIONS

FIELD OF THE INVENTION:

The present invention is in the field of microbiology. More particularly, the invention relates to methods and composition for preventing or treating infections.

BACKGROUND OF THE INVENTION:

Infection is defined as invasion and multiplication of an infectious agent in body tissues of the host. Infectious diseases are caused by pathogenic microorganisms. Pathogenic microorganisms, also called infectious agents can be: viruses, viroids, prions, bacteria, nematodes such as parasitic roundworms and pinworms, arthropods such as ticks, mites, fleas, and lice, fungi such as ringworm, and other macroparasites such as tapeworms and other helminths. In most cases, microorganisms live in harmony with their hosts via mutual or commensal interactions. Diseases can emerge when existing microorganisms become pathogenic or when new pathogenic microorganisms enter in a host. Infection may be transmitted by direct contact, indirect contact, or vectors. Direct contact may be with body excreta such as urine, feces, or mucus, or with drainage from an open sore, ulcer, or wound. Indirect contact refers to transmission via inanimate objects such as bed linens, bedpans, drinking glasses, or eating utensils. Vectors are flies, mosquitoes, or other insects capable of harboring and spreading the infectious agent. In many cases, the infectious agent uses the host' s receptors to enter in the host. For example, HIV uses the CD4 receptor which is found on T- cells and macrophages. To enter in the host, the HIV uses gpl20 to attach to a CD4 receptor.

Dendritic Cell-Specific Icam-3 Grabbing Non integrin (DC-SIGN or CD209) is a calcium-dependent type II lectin receptor (11). It is mostly expressed by immature MD-DCs and macrophage subsets (24, 45, 63). On DCs, it has been shown to act as an endocytic receptor thus promoting antigen capture and presentation to T-cells and most likely long-lasting protection for HCMV virions (13, 29, 69). Moreover it allows recognition of the endogenous adhesion molecules ICAM-2 and -3 stabilizing by the way DC interactions with endothelial cells and naive T-cells respectively (22, 24). DC-SIGN also functions as an attachment receptor for a plethora of phylogenetically diverse viral envelope glycoproteins like HIV- 1/2 gpl20, Hepatitis C virus E2, Ebola virus GP, Dengue virus E glycoprotein and HCMV gB (11, 23, 28, 52) (1, 38, 70). DC-SIGN is composed of one extra-cytoplasmic domain composed of a C- terminal calcium-dependent carbohydrate-recognition domain (CRD) linked to a neck region comprising several highly conserved 23-amino acid repeats known to participate to the lectin tetramerization (16, 44). From a molecular point of view, DC-SIGN tetramers display a high avidity for fucose- or high mannose-containing sugar residues decorating their cognate ligands (17, 71). To our knowledge, only few studies afforded to investigate the fine specificities of DC-SIGN interactions with viral glycoproteins. Accordingly, based on mutagenesis approaches HIV-1 gpl20 and human herpesvirus-8 (HHV-8) gB were shown to interact with DC-SIGN through amino acid residues involved in Ca2+ binding, mostly E347 and N349, or located within the glycan binding pocket, ie V351 and D367 (25, 31, 68).

As microorganisms develop new strategies to enter in the host and there are more and more infectious diseases, it is important to understand the interaction of infectious agents with host's receptors. Understanding their interaction could lead to find new therapeutic way to prevent or treat infections.

SUMMARY OF THE INVENTION:

The present invention relates to a method for treating an infection in a subject in need thereof comprising a step of administering to said subject an agent that blocks the interaction between DC-SIGN and an infectious ligand. In particular, the present invention is defined by claims.

DETAILED DESCRIPTION OF THE INVENTION:

Inventors described for the first time how HCMV gB and DC-SIGN interact with each other. By using various four anti-human DC-SIGN monoclonal antibodies, they demonstrated that DC-SIGN might be considered as the most important receptor for both pre- and post-fusion HCMV gB on MDDCs. They identified antigenic domain 4 as the most probable HCMV gB region contacted by DC-SIGN. Altogether these results will pave the way to the characterization of a structural model for the HCMV gB/DC-SIGN complex. Thus, these results give a new therapeutic way to prevent or treat infections in which DC-SIGN is involved as a receptor for an infectious agent.

Accordingly, the present invention relates to a method for treating an infection in a subject in need thereof comprising a step of administering to said subject a therapeutically effective amount of an agent that blocks the interaction between DC-SIGN and an infectious ligand.

As used herein, the terms "treating" or "treatment" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

As used herein, the term "infection" is defined as invasion and multiplication of an infectious agent in body tissues of the host. Infectious disease is intended to encompass any disease which results from an infection mediated by a virus, a bacteria, a parasite or a fungus.

In a particular embodiment, the infection is caused by a virus. As used herein, the term "virus" has its general meaning in the art and refers to an infectious agent which multiplies or replicates in cells of other organisms such as animals, plants and bacteria. When the viruses are not inside of an infected cell or in the process of infecting a cell, viruses exist in the form of independent particles. Typically, the viral particles, also known as virions, consist of two or three parts: (i) the genetic material made from either DNA or RNA, long molecules that carry genetic information; (ii) a protein coat, called the capsid, which surrounds and protects the genetic material; and in some cases (iii) an envelope of lipids that surrounds the protein coat when they are outside a cell. In the context of the invention, the term "virus" includes but is not limited to DNA viruses, such as adenoviruses, herpesviruses, poxviruses, parvoviruses, papillomaviruses, polyomaviruses and hepadnaviruses, and RNA viruses such as reoviruses, picornaviruses, togaviruses, coronaviruses, flaviviruses (e.g Ebola), paramyxoviruses, filoviruses, orthomyxoviruses, rhabdoviruses, and retroviruses(e.g VIH).

In a particular embodiment, the infection is caused by herpesviruses. More particularly, the infection is caused by Human cytomegalovirus (HCMV). HCMV is a prototypical beta- herpesvirus. Due to its relatively high prevalence in the worldwide population (40-90%) it is a leading cause of morbidities in newborns and immunocompromised hosts such as allo-SCT or solid organ recipients, cancer or AIDS patients while primary infections or reactivations are usually asymptomatic in immunocompetent individuals. HCMV uses its envelope glycoprotein B (HCMV gB) to take part of the membrane fusion machinery leading to the release of viral materials inside the cellular host.

In a particular embodiment, the infection is caused by a bacteria. As used herein, the term "bacteria" has its general meaning in the art and refers to prokaryotic microorganisms. Bacteria are single celled microbes. The cell structure is simpler: there is no nucleus or membrane bound organelles. Instead their control centre containing the genetic information which is contained in a single loop of DNA. Bacteria are characterised by the structural characteristics of their cell walls. Bacteria "gram-positive" have thick layers of peptidoglycan while bacteria "gram-negative" have the thin layers. In the context of the invention, the term "bacteria" includes but is not limited to Abiotrophia adiacens, Acinetobacter baumanii, Actinomycetae, Aquaspirillum family, Azospirillum family, Azotobacteraceae family,Bacteroides, Cytophaga and Flexibacter phylum, Bacteroides fragilis, Bordetella pertussis, Bordetella sp., Bacteroidaceae family, Bartonella species, Bdellovibrio family, Campylobacter species, Chlamydia species Campylobacter jejuni and C. coli, Candida albicans, Candida dubliniensis, Candida glabrata, Candida guiUiermondii, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Candida zeylanoides, Candida sp., Chlamydia pneumoniae, Chlamydia trachomatis, Clostridium sp., Corynebacterium sp., Crypococcus neoformans, Cryptococcus sp., Cryptosporidium parvum, Entamoeba sp., Enterobacteriaceae group, Enterococcus casseliflavus-flavescens-gallinarum group, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus sp., Escherichia coli and Shigella sp. group, Gemella sp., Giardia sp., Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Legionella sp., Leishmania sp., Mycobacteriaceae family, Mycoplasma pneumoniae, Neisseria gonorrhoeae, platelets contaminants group, Pseudomonas aeruginosa, Pseudomonads group, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saprophyticus, Staphylococcus sp., Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus sp., Trypanosoma brucei, Trypanosoma cruzi, Trypanosoma sp., Trypanosomatidae Vampirovibr Helicobacter Family, and Vampirovibrio family.

In a particular embodiment, the infection is caused by a parasite. As used herein, the term "parasite" has its general meaning in the art and refers to a living organism which receives nourishment and shelter from another organism where it lives. In the context of the invention, the term "parasite" includes but is not limited to parasitic protozoa, parasitic helminths (worms, e.g Nemathelminthes; Platyhelminthes), and those arthropods that directly cause disease or act as vectors of various pathogens (e.g P. falciparum).

In a particular embodiment, the infection is caused by a fungus. As used herein, the term

"fungus" has its general meaning in the art and refers to a group of eukaryotic protists, including mushrooms, yeasts, rusts, moulds, smuts, which are characterised by the absence of chlorophyll and by the presence of a rigid cell wall composed of chitin, mannans and sometimes cellulose. Typically, the term "fungus" includes but is not limited to aspergillosis, candidiasis, coccidioidomycosis, cryptococcosis, histoplasmosis, mycetomas, and paracoccidioidomycosis.

As used herein, the term "infectious ligands" refers to a product from an infectious agent as described above. Typically, the ligand comprises an antigen, e.g. a peptide antigen, from an infectious agent. For example, the ligand may be gpl20 (HIV); lipopolysaccharides, flagellin (bacteria); zymosan, mannan (fungus). In a particular embodiment, the ligand is HCMV envelope glycoprotein B. The ligand can be as a soluble recombinant or as a native viral envelope glycoprotein (HCMV gB).

As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, the subject according to the invention is a human. More particularly, the subject according to the invention has or susceptible to have at least one infection as described above.

As used herein the term "agent that blocks the interaction between DC-SIGN and infectious ligand" refers to any agent that is currently known in the art or that will be identified in the future. It includes any chemical entity that, upon administration to a subject, results in inhibition and blocking of the interaction between DC-SIGN and infectious ligand. The terms "blocking the interaction", "inhibiting the interaction" or "inhibitor of the interaction" are used herein to mean preventing or reducing the direct or indirect association of one or more molecules, peptides, proteins, enzymes or receptors; or preventing or reducing the normal activity of one or more molecules, peptides, proteins, enzymes, or receptors. Thus, the term "agent that blocks the interaction between DC-SIGN and infectious ligand" refers to a molecule which can prevent the interaction between DC-SIGN and infectious ligand by competition or by fixing to DC-SIGN.

The agent may be a molecule which binds to DC-SIGN selected from the group consisting of antibodies, aptamers, polypeptides and small organic molecules.

In a particular embodiment, the agent is a polypeptide. The term "polypeptide" refers to a polypeptide that specifically binds to DC-SIGN, thereby preventing its binding with an infectious ligand. The term "polypeptide" refers both short peptides with a length of at least two amino acid residues and at most 10 amino acid residues, oligopeptides (11-100 amino acid residues), and longer peptides (the usual interpretation of "polypeptide", i.e. more than 100 amino acid residues in length) as well as proteins (the functional entity comprising at least one peptide, oligopeptide, or polypeptide which may be chemically modified by being glycosylated, by being lipidated, or by comprising prosthetic groups). The definition of polypeptides also comprises native forms of peptides/proteins in mycobacteria as well as recombinant proteins or peptides in any type of expression vectors transforming any kind of host, and also chemically synthesized peptides.

In a particular embodiment, the agent is a small molecule. The term "small organic molecule" as used herein, refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macro molecules (e. g. proteins, nucleic acids, etc.). Typically, small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

In a particular embodiment, the agent is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.

In a particular embodiment, the agent is an antibody against DC-SIGN. The term

"antibody" is used to refer to any antibody-like molecule that has an antigen binding region. This term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs or VHH), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds- stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is non-internalizing. As used herein the term "non-internalizing antibody" refers to an antibody, respectively, that has the property of to bind to a target antigen present on a cell surface, and that, when bound to its target antigen, does not enter the cell and become degraded in the lysosome. Particularly, in the context of the invention, the antibody is a single domain antibody. The term "single domain antibody" has its general meaning in the art and refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such single domain antibody are also called VHH or "nanobody®". For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484- 490; and WO 06/030220, WO 06/003388. In the context of the invention, the amino acid residues of the single domain antibody are numbered according to the general numbering for VH domains given by the International ImMunoGeneTics information system aminoacid numbering (http://imgt.cines.fr/). Particularly, in the context of the invention, the antibody is a single chain variable fragment. The term "single chain variable fragment" or "scFv fragment" refers to a single folded polypeptide comprising the VH and VL domains of an antibody linked through a linker molecule. In such a scFv fragment, the VH and VL domains can be either in the VH - linker - VL or VL - linker - VH order. In addition to facilitate its production, a scFv fragment may contain a tag molecule linked to the scFv via a spacer. A scFv fragment thus comprises the VH and VL domains implicated into antigen recognizing but not the immunogenic constant domains of corresponding antibody.

In a particular embodiment, the agent is a monoclonal antibody. Monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique, the human B-cell hybridoma technique and the EBV-hybridoma technique.

In a particular embodiment, the monoclonal antibody is a neutralizing antibody. As used herein, the term "neutralizing antibody" refers to an antibody that blocks or reduces at least one activity of a target comprising the epitope to which the antibody specifically binds. In the context of the invention, the neutralizing antibody refers to an antibody that will bind to the DC-SIGN, thereby preventing the binding of DC-SIGN to an infectious ligand. Thus, the neutralizing antibody inhibits the membrane fusion and the subsequent intracellular signaling that facilitates invasion of the infectious agent in the host.

The sequences of monoclonal antibodies having a neutralizing activity to inhibit the interaction between DC-SIGN and an infectious ligand are described in the following table A:

Monoclonal Sequences

antibody

105E9.01

H-CDR1 SEQ ID NO: 1

GYTFTNYG

H-CDR2 SEQ ID NO: 2

INTHTGEPT

H-CDR3 SEQ ID NO: 3

ARGYYGSHYEGYYAMDY

L-CDR1 SEQ ID NO: 4

QDIGSS

L-CDR2 SEQ ID NO:5

ATS

L-CDR3 SEQ ID NO: 6

LQYASSPWT

VH SEQ ID NO: 7

QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPG KGLKWMGWINTHTGEPTYADDFKGRFALSLETSASTAYLQIN NLKNEDTATYFCARGYYGSHYEGYYAMDYWGQGTSVTVSS AKTTPPSVYPLAPGSAAQTN

VL SEQ ID NO: 8

DIQMTQSPSSLSASLGERVSLTCRASQDIGSSLNWLQQEPDGTI KRLIYATSNLDSGVPKRFSGSRSGSDYSLTISSLESEDFVDYYC LQYASSPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASV

Monoclonal

antibody

108H8.05

H-CDR1 SEQ ID NO: 9

GFTFSSYA H-CDR2 SEQ ID NO: 10

TSSGGRI

H-CDR3 SEQ ID NO: 11

ARGHYGYEYYFDY

L-CDR1 SEQ ID NO: 12

QDVSTA

L-CDR2 SEQ ID NO: 13

SAS

L-CDR3 SEQ ID NO: 14

QQHSSIPLT

VH SEQ ID NO: 15

EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPE KRLEWVASTSSGGRIYYSDSVKGRFTISRENARNLLYLQMSSL RSEDTAMYYCARGHYGYEYYFDYWGQGTTLTVSSESQSFPN VFPLV

VL SEQ ID NO: 16

DIVMTQSHKFMSTSVGDRVSrrCKASQDVSTAVAWYQQKPG QSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVEAEDLAV Y YCQQHS S IPLTFG AGTKLELKRAD A APT VSI

Monoclonal

antibody 114F1.08

H-CDR1 SEQ ID NO: 17

GYSFTGYF

H-CDR2 SEQ ID NO: 18

INPYNGDT

H-CDR3 SEQ ID NO: 19

GRSNHGYPGYPMDY

L-CDR1 SEQ ID NO: 20

TGAVTTSNY

L-CDR2 SEQ ID NO: 21

GTS

L-CDR3 SEQ ID NO: 22

ALWYSTHYV VH SEQ ID NO: 23

EVQLQQSGPELVKPGASVKISCKASGYSFTGYFMNWVKESHG KSLEWIGRINPYNGDTFYNQRFKGRATLTVDKSSITAHMELLS LTSEDSAVYYCGRSNHGYPGYPMDYWGQGTSVTVSSAKTTP PSVNSTG

VL SEQ ID NO: 24

QAVWTQESALTTSPGGTVILTCRSSTGAVTTSNYANWVQEKP

DHLFTGLIGGTSNRAPGVPVRFSGSLIGDKAALTITGAQTEDD

AMYFCALWYSTHYVFGGGTKVTVLGQPK

Monoclonal

antibody

120E12.03

H-CDR1 (A) SEQ ID NO: 25

GFTFSSYA

H-CDR2 (B) SEQ ID NO: 26

ISSGGST

H-CDR3 (C) SEQ ID NO: 27

ARGQYGYEYYFDY

H-CDR1 (D) SEQ ID NO: 28

GFSLTDYS

H-CDR2 (E) SEQ ID NO: 29

Mr GYGST

H-CDR3 (F) SEQ ID NO: 30

ARDGGYWAMDY

L-CDR1 SEQ ID NO: 31

QDVSTA

L-CDR2 SEQ ID NO: 32

SAS

L-CDR3 SEQ ID NO: 33

QQHSSIPLT

VH 1 SEQ ID NO: 34

EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPE KRLEWVASISSGGSTYYSDSVKGRFIISRDIARNILYLQMSSLR SEDTAMYYCARGQYGYEYYFDYWGQGTTLTVSSAKTTPPSV YPLAPGCGDTTGSSVTLG

VH 2 SEQ ID NO: 35

QVQLKESGPGLVAPSQSLSITCTVSGFSLTDYSVNWVRQPPGK GLEWLGMr GYGSTDYNSALKSRLSISKDNSKSQVFLKMNSL QTDDTARYYCARDGGYWAMDYWGQGTSVIVSSAKTTPPSV YPLAPGSAAQTNSMVT

VL SEQ ID NO: 36

DIVMTQSHKFMSTSVGDRVSrrCKASQDVSTAVAWYQQKPG QSPKLLIYSASFRYTGVPDRFTGSGSGTDFTFTIISVQAEDLAV YYCQQHSSIPLTFGAGTKLELKRADAAPTVSIFPPSVS

Table A

In the context of the invention, the amino acid residues of the antibody as described above are numbered according to the IMGT numbering system. The IMGT unique numbering has been defined to compare the variable domains whatever the antigen receptor, the chain type, or the species (Lefranc M.-P., "Unique database numbering system for immunogenetic analysis" Immunology Today, 18, 509 (1997) ; Lefranc M.-P., "The IMGT unique numbering for Immunoglobulins, T cell receptors and Ig-like domains" The Immunologist, 7, 132-136 (1999).; Lefranc, M.-P., Pommie, C, Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. and Lefranc, G., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains" Dev. Comp. Immunol., 27, 55-77 (2003).). In the IMGT unique numbering, the conserved amino acids always have the same position, for instance cysteine 23, tryptophan 41, hydrophobic amino acid 89, cysteine 104, phenylalanine or tryptophan 118. The IMGT unique numbering provides a standardized delimitation of the framework regions (FR1-IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to 128) and of the complementarity determining regions: CDRl-IMGT: 27 to 38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117. If the CDR3-IMGT length is less than 13 amino acids, gaps are created from the top of the loop, in the following order 111, 112, 110, 113, 109, 114, etc. If the CDR3-IMGT length is more than 13 amino acids, additional positions are created between positions 111 and 112 at the top of the CDR3-IMGT loop in the following order 112.1,111.1, 112.2, 111.2, 112.3, 111.3, etc. (http://www.imgt.org/IMGTScientificChart/Nomenclature/IMGT-FRCDRdefinition.html). In a particular embodiment, the method according to the invention, wherein, the monoclonal antibody having the specificity for DC-SIGN comprises:

a) a heavy chain having at least 70% of identity with the sequence set forth as SEQ ID NO:7; and a light chain having at least 70% of identity with the sequence set forth as SEQ ID NO: 8;

b) a heavy chain having at least 70% of identity with the sequence set forth as SEQ ID NO: 15; and a light chain having at least 70% of identity with the sequence set forth as SEQ ID NO: 16;

c) a heavy chain having at least 70% of identity with the sequence set forth as SEQ ID NO:23 and a light chain having at least 70% of identity with the sequence set forth as SEQ ID NO:24;

d) a heavy chain having at least 70% of identity with the sequence set forth as SEQ ID NO:34 and a light chain having at least 70% of identity with the sequence set forth as SEQ ID NO:36; or

e) a heavy chain having at least 70% of identity with the sequence set forth as SEQ ID NO:35 and a light chain having at least 70% of identity with the sequence set forth as SEQ ID NO:36.

According to the invention a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; or 99% of identity with the second amino acid sequence. Amino acid sequence identity is typically determined using a suitable sequence alignment algorithm and default parameters, such as BLAST P (Karlin and Altschul, 1990).

In a particular embodiment, the method according to the invention, wherein, the monoclonal antibody having the specificity for DC-SIGN comprises:

a) i) a heavy chain wherein the variable domain comprises: a H-CDR1 having the sequence set forth as SEQ ID NO: 1; a H-CDR2 having the sequence set forth as SEQ ID NO: 2; a H-CDR3 having the sequence set forth as SEQ ID NO: 3; ii) a light chain wherein the variable domain comprises L-CDR1 having the sequence set forth as SEQ ID NO: 4; a L-CDR2 having the sequence set forth as SEQ ID NO:5; a L-CDR3 having the sequence set forth as SEQ ID NO:6;

b) i) a heavy chain wherein the variable domain comprises: a H-CDR1 having the sequence set forth as SEQ ID NO: 9; a H-CDR2 having the sequence set forth as SEQ ID NO: 10; a H-CDR3 having the sequence set forth as SEQ ID NO: 11; ii) a light chain wherein the variable domain comprises L-CDRl having the sequence set forth as SEQ ID NO: 12; a L-CDR2 having the sequence set forth as SEQ ID NO: 13; a L-CDR3 having the sequence set forth as SEQ ID NO: 14;

c) i) a heavy chain wherein the variable domain comprises: a H-CDR1 having the sequence set forth as SEQ ID NO: 17; a H-CDR2 having the sequence set forth as SEQ ID NO: 18; a H-CDR3 having the sequence set forth as SEQ ID NO: 19; ii) a light chain wherein the variable domain comprises L-CDRl having the sequence set forth as SEQ ID NO: 20; a L-CDR2 having the sequence set forth as SEQ ID NO:21; a L-CDR3 having the sequence set forth as SEQ ID NO:22;

d) i) a heavy chain wherein the variable domain comprises: a H-CDR1 having the sequence set forth as SEQ ID NO: 25; a H-CDR2 having the sequence set forth as SEQ ID NO: 26; a H-CDR3 having the sequence set forth as SEQ ID NO: 27; ii) a light chain wherein the variable domain comprises L-CDRl having the sequence set forth as SEQ ID NO: 31; a L-CDR2 having the sequence set forth as SEQ ID NO:32; a L-CDR3 having the sequence set forth as SEQ ID NO:33; or

e) i) a heavy chain wherein the variable domain comprises: a H-CDR1 having the sequence set forth as SEQ ID NO: 28; a H-CDR2 having the sequence set forth as SEQ ID NO: 29; a H-CDR3 having the sequence set forth as SEQ ID NO: 30; ii) a light chain wherein the variable domain comprises L-CDRl having the sequence set forth as SEQ ID NO: 31; a L-CDR2 having the sequence set forth as SEQ ID NO:32; a L-CDR3 having the sequence set forth as SEQ ID NO:33.

In a further embodiment, the method according to the invention, wherein, the monoclonal antibody having the specificity for DC-SIGN comprises:

a) i) a heavy chain having at least 70% of identity with the sequence set forth as SEQ ID NO:7; ii) a light chain having at least 70% of identity with the sequence set forth as SEQ ID NO:8; iii) wherein the heavy chain having a variable domain comprises: a H-CDR1 having the sequence set forth as SEQ ID NO: 1; a H-CDR2 having the sequence set forth as SEQ ID NO: 2; a H-CDR3 having the sequence set forth as SEQ ID NO: 3; iv) wherein the light chain having a variable domain comprises L- CDR1 having the sequence set forth as SEQ ID NO: 4; a L-CDR2 having the sequence set forth as SEQ ID NO:5; a L-CDR3 having the sequence set forth as SEQ ID NO:6;

b) i) a heavy chain having at least 70% of identity with the sequence set forth as SEQ ID NO: 15; ii) a light chain having at least 70% of identity with the sequence set forth as SEQ ID NO: 16; iii) wherein the heavy chain having a variable domain comprises: a H-CDR1 having the sequence set forth as SEQ ID NO: 9; a H-CDR2 having the sequence set forth as SEQ ID NO: 10; a H-CDR3 having the sequence set forth as SEQ ID NO: 11; iv) wherein the light chain having a variable domain comprises L- CDR1 having the sequence set forth as SEQ ID NO: 12; a L-CDR2 having the sequence set forth as SEQ ID NO: 13; a L-CDR3 having the sequence set forth as SEQ ID NO: 14;

c) i) a heavy chain having at least 70% of identity with the sequence set forth as SEQ ID NO:23 ii) a light chain having at least 70% of identity with the sequence set forth as SEQ ID NO:24; iii) wherein the heavy chain having a variable domain comprises: a H-CDR1 having the sequence set forth as SEQ ID NO: 17; a H-CDR2 having the sequence set forth as SEQ ID NO: 18; a H-CDR3 having the sequence set forth as SEQ ID NO: 19; iv) wherein the light chain having a variable domain comprises L- CDR1 having the sequence set forth as SEQ ID NO: 20; a L-CDR2 having the sequence set forth as SEQ ID NO:21; a L-CDR3 having the sequence set forth as SEQ ID NO:22;

d) i) a heavy chain having at least 70% of identity with the sequence set forth as SEQ ID NO:34 ii) a light chain having at least 70% of identity with the sequence set forth as SEQ ID NO:36; iii) wherein the heavy chain having a variable domain comprises a H-CDR1 having the sequence set forth as SEQ ID NO: 25; a H-CDR2 having the sequence set forth as SEQ ID NO: 26; a H-CDR3 having the sequence set forth as SEQ ID NO: 27; iv) wherein the light chain having a variable domain comprises L-CDR1 having the sequence set forth as SEQ ID NO: 31; a L-CDR2 having the sequence set forth as SEQ ID NO:32; a L-CDR3 having the sequence set forth as SEQ ID NO:33; or

e) i) a heavy chain having at least 70% of identity with the sequence set forth as SEQ ID NO:35; ii) a light chain having at least 70% of identity with the sequence set forth as SEQ ID NO:36; iii) wherein the heavy chain having a variable domain comprises a H-CDR1 having the sequence set forth as SEQ ID NO: 28; a H-CDR2 having the sequence set forth as SEQ ID NO: 29; a H-CDR3 having the sequence set forth as SEQ ID NO: 30; iv) the light chain having a variable domain comprises L-CDR1 having the sequence set forth as SEQ ID NO: 31; a L-CDR2 having the sequence set forth as SEQ ID NO:32; a L-CDR3 having the sequence set forth as SEQ ID NO:33. In a particular embodiment, the method according to the invention, wherein the monoclonal antibody having a heavy chain consisting of the sequence given in SEQ ID NO: 7.

In a particular embodiment, the method according to the invention, wherein the monoclonal antibody having a heavy chain consisting of the sequence given in SEQ ID NO: 15.

In a particular embodiment, the method according to the invention, wherein the monoclonal antibody having a heavy chain consisting of the sequence given in SEQ ID NO:23.

In a particular embodiment, the method according to the invention, wherein the monoclonal antibody having a heavy chain consisting of the sequence given in SEQ ID NO:34.

In a particular embodiment, the method according to the invention, wherein the monoclonal antibody having a heavy chain consisting of the sequence given in SEQ ID NO: 35.

In another embodiment, the method according to the invention, wherein the monoclonal antibody having a light chain consisting of the sequence given in SEQ ID NO: 8.

In a particular embodiment, the method according to the invention, wherein the monoclonal antibody having a light chain consisting of the sequence given in SEQ ID NO: 16.

In a particular embodiment, the method according to the invention, wherein the monoclonal antibody having a light chain consisting of the sequence given in SEQ 24.

In a particular embodiment, the method according to the invention, wherein the monoclonal antibody having a light chain consisting of the sequence given in SEQ 36.

In another embodiment, the method according to the invention, wherein the monoclonal antibody has a heavy chain wherein the variable domain consisting of the sequence given in: SEQ ID NO: 1; SEQ ID NO: 2 and SEQ ID NO: 3.

In another embodiment, the method according to the invention, wherein the monoclonal antibody has a heavy chain wherein the variable domain consisting of the sequence given in: SEQ ID NO: 9; SEQ ID NO: 10 and SEQ ID NO: 11.

In another embodiment, the method according to the invention, wherein the monoclonal antibody has a heavy chain wherein the variable domain consisting of the sequence given in: SEQ ID NO: 17; SEQ ID NO: 18 and SEQ ID NO: 19.

In another embodiment, the method according to the invention, wherein the monoclonal antibody has a heavy chain wherein the variable domain consisting of the sequence given in: SEQ ID NO: 25; SEQ ID NO: 26 and SEQ ID NO: 27.

In another embodiment, the method according to the invention, wherein the monoclonal antibody has a heavy chain wherein the variable domain consisting of the sequence given in: SEQ ID NO: 28; SEQ ID NO: 29 and SEQ ID NO: 30. In another embodiment, the method according to the invention, wherein the monoclonal antibody has a light chain wherein the variable domain consisting of the sequence given in: SEQ ID NO: 4; SEQ ID NO:5 and SEQ ID NO:6.

In another embodiment, the method according to the invention, wherein the monoclonal antibody has a light chain wherein the variable domain consisting of the sequence given in: SEQ ID NO: 12; SEQ ID NO: 13 and SEQ ID NO: 14.

In another embodiment, the method according to the invention, wherein the monoclonal antibody has a light chain wherein the variable domain consisting of the sequence given in: SEQ ID NO: 20; SEQ ID NO: 21 and SEQ ID NO: 22.

In another embodiment, the method according to the invention, wherein the monoclonal antibody has a light chain wherein the variable domain consisting of the sequence given in: SEQ ID NO: 31; SEQ ID NO:32 and SEQ ID NO:33.

In a particular embodiment, the method according to the invention, wherein, the monoclonal antibody is an antibody which competes for binding to DC-SIGN with at least one antibody as described above.

The invention further relates to a nucleic acid encoding molecule encoding the monoclonal antibody as described above. Nucleic acids of the invention may be produced by any technique known per se in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination(s).

In another embodiment, the invention relates to an expression vector comprising a nucleic acid sequence encoding the monoclonal antibody as described above. According to the invention, expression vectors suitable for use in the invention may comprise at least one expression control element operationally linked to the nucleic acid sequence. The expression control elements are inserted in the vector to control and regulate the expression of the nucleic acid sequence. Examples of expression control elements include, but are not limited to, lac system, operator and promoter regions of phage lambda, yeast promoters and promoters derived from polyoma, adenovirus, retrovirus, lentivirus or SV40. Additional preferred or required operational elements include, but are not limited to, leader sequence, termination codons, polyadenylation signals and any other sequences necessary or preferred for the appropriate transcription and subsequent translation of the nucleic acid sequence in the host system. It will be understood by one skilled in the art that the correct combination of required or preferred expression control elements will depend on the host system chosen. It will further be understood that the expression vector should contain additional elements necessary for the transfer and subsequent replication of the expression vector containing the nucleic acid sequence in the host system. Examples of such elements include, but are not limited to, origins of replication and selectable markers. It will further be understood by one skilled in the art that such vectors are easily constructed using conventional methods or commercially available.

In some embodiments, the invention relates to a host cell comprising the expression vector as descried above. Typically, the invention relates to a host cell which has been transfected, infected or transformed by the nucleic acid and/or the vector as described above. The Examples of host cells that may be used are eukaryote cells, such as animal, plant, insect and yeast cells and prokaryotes cells, such as E. coli. The means by which the vector carrying the gene may be introduced into the cells include, but are not limited to, microinjection, electroporation, transduction, or transfection using DEAE-dextran, lipofection, calcium phosphate or other procedures known to one skilled in the art. In another embodiment, eukaryotic expression vectors that function in eukaryotic cells are used. Examples of such vectors include, but are not limited to, viral vectors such as retrovirus, adenovirus, adeno- associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus; lentivirus, bacterial expression vectors, plasmids, such as pcDNA3 or the baculovirus transfer vectors. Preferred eukaryotic cell lines include, but are not limited to, COS cells, CHO cells, HeLa cells, NIH/3T3 cells, 293 cells (ATCC# CRL1573), T2 cells, dendritic cells, or monocytes.

As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an agent that blocks the interaction between DC-SIGN and infectious ligand) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

By a "therapeutically effective amount" is meant a sufficient amount of an agent that blocks the interaction between DC-SIGN and infectious ligand to prevent for use in a method for the treatment of infections at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

The present invention relates also to a pharmaceutical composition comprising the monoclonal antibody as described above. Typically, the agent that blocks the interaction between DC-SIGN and an infectious ligand, notably the monoclonal antibodies or the vector comprising monoclonal antibodies as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.

As used herein, the terms "pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The peptide or the drug conjugate (or the vector comprising peptide or the drug conjugate) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. The drug conjugate (or the vector containing the drug conjugate) may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 100 milligrams per dose. Multiple doses can also be administered. The invention will be further illustrated by the following figures and examples.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: AD-4- and AD-5- but not AD-l/2-specific antibodies inhibit only partially the DC-SIGN/HCMV gB interaction. (A) Anti-AD-1 (ITC33, ITC39, ITC48, ITC52 and rrC63B), -AD-2 (ITC88) mAbs or a polyclonal anti-HCMV gB were used as potential inhibitors in a HCMV gB binding assay on immature DC-SIGN+ monocyte-derived dendritic cells (MD-DCs; day 6 of differentiation). Binding of biotinylated HCMV gB followed by a fluorochrome-conjugated streptavindin was assessed by flow cytometry. Binding intensities are represented as mean percentages of the maximum binding (n=3), i.e. without any antibody, and compared to an irrelevant isotypic control (ctrl).HCMV gB binding assay on parental or DC- SIGN-expressing U937 cell lines (B) or on day-6 immature MD-DCs (C). Fifteen non- commercially available anti-HCMV gB antibodies (Dendritics SA) were compared to the 1G2 (against AD-5), SM5-1 (against AD-4) mAbs or a commercially available anti-HCMV gB pAb for their ability to block the DC-SIGN/HCMV gB interaction. Binding intensities are represented as mean percentages of the maximum binding, i.e. without any antibody (n=5). Asterisks represent significant results (*=p<0.05 and **=p<0.01; Mann-Whitney t-test).

Figure 2: HCMV trans-infection by DC-SIGN+ transfectants or MD-DCs is drastically impaired by an anti-AD-4 monoclonal antibody. (A) The fifteen non- commercially available anti-HCMV gB antibodies (Dendritics SA) used in the Figures 4C and 4D were compared to the 1G2 (against AD-5), SM5-1 (against AD-4) mAbs or a commercially available anti-HCMV gB pAb for their ability to block the transmission of HCMV (TB40/E- GFP strain) by DC-SIGN+ U937 cells to highly permissive MRC-5 cells (=trans-infection; MOI=2). After 72 hours, MRC-5 cells were washed, fixed with 90% acetone and stained with an Alexa 488®-conjugated anti-IE antigen (clone 8B1.2). The percentage of infected MRC-5 was determined by comparing total cell (DAPI+) to IE+ cell numbers per image. Images were acquired on an Axioscop inverted fluorescence microscope (Zeiss, Germany). Results are expressed as a mean percentages of infected cells (MRC-5, n=4). (B) As described in the Figure 5A, trans-infection of various HCMV strains (TRI, a clinical isolate, VHL/E, Toledo and TB40/E-GFP) by day-6 immature MD-DCs was determined (MOI=2). Results are expressed as mean percentages of infected cells (MRC-5, n=4). Asterisks indicate significant P-values (*=P<0.05 and **=P<0.01; ANOVA with Welch correction).

Figure 3: Anti-DC-SIGN CRD antibodies totally abrogate the DC-SIGN/HCMV gB interaction. As in Figures 4C and 4D, the binding of biotinylated HCMV gB ^g/ml) coupled to a fluorochrome-conjugated streptavidin detection on (A) parental or DC-SIGN- expressing U937 cell lines or (B) day-6 immature MD-DCs was assessed by flow cytometry. Fifteen non-commercially available anti-DC-SIGN antibodies against the neck, ECPR or CRD regions (Dendritics SA) were compared to the H200 (neck), IB 10, AZN-D1 and MR-1 (CRD) commercially available mAbs for their ability to block the DC-SIGN/HCMV gB interaction. Binding intensities are represented as mean percentages of the maximum binding (n=5), i.e. without any antibody, and compared to an irrelevant isotypic control (ctrl). Asterisks represent significant results (*=p<0.05, **=p<0.01, ***=p<0.005, ****=p<0.001; Mann-Whitney t-test); nd=not detected. Figure 4: Blocking DC-SIGN strongly impairs the HCMV trans-infection by DC- SIGN+ transfectants or MD-DCs to highly permissive MRC-5 cells. (A) Antibody-mediated blockade of HCMV trans-infection by parental or DC-SIGN+ U937 cells. These experiments were conducted as described in the legend of the Figure 5A. Briefly, anti-DC-SIGN 3/4/6 (anti- neck) and 8/10/12/13/14/15/MR-l (anti-CRD) were used at 2(Vg/ml to block the capture and further transmission of HCMV (TB40/E-GFP strain, MOI=2) to highly permissive MCR-5 cells by U937 DC-SIGN transfectants. Results are expressed as mean percentages of infected cells (n=5). Antibody-mediated blockade of HCMV trans-infection by MD-DCs to MRC-5 cells. Various HCMV strains were used in this test: TB40/E-GFP, VHL/E, Toledo and a clinical isolate (TRI). Two anti-neck (1/2) and seven anti-CRD antibodies were incubated at 20μg/ml with viruses before an additional incubation step on MD-DCs prior to being applied on a MRC- 5 subconfluent monolayer. These experiments were conducted as described in the legend of the Figure 5A. Results are expressed as mean percentages of infected cells (n=5). Asterisks represent significant results (*=p<0.05, **=p<0.01, ***=p<0.005; Mann- Whitney).

EXAMPLE:

Material & Methods

Ethic statements

Human fresh blood samples from healthy volunteers were obtained from the Etablissement Francais du Sang, the French blood donor bank (EFS, Nantes, France). As a consequence no ethics statement is required for this work.

Cells

Adult blood monocytes were isolated by negative immune-magnetic separation (Miltenyi Biotec, Bergisch Gladbach, Germany) or by elutriation (DTC cell-sorting facility; Nantes University Hospital/Biogen Ouest, Nantes, France) from healthy blood donors. Cell isolation usually yielded more than 95% purity of CD 14+ cells as assessed by flow cytometry. Monocytes were differentiated into MD-DCs in the presence of 20ng/ml recombinant human (rh) rhIL-4 (Cellgenix, Freiburg, Germany) and 100 ng/ml rhGM-CSF (Gentaur, Paris, France) (58) in 2mM glutamine RPMI 1640 (Life Technologies™, Thermo Fisher Scientific, Waltham, MA), 2% human serum albumin (HSA; Vialbex™, LFB, France). U937 cells stably expressing the full length DC-SIGN were generated by lentiviral transduction and propagated as already described elsewhere (28). HEK293T cells and MRC-5 fibroblasts (RD Biotech, France) were propagated in DMEM, 2mM glutamine, 10% FCS. MRC-5 cells were used at 70-80% confluency for HCMV propagation and trans-infection assays (described below in this section).

Viruses and reagents One in-house clinical isolate (TRI) generated as described previously (21) and three low passage HCMV laboratory strains (Toledo, TB40/E-GFP and VHL/E) (5, 51, 62) were propagated on MRC-5 cells and used as clarified viral supernatants for trans-infection experiments at a multiplicity of infection (MOI) of 2 (29). CHO-derived recombinant CMV glycoprotein B was kindly provided by Sanofi Pasteur (Marcy l'Etoile, France). Recombinant HIV-1 IIIB gpl20 also derived from CHO cells was purchased from ImmunoDx (Woburn, MA). Both envelope glycoproteins were used either as purified or conjugated reagents, ie with Alexa-488, Alexa-647 or biotin microscale protein labeling kits (Molecular Probes, Thermo Fisher Scientific, Waltham, MA) depending on the type of assay to perform. Fifteen anti- HCMV gB antibodies were generated by Dendritics (Lyon, France). They were used with two other anti-HCMV gB mAbs (clones 1G2 and SM5-1) provided by Pr. Michael Mach (53) and a commercially available rabbit polyclonal antibody (Sinobiological, China) for HCMV gB binding or HCMV trans-infection inhibition assays. Anti-AD-1/2 antibodies were kindly provided by Dr Mats Ohlin (47). Eighteen anti-human DC-SIGN from Dendritics used in this study are described in the Table 1. Their reactivities against DC-SIGN subdomains or regions were assessed by flow cytometry using stably transfected HEK293T cells. Four other anti-DC- SIGN mAbs and the commercially available MR1 (Bio-Rad AbD Serotec, Oxford, UK), AZN- Dl (Beckman Coulter France, Villepinte, France), IB 10 clones as well as an anti-neck polyclonal antibody (clone H200, Santa Cruz Biotechnology Inc., Heidelberg, Germany) were also used in this work. All antibodies were used at 20μg/ml or ^g/ml for blockade and flow cytometry analyses respectively.

Establishment of stably transfected cell lines expressing wild type or mutant DC- SIGN

HEK 293T cells were transfected with Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Waltham, MA) and plasmids encoding the wild type (wt) DC-SIGN (Genbank: M98457) or deletional mutants, ie either without the neck region (AA 81-252, Aneck) or the carbohydrate binding domain (CRD, AA 253-404, ACRD) DC-SIGN in fusion with GFP as described elsewhere (36). These plasmids were kindly provided by Pr Kenneth Jacobson (University of North Carolina, USA). A pEGFP plasmid was use as a negative control. Single point mutants of the DC-SIGN CRD were also stably expressed in HEK293T cells (N311A, E347A, N349A, V351A, D367A and S360A; kindly gifted by Dr Frank Jenkins, University of Pittsburgh, USA). Selection of stable DC-SIGN transfectants was performed by subculturing cell lines with geneticin (G-418 disulfate salt, Sigma- Aldrich). The DC-SIGN surface expression was assessed by flow cytometry using either an anti-neck antibody (pAb, H200) or an anti-CRD (MR-1 clone) subsequently revealed by an APC-conjugated secondary anti-IgG (H+L) (BD Biosciences, Franklin Lakes, NJ).

Viral envelope glycoprotein binding assay

MD-DCs or DC-SIGN-expressing U937 cells were resuspended in TBS, ImM CaC12, 2mM MgC12, 0.1% bovine serum albumin (BSA) and then seeded in 96-well plates at 1.10 ^Λ 5 cells / wells. DC-SIGN or HCMV gB antibody-mediated blockade was performed by incubating cells with specific antibodies or with an irrelevant antibody (20μg/ml) for 30 min at 4°C. Biotin-labelled recombinant HCMV gB or HIV-1 IIIB gpl20 ^g/ml) were added to cells for 20min at 4°C. Cells were then washed three times with cold TBS, ImM CaC12, 2mM MgC12, 0.1% BSA and further stained with APC- or PE-conjugated streptavidin (BD Biosciences, Franklin Lakes, NJ) before being analyzed on a LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ) and the FlowJo software (Tree Star, Ashland, OR).

Enzymatic or chemical HCMV gB deglycosylation

When required, recombinant HCMV gB or HIV-1 gpl20 ^g per condition) were enzymatically de glycosylated with 3000U PNGaseF (Peptide-N-Glycosidase F) or 600U a(2 to 3,6)-neuraminidase (New England BioLabs Inc., Ipswich, MA) or 2U a(l-2,3,6)-mannosidase (Prozyme, Hayward, CA) for at least 18 hours at 37°C. Enzymatic treatments were performed according to manufacturer's instructions. However denaturing conditions, ie addition of SDS, were only applied when required. Enzymes were removed by dialysis of the sample (0.5 ml Amicon Ultra, Ultracel 50k Millipore Merck, Cork IRL) against PBS after treatment. For a complete deglycosyation, HCMV gB was treated with trifluoromethanesulfonic acid (TFMS Chemical Deglycosylation Kit; Sigma-Aldrich, St. Louis, MO) and pyridine solutions according to manufacturer's instructions.

Trans-infection experiments

To assess the blocking properties of commercially and non-commercially anti-gB or

DC-SIGN antibodies in a relevant in vitro infection assay, a DC-SIGN-expressing U937 cell line as well as immature MD-DCs were used in a trans-infection assay described elsewhere (28). Briefly, cells were incubated with blockers for 30min at 4°C before adding viruses without washing (MOI=2). After subsequent 2 hours incubation at 37°C, cells were washed and put in close contact with a highly permissive MRC-5 cell monolayer for 72 hours. MRC-5 cells were washed three times, fixed with acetone/water 9: 1 vokvol and stained with an Alexa 488®- conjugated anti-IE/E cytomegalovirus antigens (clone 8B1.2, Millipore, MA). The percentage of infected MRC-5 cells was determined among the total cell number (DAPI positive cells) on digitalized images treated with the Fiji software. For each experiment, at least four images per condition were analyzed on Fiji. Results are represented as mean percentages of infected cells.

Lectin adhesion assay

Plates on which were adsorbed recombinant plant or animal lectins (DC-SIGN=DC- Specific ICAM-3 Grabbing Non-integrin, ConA=concanavalin A, DSA= Datura Stramonium Agglutinin, WGA=Wheat Germ Agglutinin, MPA=Maclura Pomifera Agglutinin and SNA=Sambucus Nigra Agglutinin) were supplied by GLYcoDiag (Orleans, France). Lectin- functionalized plates were saturated during 30 min at room temperature with TBS solution containing 0.05% Tween, 0.5% BSA, ImM CaC12 and 2mM MgC12. After washing biotin- conjugated HCMV gB or HIV-1 IIIB gpl20 were added at 10, 2 and 0.4 μg/ml in triplicate for 1 hour at 4°C with gentle shaking. Envelope glycoprotein binding was revealed by a final incubation step with Alexa® 488-streptavidin (BD Biosciences, Franklin Lakes, NJ). Unbound reagents were removed by a thorough washing step and fluorescence was acquired with a VICTOR X fluorimeter and analyzed with the PerkinElmer 2030 Manager software (Perkin Elmer, Norwalk, CT). Similar assays were performed with whole infectious HCMV particles (TB40E strain). Viral inputs were normalized on the basis of the genome copy numbers for each viral preparation used in this study. Lectin-bound viruses were quantified by qPCR on a StepOne Plus apparatus (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA) as described elsewhere (6).

Kd measurement by surface plasmon resonance

Surface plasmon resonance (SPR) experiments were performed on a Biacore T200 using a CM3 chip, functionalized at 5 μί/ηιίη. Streptactin (IBA company) and then lectins were immobilized on flow cells using amine-coupling method. Flow cell (Fc) 1 and 3 were prepared as reference surface. Fc 1 to 4 were activated with 50 μΐ_^ of a 0.2M EDC/ 0.05 M NHS mixture. After this step, Fc 1 to 4 were functionalized with 170 μg/mL streptactin, and then remaining activated groups in all cells were blocked with 80 μΐ_^ 1M ethanolamine. After blocking, the four Fc were treated with 5 μΐ_^ of 10 mM HCl to remove unspecific bound protein and 5 μΐ_^ of 50 mM NaOH/lM NaCl to expose surface to regeneration protocol. Finally, an average of 2300 RU of streptactin was immobilized on each surface. This procedure was repeated for the functionalization of DC-SIGN ECD (2421 RU) on Fc2. For direct interaction studies, increasing HCMV gB concentrations were prepared in a running buffer composed of 25 mM Tris pH 8, 150 mM NaCl, 4 mM CaC12, 0.05% P20 surfactant, and 85 DL of each sample was injected onto the surfaces at 30 μΕ/ηιίη flow rate. Resulting sensorgrams were reference surface corrected. The apparent HCMV gB affinity for the DC-SIGN ECD-coated surface was determined by fitting the Langmuir model to the plots of glycoprotein binding responses versus concentration. In this simplified modeling approach, we consider the surface, as a whole, as being the ligand. Thus, the dissociation constant (Kd) obtained reflects the affinity for the surface and not for individual lectin receptor. However, this mode of multisite interaction onto a surface is closer to the real interaction mode at the cell surface than considering a stoichiometric interaction with a lectin receptor. Doing so, fits of very good quality have been obtained to qualify the interaction.

Western Blot analyses

Equal amounts (300μg) of untreated or treated HCMV gB were loaded on a 4-20% gradient gel (Bio-Rad Laboratories, Hercules, CA). After separation by electrophoresis, proteins were transferred onto a nitrocellulose membrane which was subsequently saturated in 5% w/v nonfat dry milk, IX TBS, 0.1% Tween® 20. An overnight incubation at 4°C with an anti-HCMV gB pAb (0. ^g/ml Sino Biological Inc., China) or with biotin-conjugated ConA, WGA or PNA (Peanut Agglutinin; all from Sigma Aldrich) in IX TBS, 0.1% Tween® 20 followed by a HRP-conjugated streptavidin or anti-rabbit antibody (Jackson Immunoresearch Europe Ltd, UK) were performed to reveal HCMV gB and its molecular weight alterations by enzymatic treatments. A SuperSignal West Pico chemiluminescent substrate was added to ensure HCMV gB detection (Thermo Fisher Scientific, Waltham, MA).

Statistical analysis

Statistical tests were performed using the GraphPad Prism 5.0 software (GraphPad Software Inc., La Jolla, CA, USA). Comparison of unpaired samples was performed using a non-parametric Mann-Whitney rank test or a one-way ANOVA including a Welch correction as an alternative when compared had unequal variances (14). P-values below or equal to 0.05 were considered as significant.

Table 1 : DC-SIGN specificity determination for mAbs used in this study by flow cytometry

ECPR=extra-cytoplasmic proximal region (AA 59-95); it is comprised between the transmembrane part and the neck region (AA 96-257} of DC- SIGN. CRD=cart3ohydtate recognition domain or lectin domain (AA 258-404), MAbs were used as purified or hybridoma supernatants at 2fig ml or four-times diluted respectively; the * mark means that mAbs were used at 5pg ml {purified) or pure hybridoma supernatants. One representative determination is shown out of two; similar results were obtained in both experiments.

Results

DC-SIGN interacts with the HCMV envelope glycoprotein B only through its CRD

We previously demonstrated by SPR that the HCMV gB or the HIV-1 IIIB gpl20 and the DC-SIGN lectin domain could interact with a comparable affinity (28). However whether other DC-SIGN regions play a role in this interaction remained largely unknown. To start a deeper characterization of the HCMV gB/DC-SIGN interaction, we first established cell lines ectopically expressing the wild type (wt) human DC-SIGN (AA 1-404; UnitProtKB: Q9NNX6) or two deletion mutants respectively lacking the neck repeat (AA 1-80 in frame with AA 253- 404, Aneck) or the CRD (AA 1-252, ACRD) regions in a stable manner. To facilitate DC-SIGN expression monitoring, it was expressed as a fusion protein with the eGFP as already described elsewhere (36). We first showed that all cell lines expressed similar eGFP signal intensities reflecting very similar amounts of full-length or truncated proteins at their surface. We confirmed this by performing staining with mAbs against either the CRD or the neck (data not shown). We then analyzed the binding of a soluble recombinant HCMV gB to these various cell lines by flow cytometry. As expected, the absence of the lectin domain almost totally abrogated the fixation whereas the Aneck mutant DC-SIGN was still capable of binding the HCMV gB as efficiently as the full length lectin strongly suggesting that the CRD was the unique region of DC-SIGN to be responsible of this interaction with the HCMV gB. It also suggested that major deletions such as the neck removal (Aneck) did not alter recognition of DC-SIGN ligands. Based on these results we sought to identify more precisely the CRD AA residues involved in the DC-SIGN interaction with the HCMV gB. Some AA residues were already known as direct interacting partners for the ΗΓΥ - 1 gp 120. Single point mutants for some of these AA residues (E311A, E347A, N349A, V351A, D367A and S360A where A indicates an alanine instead of the mentioned AA) were ectopically expressed in HEK293T cells. Binding of HCMV gB or HIV-1 IIIB gpl20 on these transfectants was analyzed by flow cytometry with respect to the basal binding of both envelope glycoproteins on parental HEK293T cells. Interestingly, both viral ligands interacted with wt or mutated DC-SIGN-expressing cell lines in a very similar fashion suggesting that both glycoproteins share an almost completely overlapping interaction zone on DC-SIGN. Most notably, E347A and N349A mutations significantly impaired viral glycoprotein fixation whereas the N311A and S360A had no detectable effect. These results were in total agreement with was has been already reported by others with the HIV-1 gpl20 (25). Moreover the DC-SIGN expression for all these constructs was assessed with two anti-CRD or -neck repeat antibodies (H200, data not shown) allowing us to cautiously state that differential glycoprotein binding activities were not due to significant DC-SIGN expression variations on transfectants. Conversely, V351A and D367A mutants displayed an increase potential to bind both viral ligands suggesting that these AA positions could also play an important role in the interaction with exogenous ligands by slightly but significantly increasing affinity. Taken together, these results demonstrated that DC-SIGN recognized the HCMV gB only through its lectin domain and that the recognition of HIV-1 IIIB gpl20 and HCMV gB appeared to occur via conserved AA residues within the CRD.

CHO-derived HCMV gB contains high mannose sugars which are mandatory for its binding to DC-SIGN

We previously demonstrated that HCMV gB and HIV-1 IIIB gpl20 shared the same interacting AA on DC-SIGN. However both envelope glycoproteins differ in their three- dimensional structure. We postulated that these two heavily glycosylated viral glycoproteins might harbor similar glycan covers; (such as prototypical N- or O-glycans). To address this point lectin-functionalized flat bottom 96-well plates (ConA, DSA, WGA, MPA and SNA or DC-SIGN, respectively from plant or mammalian origins) were used to assess their ability to retain fluorescently labelled HCMV gB or HIV-1 IIIB gpl20. We observed a dose-dependent binding of both glycoproteins on DC-SIGN, ConA, DSA and to a lesser extent on WGA suggesting that high mannose-containing N-glycans were mostly retrieved on both CHO- derived viral glycoproteins (8, 48). These results were in line with other pioneering studies (17). Interestingly, MPA only displayed a very weak binding activity suggesting that a majority of N-glycans but negligible or even no O-glycans were present on CHO-derived glycoproteins. Similarly, we could show that HCMV gB and HIV-1 IIIB gpl20 were most likely lacking a2,6- branched sialic acid residues as accounted by bearly detectable signals obtained with the Sambucus nigra agglutinin (SNA) (33) . Since the HCMV gB used here was produced in CHO cells, we wondered to what extent its glycosylation cover could resemble the one of whole HCMV particles produced in human foreskin fibroblasts (HFF). Then lectin-binding assays were performed with two doses of infectious HCMV (2.106 and 8.106 viral genome or vg/well). Viral genomes were extracted from lectin-bound virions and further quantified by qPCR. Here again DC-SIGN, WGA and ConA allowed HCMV retention in a dose-dependent manner whereas DSA did not permit HCMV attachment confirming that high mannose sugars present on the whole particle were the main contributors of these lectin-dependent interactions. Interestingly, MPA, mainly involved in interactions with O-glycans, was able to retain HCMV particles in this setting strongly suggesting that other O-glycosylated envelope glycoproteins distinct from gB could participate to the interaction between HCMV particles and other lectins on DCs.

In order to evaluate more precisely how HCMV gB N-glycans contribute to the interaction with DC-SIGN, we determined the dissociation constant (Kd) of the HCMV gB/DC- SIGN interaction by SPR after its enzymatically-driven deglycosylation. We first evaluated the efficacy of deglycosylation with the Peptide-N-Glycosidase F (PNGase F), known to cleave N- glycan residues close to the branched asparagine. A molecular weight loss of approximately 30kDa after applying such a treatment suggesting that the HCMV gB was heavily N- glycosylated. Most of the putative N-glycosylation sites (18/19 according to viral strains) are thought to be effectively glycosylated. Given that the mean molecular weight for a N-glycan residue is around 2 kDa we considered that approximately 15 to 16 putative glycosylation sites were indeed glycosylated. This was in accordance with previous study (7). Unexpectedly removal of a(l-2,3,6)-branched mannose residues by the a(l-2,3,6)-mannosidase treatment led to a limited molecular weight loss suggesting that only few mannose residues were present on HCMV gB. Conversely, treatment with a broad spectrum neuraminidase consistently altered the apparent molecular weight in gel indicating that sialic acids readily trimmed CHO-derived HCMV gB. We then measured the affinity of DC-SIGN for de glycosylated (same as above) compared to untreated HCMV gB by SPR. We could calculate the apparent Kd for all conditions. Interestingly, we could demonstrate that untreated HCMV gB had an apparent Kd (Kdapp) of 14 pM which characterized a higher affinity interaction compared to what we described in a previous study (28). This difference was most likely due to the fact we used a distinct extrapolation method to modelize experimental values and extrapolate Kds from the binding curves (cf. Materials and Methods section). Whereas removal of sialic acids on HCMV gB did not readily alter its binding to immobilized DC-SIGN (Kdapp=25 pM), the mannosidase treatment caused a marked drop of the Kdapp demonstrating that a limited mannose content in HCMV gB mediated a major part of the interaction with DC-SIGN (Kdapp=149 pM). Finally, we assessed the Kdapp for the PNGase F-treated gB. Surprisingly, we observed an even bigger decrease of the Kdapp compared to the one obtained with the mannosidase-treated gB even though we were expecting a huge increase of the Kdapp. We hypothesized that the PNGase F treatment did not remove all N-glycans from the HCMV gB as already shown by others for the HIV-1 gpl20 (39). To verify our hypothesis, sodium dodecyl sulphate was added to the PNGase digestion to increase accessibility of the enzyme to hidden glycosylation sites. We could observe a sharper band revealed by western blot with a polyclonal anti-gB antibody. In addition, a complete chemical de glycosylation with TFMS led to a similar molecular weight band in gel. All this suggested that the sole PNGase F deglycosylation was uncomplete thus leaving sufficient accessible N-glycans to permit binding to DC-SIGN thus confirming our hypothesis. Finally, untreated or deglycosylated gB was run on a SDS-PAGE gel before being transferred on a membrane and blotted with biotinylated ConA, WGA and Peanut agglutinin (PNA), known to react with O-glycans. Whereas almost no signal was obtained with PNA confirming the absence of O-glycans on the CHO-derived HCMV gB, ConA and WGA allowed for detection of untreated gB in accordance with its heavy N-glycan content. Biotinylated WGA failed to reveal deglycosylated gB regardless of the deglycosylation method employed. Interestingly, the SDS addition led to a tiny band of lower molecular weight demonstrating that very few N- glycan still remained after the PNGase F digestion in this setting. Only TFMS treatment of the gB allowed for a complete deglycosylation as shown by the ConA signal extinction but its stringency was expected to strongly denature the HCMV gB thus preventing its ability to interact with DC-SIGN though this remains to be clearly shown . Altogether, these results fully support that the HCMV gB whether as a CHO-derived recombinant glycoprotein or on a HCMV particle binds DC-SIGN through its mannose containing N-glycans with most likely a poor contribution of the sialic acids if any.

Antibodies against the antigenic domains 4 and 5 are solely able to partially neutralize the HCMV gB/DC-SIGN interaction

In an attempt to map the interaction zone between DC-SIGN and HCMV gB we next took advantage of a set of antibodies directed against the HCMV gB antigenic domains (AD) 1, 2, 4 and 5 and some with unknown specificities. We first used mAbs against AD-1 (clones ITC33, ITC39, ITC48, ITC52 and ITC63B) and AD-2 (clone ITC88) from Dr Mats Ohlin (47) as potential blockers of the HCMV gB binding on DC-SIGN+ cells. None of these mAbs exhibit a neutralizing activity except the commercially available polyclonal antibody that we used to reveal gB in western blot experiments. Interestingly, this pAb always displayed a strong neutralizing activity of the DC-SIGN/HCMV gB interaction suggesting that neutralizing epitopes do really exist. Then, fifteen mAbs of unknown specificities as well as the 1G2 and SM5-1 clones, respectively recognizing AD-5 and AD-4 (53), were tested as blockers in a HCMV gB binding assays on DC-SIGN+ U937 cells (Figure 4C) and immature MD-DCs (Figure 1C). Similar results were obtained for both cell types. These results showed again an almost complete neutralizing activity of the pAb (84-100% mean neutralization; Figures IB and 1C) supporting the idea that DC-SIGN is most likely the only receptor for the HCMV gB on U937 transfectants but also on MD-DCs. Moreover, mAbs 14 and 15 as well as the 1G2 and SM5-1 clones, already known to block HCMV infection of fibroblasts (66, 67), were solely capable of a partial but significant blockade of the gB binding (no more than 48% of mean neutralization; Figures IB and 1C). Based on all these results we concluded that only anti-AD- 4 or AD-5 antibodies could block the HCMV gB/DC-SIGN interaction in our setting suggesting that the contact zone could be located somewhere between both ADs. Noticeably, both domains contain most of known glycosylation sites arguing in favour of their involvement in this interaction (7). Previous studies have proposed that soluble HCMV gB could mainly exist in a post-fusion state even though no formal proof was provided yet (61). Next we wondered whether those antibodies could affect the interaction of DC-SIGN with HCMV gB inserted in the viral envelope. This was tested by analyzing the capture and transmission, so-called trans- infection, of various infectious HCMV strains from a DC-SIGN+U937 cells to highly permissive MRC-5 fibroblasts. Doing this we could show that the anti-HCMV gB pAb kept its blocking activity toward the TB40/E endotheliotropic strain (65-74% mean neutralization) whereas the mAbs 14 and 15 lost their neutralizing properties with whole infectious HCMV particles (Figure 2A). Interestingly, based on structural analyses the SM5-1 mAb is thought to recognize both the pre- and post-fusion HCMV gB conformations (66). This is in line with our current results since both soluble and virion-associated gB were blocked with this mAb. It has been proposed that the SM5-1 mAb could block the pre- to post- fusion conformation transition (76). We obtained very similar results by extending our analysis to other HCMV strains, TRI, an in-house prepared clinical isolate, and VHL/E or Toledo, two low passage laboratory strains (Figure 2B). Here again, SM5-1 -mediated inhibition ranged between 96% to near 100% irrespective of the viral strain. Taken together, these results demonstrated that the HCMV gB blockade may not be the most efficient approach to inhibit the DC-SIGN-mediated capture of the virus and this is meant to be due to different HCMV gB conformational states. However, these results allowed us to define AD-4 and/or AD-5 as the most probable HCMV gB domains interacting with DC-SIGN.

Antibody-mediated targeting of the DC-SIGN CRD totally abrogates interaction with gB and HCMV trans-infection

We next investigated the blockade of DC-SIGN with a set of sixteen non-commercially available anti-DC-SIGN antibodies. First we assessed the fine specificity for each antibody by flow cytometry on the wt DC-SIGN, Aneck or ACRD-expressing cell lines. Based on this we could identify ten anti-CRD mAbs (mAbs 7 to 15), four anti-neck (mAbs 3 to 6) and two antibodies targeting the extra-cellular proximal region (mAbs 1 and 2) which were staining all wt or mutated DC-SIGN-expressing cells (Table 1). In addition, we also tested one anti-neck (H200) and three anti-CRD (1B10, AZN-D1 and MR-1), all commercially available, as blockers of the HCMV gB binding as already done and presented in Figures IB and 1C. We first evaluated the ability of DC-SIGN+ U937 cells to bind recombinant soluble HCMV gB. None of the anti-neck mAbs were able to significantly neutralize gB binding confirming that this part of the DC-SIGN molecule was definitely not involved in this interaction. Almost all anti-CRD mAbs significantly displayed a moderate to high neutralizing activity reinforcing our conclusions on the CRD involvement in the HCMV gB/DC-SIGN interaction. For some CRD- specific mAbs (11 to 15), a total abrogation of the gB binding was even observed (Figure 3A). Those results were confirmed by the blocking capacity of three commercial anti-CRD mAbs, the 1B10, AZN-D1 and MR-1 clones, all known to block interactions between DC-SIGN and various viral ligands. This strongly support that DC-SIGN is most likely the only attachment receptor for the HCMV gB on U937 tranfectants. Interestingly, mAb 1, which was shown to recognize the small extra-cellular proximal region of DC-SIGN (ECPR; AA 59-95), also exhibited a high neutralizing activity. However this epitope was not proximal to the CRD we thought that the mAb fixation could disturb either the DC-SIGN oligomerization or its conformation at the cell surface. To ensure that all this was also true on more relevant cells, similar experiments were performed with immature MD-DCs. Very similar results were obtained in this setting confirming by the way that mAbs from 11 to 15 displayed a strong and reliable capacity to totally block HCMV gB binding to DC-SIGN on MD-DCs (Figure 3B). As a consequence, it could be stated from this that DC-SIGN might be the major if not the only one attachment receptor for the HCMV gB on MD-DCs. Similar results were obtained with L- SIGN, another C-type lectin exhibiting a high degree of homology with DC-SIGN (70%; data not shown). A restricted set of the previously tested anti-DC-SIGN mAbs was used to block the HCMV trans-infection by DC-SIGN-expressing cells to highly permissive MRC-5 cells. We first showed that two out of the three tested anti-neck antibodies inhibited trans-infection suggesting that DC-SIGN tetramer or microdomain organization disruption that most likely occurred could also represent an efficient blocking mechanism for the viral transmission beside the competition for the glycan binding site (Figure 4). All anti-CRD antibodies were shown to drastically impair the TB40/E-GFP trans-infection by DC-SIGN+ U937 cells confirming that DC-SIGN was most likely the only attachment receptor for this HCMV strain on tested cells and also that early antibody-mediated DC-SIGN CRD blockade was efficient to prevent HCMV cell-to-cell transmission (93-95% mean neutralization; Figure 4A). We also reported similar with MD-DCs but inhibition occurred to a lower extent since trans-infection with TB40/E-GFP was somehow reduced depending on mAbs used (from 62 to 75% reduction; Figure 4B). This could be explained by a lower DC-SIGN expression on MD-DCs compared to U937 cells. Trans-infection with three other viral strains, namely VHL/E, Toledo and TRI, a clinical isolate from our laboratory, was consistently inhibited with inhibition ratios ranging from 67-83%, 70- 86% and 79-87.5% respectively and most probably depending on mAb avidities (Figures 4C, 4D and 4E respectively) . Taken together, we demonstrated that most of the anti-CRD antibodies previously shown to block HCMV gB binding to DC-SIGN were also shown to almost completely block trans-infection of various laboratory or clinically-relevant HCMV strains. These results strongly support the idea that DC-SIGN maybe a major attachment receptor if not the only one on MD-DCs for HCMV.

Discussion

In this study, we have shown that the lectin domain is the sole part of DC-SIGN reacting with HCMV gB. Point mutations also provided consistent results showing that most likely DC- SIGN recognizes all its viral ligands, mostly envelope glycoproteins, in a similar manner since amino acid residues known to directly interact with HIV gpl20 (25, 68) or HHV-8 gB (31) were involved in the DC-SIGN/HCMV gB interaction as well with the exception of the V351 A mutant. Indeed in our hands this mutation also increased adhesion of HCMV gB similarly to the already described V351A DC-SIGN mutant towards HIV-1 gpl20 (68). These observations suggest that highly conserved mechanisms of recognition by DC-SIGN could result from a convergence in viral evolution despite the wide array of recognized viruses. The most probable explanation for this relies on the fact that DC-SIGN mainly recognizes flexible high-mannose or fucose-containing sugars on viral glycoproteins (17) thus enabling interaction formation between a non-polymorphic lectin and various ligands. The relatively low affinity binding of DC-SIGN monomers towards sugar residues was shown to be compensated by the lectin multimerization (44). Interestingly, our results demonstrated that when the neck repeat region was deleted in DC-SIGN its binding to HCMV gB was not dramatically altered compared to wt DC-SIGN. Although some previous works showed that recombinant DC-SIGN CRDs alone were unable to multimerize (16, 44) others brought evidence showing that membrane-bound DC-SIGN CRDs but not the neck-repeat-deleted lectin was still capable of forming small microdomains at the cell surface suggesting that clustered DC-SIGN CRDs may keep a certain level of interaction with low-affinity sugar moieties harbored by viral glycoproteins (36). This could be reinforced by the fact that DC-SIGN can bind oligosaccharides through two distinct interaction sites thus potentially increasing affinity for monomeric ligands (15). A recent study reported that unlike HSV-1 or EBV, the HCMV gB was highly N-glycosylated as determined by mass spectrometry with virtually all putative N-glycosylation sites branched with glycans corresponding to a 23kDa approximative molecular weight (61). An additional work relying on the crystal structure of HCMV gB led also to the conclusion that almost all putative N- glycosylation sites were decorated with glycans (7). Our data are in total agreement with these findings. Moreover, we could demonstrate that either the CHO-derived soluble HCMV gB or the whole virion were capable of interacting with WGA reinforcing previous data. Nevertheless, differences were observed regarding their respective DSA binding ability. This suggested a potential bias due to the gB environment itself, ie soluble or virion- associated, leading to barely distinct glycosylation profiles and/or steric hindrance thus preventing virion- associated gB to complex with some lectins. Here, we went deeper in the qualitative analysis of HCMV gB- bearing N-glycans by showing that they were mostly constituted by high-mannose residues in accordance with their behavior towards DC-SIGN or ConA. Interestingly, virions were also strongly able to interact with DC-SIGN suggesting that high-mannose residues decorating HCMV gB might definitely play a prominent role in the viral attachment as already proposed by our team (28). We reported here that unlike recombinant HCMV gB, entire viral particles bound to MPA suggesting that O-glycans were also present on virions but not on the gB itself. These results were in line with a recent report showing that HCMV gB was bearing only two occupied O-glycosylation sites. Conversely, other envelope glycoproteins (gH or gN) or virally- encoded Fey receptors (RL11, RL12 and UL119) were heavily O-glycosylated supporting our findings (3). Despite CHO cells are known to produce highly sialylated glycoproteins we have been surprised to observe an almost undetectable gB or virions binding to SNA suggesting a poor involvement of sialic acids in the overall binding capacity of the virus even though they accounted for a substantial glycan amount on recombinant gB (Figure 3 A and (4). In an attempt to demonstrate the importance of glycans in the HCMV gB/DC-SIGN interaction, we deglycosylated gB with various glycosidases before assessing the ability of treated gB to bind immobilized DC-SIGN CRD by SPR. We could show that the PNGase treatment was unable to completely remove all N-glycans on gB. This was consistent with similar studies on HIV-1 gpl20 (39). A complete deglycosylation could be only obtained using TFMS but such denaturing treatment precluded us to assess the binding of the fully deglycosylated gB to DC- SIGN by SPR. This prevented any permanent conclusion on this point even though we could state from our work that high mannose-containing N-glycans were participating to this interaction.

In an attempt to characterize the HCMV/DC-SIGN interaction, we sought for blocking antibodies either directed against the gB or DC-SIGN. We first looked at the possibility to neutralize the gB itself. Using antibodies of unknown fine specificity, ie targeting a defined AD of HCMV gB, we could show that gB blockade might not be the most efficient way to block its interaction with DC-SIGN although we could entirely neutralize HCMV transmission from MD-DCs to permissive fibroblasts with polyclonal or monoclonal antibodies suggesting that neutralizing epitopes exist and that the overall strategy to specifically block HCMV gB is somehow promising. Interestingly, trans-infection blockades mediated by the anti-AD-4 SM5- 1 and to a lesser extent the anti-AD-5 1G2 mAbs were the most pronounced in our setting. However, trans-infection is a two-step process including the viral binding to DC-SIGN- expressing cells followed by a cell-to-cell close contact allowing viral transmission from the HCMV loaded cells to permissive targets. As a consequence we have not been able to establish precisely how both mAbs functioned. Nevertheless, our work strongly suggests that most likely AD-4 which is the most abundantly glycosylated part of HCMV gB interact with DC-SIGN either as a soluble recombinant or virion- associated ligand (7). Based on structural homologies and functional studies HCMV gB (61) has been assigned to the class III fusion protein family (41) which includes HSV-1 (30) and EBV (2) gBs as well as other phylogenetically divergent viral envelope glycoproteins like the well-known VSV-G (55). Class III fusion proteins exist as pre- and post-fusion states (56) and conversion from one to the other has been shown to occur upon pH drop thus facilitating fusion between the viral envelope and the host cell plasma membrane (12). Based on the pre-fusion VSV-G three dimensional structure, a first model of the pre-fusion HCMV gB was proposed by Spindler and colleagues (66). In this model, AD-4 was located at the most external part of HCMV gB suggesting a huge conformational change to occur. More recently, an electron cryotomography-based approach was implemented to clearly show that membrane- associated HSV-1 gB existed as two distinct conformations (77). These results represented the first demonstration of the co-existence on exosomes mimicking the viral envelope environment of pre- and post-fusion HSV-1 gB. Due to the high homology between HSV-1 and HCMV gBs, it is tempting to speculate that the latter might behave as the former thus providing one possible explanation for why some of the anti-HCMV gB antibodies similarly blocked the interaction with DC-SIGN either using the recombinant post- fusion or the virion-associated pre- and post-fusion gB molecules. Interestingly, this study also provided a distinct pre-fusion structure for HSV-1 gB compared to what was reported by Spindler et al (66) characterized by a more protruding but still laterally located AD-4 domain. Of note, whether this is true for HCMV gB deserves to be demonstrated. However, on this basis, we assume that such a pre-fusion HCMV gB structure also exists thus providing a fair platform to interact with DC-SIGN otherwise known to bind at least post-fusion gB but maybe also the pre- fusion conformer. Given that the DC-SIGN ectodomain size is slightly decreased upon contact with its cognate ligands we also propose to consider DC-SIGN as playing a dual role of attachment receptor and of a cell-specific help for fusion as well (42).

From the specific antibody-mediated blockade experiments presented in this study, we could state that DC-SIGN is the sole receptor for recombinant HCMV gB on MD-DCs as well as on the DC-SIGN+ U937 cell line. A similar efficiency was also shown for the trans-infection of the TB40/E strain by DC-SIGN+ U937 cells suggesting that on those myeloid cells, DC- SIGN was most likely the only virions receptor on the cell surface. This observation was consistent with previous works showing that U937 cells do not express high-mannose specific lectins like the mannose receptor that could interfere with DC-SIGN functions (73). However, we also brought strong indications that the transmission of various HCMV strains by MD-DCs was not totally impaired by the anti-DC-SIGN CRD mAbs though the neutralizing activity for most of these was exceeding 60% (62 to 87.5% reduction rates) depending on viral strains. This may suggest the existence of minor interactions between other viral envelope glycoproteins and cellular receptors distinct from DC-SIGN. A more comprehensive analysis of the potential role played by the pentameric complex composed of gH/gL and UL128L products should be undertaken to better delineate this residual binding of the virus to MD-DCs.

Here we provided keys to understand how the lectin DC-SIGN can directly interact with the HCMV gB, one of the most abundant envelope glycoprotein on HCMV particles. Importantly, this work will pave the way to the generation of a structural model of the HCMV gB/DC-SIGN complex thus providing a basis for the rationalized design of new antivirals. Unlike what was shown previously by others with HIV-1, we propose that this high-mannose- dependent interaction represent the most efficient one to allow for binding of various HCMV laboratory strains or isolates on MD-DCs (26); whether this is also the case with other human tissue-resident or blood circulating DC subsets or macrophages expressing DC-SIGN remains to be established.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

1. Alvarez, C. P., F. Lasala, J. Carrillo, O. Muniz, A. L. Corbi, and R. Delgado. 2002. C-type lectins DC-SIGN and L-SIGN mediate cellular entry by Ebola virus in cis and in trans. J Virol 76:6841-4.

2. Backovic, M., R. Longnecker, and T. S. Jardetzky. 2009. Structure of a trimeric variant of the Epstein-Barr virus glycoprotein B. Proc Natl Acad Sci U S A 106:2880-5.

3. Bagdonaite, I., R. Norden, H. J. Joshi, S. L. King, S. Y. Vakhrushev, S. Olofsson, and H. H. Wandall. 2016. Global Mapping of O-Glycosylation of Varicella Zoster Virus, Human Cytomegalovirus, and Epstein-Barr Virus. J Biol Chem 291: 12014-28.

4. Bohm, E., B. K. Seyfried, M. Dockal, M. Graninger, M. Hasslacher, M. Neurath,

C. Konetschny, P. Matthiessen, A. Mitterer, and F. Scheiflinger. 2015. Differences in N- glycosylation of recombinant human coagulation factor VII derived from BHK, CHO, and HEK293 cells. BMC Biotechnol 15:87.

5. Borst, E. M., L. Standker, K. Wagner, T. F. Schulz, W. G. Forssmann, and M. Messerle. 2013. A peptide inhibitor of cytomegalovirus infection from human hemofiltrate.

Antimicrob Agents Chemother 57:4751-60.

6. Bressollette-Bodin, C, M. Coste-Burel, B. Besse, E. Andre-Garnier, V. Ferre, and B. M. Imbert-Marcille. 2009. Cellular normalization of viral DNA loads on whole blood improves the clinical management of cytomegalovirus or Epstein Barr virus infections in the setting of pre-emptive therapy. J Med Virol 81:90-8.

7. Burke, H. G., and E. E. Heldwein. 2015. Crystal Structure of the Human Cytomegalovirus Glycoprotein B. PLoS Pathog l l:el005227.

8. Chabrol, E., A. Nurisso, A. Daina, E. Vassal-Stermann, M. Thepaut, E. Girard, R. R. Vives, and F. Fiesch. 2012. <Chabrol-Fieschi-12-PlosOne-glycosaminoglycans and langerin and gpl20.pdf>. PLOS One

9. Compton, T., D. M. Nowlin, and N. R. Cooper. 1993. Initiation of human cytomegalovirus infection requires initial interaction with cell surface heparan sulfate. Virology 193:834-41. 10. Cranage, M. P., T. Kouzarides, A. T. Bankier, S. Satchwell, K. Weston, P. Tomlinson, B. Barrell, H. Hart, S. E. Bell, A. C. Minson, and et al. 1986. Identification of the human cytomegalovirus glycoprotein B gene and induction of neutralizing antibodies via its expression in recombinant vaccinia virus. EMBO J 5:3057-63.

11. Curtis, B. M., S. Scharnowske, and A. J. Watson. 1992. Sequence and expression of a membrane- associated C-type lectin that exhibits CD4-independent binding of human immunodeficiency virus envelope glycoprotein gpl20. Proc Natl Acad Sci U S A 89:8356-60.

12. Dollery, S. J., M. G. Delboy, and A. V. Nicola. 2010. Low pH-induced conformational change in herpes simplex virus glycoprotein B. J Virol 84:3759-66.

13. Engering, A., T. B. Geijtenbeek, S. J. van Vliet, M. Wijers, E. van Liempt, N.

Demaurex, A. Lanzavecchia, J. Fransen, C. G. Figdor, V. Piguet, and Y. van Kooyk. 2002. The dendritic cell-specific adhesion receptor DC-SIGN internalizes antigen for presentation to T cells. J Immunol 168:2118-26.

14. Fagerland, M. W. 2012. t-tests, non-parametric tests, and large studies— a paradox of statistical practice? BMC Med Res Methodol 12:78.

15. Feinberg, H., R. Castelli, K. Drickamer, P. H. Seeberger, and W. I. Weis. 2007. Multiple modes of binding enhance the affinity of DC-SIGN for high mannose N-linked glycans found on viral glycoproteins. J Biol Chem 282:4202-9.

16. Feinberg, H., Y. Guo, D. A. Mitchell, K. Drickamer, and W. I. Weis. 2005. Extended neck regions stabilize tetramers of the receptors DC-SIGN and DC-SIGNR. J Biol

Chem 280: 1327-35.

17. Feinberg, H., D. A. Mitchell, K. Drickamer, and W. I. Weis. 2001. Structural basis for selective recognition of oligosaccharides by DC-SIGN and DC-SIGNR. Science 294:2163-6.

18. Feire, A. L., H. Koss, and T. Compton. 2004. Cellular integrins function as entry receptors for human cytomegalovirus via a highly conserved disintegrin-like domain. Proc Natl Acad Sci U S A 101: 15470-5.

19. Feire, A. L., R. M. Roy, K. Manley, and T. Compton. 2010. The glycoprotein B disintegrin-like domain binds beta 1 integrin to mediate cytomegalovirus entry. J Virol 84: 10026-37.

20. Fernandez-Ruiz, M., I. Corrales, M. Arias, J. M. Campistol, E. Gimenez, J. Crespo, M. O. Lopez-Oliva, I. Beneyto, P. L. Martin-Moreno, F. Llamas-Fuente, A. Gutierrez, T. Garcia-Alvarez, R. Guerra-Rodriguez, N. Calvo, A. Fernandez-Rodriguez, J. M. Tabernero- Romo, M. D. Navarro, A. Ramos- Verde, J. M. Aguado, and D. Navarro. 2015. Association between individual and combined SNPs in genes related to innate immunity and incidence of CMV infection in seropositive kidney transplant recipients. Am J Transplant 15: 1323-35.

21. Frascaroli, G., and C. Sinzger. 2014. Distinct properties of human cytomegalovirus strains and the appropriate choice of strains for particular studies. Methods Mol Biol 1119:29-46.

22. Geijtenbeek, T. B., D. J. Krooshoop, D. A. Bleijs, S. J. van Vliet, G. C. van Duijnhoven, V. Grabovsky, R. Alon, C. G. Figdor, and Y. van Kooyk. 2000. DC-SIGN-ICAM- 2 interaction mediates dendritic cell trafficking. Nat Immunol 1:353-7.

23. Geijtenbeek, T. B., D. S. Kwon, R. Torensma, S. J. van Vliet, G. C. van Duijnhoven, J. Middel, I. L. Cornelissen, H. S. Nottet, V. N. KewalRamani, D. R. Littman, C.

G. Figdor, and Y. van Kooyk. 2000. DC-SIGN, a dendritic cell-specific HIV-l-binding protein that enhances trans-infection of T cells. Cell 100:587-97.

24. Geijtenbeek, T. B., R. Torensma, S. J. van Vliet, G. C. van Duijnhoven, G. J. Adema, Y. van Kooyk, and C. G. Figdor. 2000. Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses. Cell 100:575-85.

25. Geijtenbeek, T. B., G. C. van Duijnhoven, S. J. van Vliet, E. Krieger, G. Vriend, C. G. Figdor, and Y. van Kooyk. 2002. Identification of different binding sites in the dendritic cell-specific receptor DC-SIGN for intercellular adhesion molecule 3 and HIV-1. J Biol Chem 277: 11314-20.

26. Granelli-Piperno, A., A. Pritsker, M. Pack, I. Shimeliovich, J. F. Arrighi, C. G.

Park, C. Trumpfheller, V. Piguet, T. M. Moran, and R. M. Steinman. 2005. Dendritic cell- specific intercellular adhesion molecule 3-grabbing nonintegrin/CD209 is abundant on macrophages in the normal human lymph node and is not required for dendritic cell stimulation of the mixed leukocyte reaction. J Immunol 175:4265-73.

27. Guo, Y., H. Feinberg, E. Conroy, D. A. Mitchell, R. Alvarez, O. Blixt, M. E.

Taylor, W. I. Weis, and K. Drickamer. 2004. Structural basis for distinct ligand-binding and targeting properties of the receptors DC-SIGN and DC-SIGNR. Nat Struct Mol Biol 11:591-8.

28. Halary, F., A. Amara, H. Lortat-Jacob, M. Messerle, T. Delaunay, C. Houles, F. Fieschi, F. Arenzana-Seisdedos, J. F. Moreau, and J. Dechanet-Merville. 2002. Human cytomegalovirus binding to DC-SIGN is required for dendritic cell infection and target cell trans-infection. Immunity 17:653-64.

29. Haspot, F., A. Lavault, C. Sinzger, K. Laib Sampaio, Y. D. Stierhof, P. Pilet, C. Bressolette-Bodin, and F. Halary. 2012. Human cytomegalovirus entry into dendritic cells occurs via a macropinocytosis-like pathway in a pH-independent and cholesterol-dependent manner. PLoS One 7:e34795.

30. Heldwein, E. E., H. Lou, F. C. Bender, G. H. Cohen, R. J. Eisenberg, and S. C. Harrison. 2006. Crystal structure of glycoprotein B from herpes simplex virus 1. Science 313:217-20.

31. Hensler, H. R., M. J. Tomaszewski, G. Rappocciolo, C. R. Rinaldo, and F. J. Jenkins. 2014. Human herpesvirus 8 glycoprotein B binds the entry receptor DC-SIGN. Virus Res 190:97-103.

32. Isaacson, M. K., and T. Compton. 2009. Human cytomegalovirus glycoprotein B is required for virus entry and cell-to-cell spread but not for virion attachment, assembly, or egress. J Virol 83:3891-903.

33. Knibbs, R. N., I. J. Goldstein, R. M. Ratcliffe, and N. Shibuya. 1991. Characterization of the carbohydrate binding specificity of the leukoagglutinating lectin from Maackia amurensis. Comparison with other sialic acid-specific lectins. J Biol Chem 266:83-8.

34. Kuo, C. P., C. L. Wu, H. T. Ho, C. G. Chen, S. I. Liu, and Y. T. Lu. 2008.

Detection of cytomegalovirus reactivation in cancer patients receiving chemotherapy. Clin Microbiol Infect 14:221-7.

35. Lantto, J., Y. Lindroth, and M. Ohlin. 2002. Non-germ-line encoded residues are critical for effective antibody recognition of a poorly immunogenic neutralization epitope on glycoprotein B of human cytomegalovirus. Eur J Immunol 32: 1659-69.

36. Liu, P., X. Wang, M. S. Itano, A. K. Neumann, K. Jacobson, and N. L. Thompson. 2012. The formation and stability of DC-SIGN microdomains require its extracellular moiety. Traffic 13:715-26.

37. Liu, Y., A. Tai, K. I. Joo, and P. Wang. 2013. Visualization of DC-SIGN- mediated entry pathway of engineered lentiviral vectors in target cells. PLoS One 8:e67400.

38. Lozach, P. Y., H. Lortat-Jacob, A. de Lacroix de Lavalette, I. Staropoli, S. Foung, A. Amara, C. Houles, F. Fieschi, O. Schwartz, J. L. Virelizier, F. Arenzana-Seisdedos, and R. Altmeyer. 2003. DC-SIGN and L-SIGN are high affinity binding receptors for hepatitis C virus glycoprotein E2. J Biol Chem 278:20358-66.

39. Ma, B. J., S. M. Alam, E. P. Go, X. Lu, H. Desaire, G. D. Tomaras, C. Bowman,

L. L. Sutherland, R. M. Scearce, S. Santra, N. L. Letvin, T. B. Kepler, H. X. Liao, and B. F. Haynes. 2011. Envelope deglycosylation enhances antigenicity of HIV- 1 gp41 epitopes for both broad neutralizing antibodies and their unmutated ancestor antibodies. PLoS Pathog 7:el002200. 40. Manicklal, S., V. C. Emery, T. Lazzarotto, S. B. Boppana, and R. K. Gupta. 2013. The "silent" global burden of congenital cytomegalovirus. Clin Microbiol Rev 26:86- 102.

41. Melnik, L. I., R. F. Garry, and C. A. Morris. 2011. Peptide inhibition of human cytomegalovirus infection. Virol J 8:76.

42. Menon, V., N. Spruston, and W. L. Kath. 2009. A state-mutating genetic algorithm to design ion-channel models. Proc Natl Acad Sci U S A 106: 16829-34.

43. Mezger, M., M. Steffens, C. Semmler, E. M. Arlt, M. Zimmer, G. I. Kristjanson, T. F. Wienker, M. R. Toliat, T. Kessler, H. Einsele, and J. Loeffler. 2008. Investigation of promoter variations in dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN) (CD209) and their relevance for human cytomegalovirus reactivation and disease after allogeneic stem-cell transplantation. Clin Microbiol Infect 14:228-34.

44. Mitchell, D. A., A. J. Fadden, and K. Drickamer. 2001. A novel mechanism of carbohydrate recognition by the C-type lectins DC-SIGN and DC-SIGNR. Subunit organization and binding to multivalent ligands. J Biol Chem 276:28939-45.

45. Mummidi, S., G. Catano, L. Lam, A. Hoefle, V. Telles, K. Begum, F. Jimenez, S. S. Ahuja, and S. K. Ahuja. 2001. Extensive repertoire of membrane-bound and soluble dendritic cell-specific ICAM- 3 -grabbing nonintegrin 1 (DC-SIGN1) and DC-SIGN2 isoforms. Inter-individual variation in expression of DC-SIGN transcripts. J Biol Chem 276:33196-212.

46. Navarro, D., P. Paz, S. Tugizov, K. Topp, J. La Vail, and L. Pereira. 1993.

Glycoprotein B of human cytomegalovirus promotes virion penetration into cells, transmission of infection from cell to cell, and fusion of infected cells. Virology 197: 143-58.

47. Ohlin, M., V. A. Sundqvist, M. Mach, B. Wahren, and C. A. Borrebaeck. 1993. Fine specificity of the human immune response to the major neutralization epitopes expressed on cytomegalovirus gp58/116 (gB), as determined with human monoclonal antibodies. J Virol 67:703-10.

48. Paessens, L. C, J. J. Garcia-Vallejo, R. J. Fernandes, and Y. van Kooyk. 2007. The glycosylation of thymic microenvironments. A microscopic study using plant lectins. Immunol Lett 110:65-73.

49. Palucka, A. K., J. Gatlin, J. P. Blanck, M. W. Melkus, S. Clayton, H. Ueno, E.

T. Kraus, P. Cravens, L. Bennett, A. Padgett- Thomas, F. Marches, M. Islas-Ohlmayer, J. V. Garcia, and J. Banchereau. 2003. Human dendritic cell subsets in NOD/SCID mice engrafted with CD34+ hematopoietic progenitors. Blood 102:3302-10. 50. Patrone, M., M. Secchi, E. Bonaparte, G. Milanesi, and A. Gallina. 2007. Cytomegalovirus UL131-128 products promote gB conformational transition and gB-gH interaction during entry into endothelial cells. J Virol 81: 11479-88.

51. Plotkin, S. A., S. E. Starr, H. M. Friedman, E. Gonczol, and R. E. Weibel. 1989. Protective effects of Towne cytomegalovirus vaccine against low-passage cytomegalovirus administered as a challenge. J Infect Dis 159:860-5.

52. Pohlmann, S., E. J. Soilleux, F. Baribaud, G. J. Leslie, L. S. Morris, J. Trowsdale, B. Lee, N. Coleman, and R. W. Doms. 2001. DC-SIGNR, a DC-SIGN homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans. Proc Natl Acad Sci U S A 98:2670-5.

53. Potzsch, S., N. Spindler, A. K. Wiegers, T. Fisch, P. Rucker, H. Sticht, N. Grieb, T. Baroti, F. Weisel, T. Stamminger, L. Martin-Parras, M. Mach, and T. H. Winkler. 2011. B cell repertoire analysis identifies new antigenic domains on glycoprotein B of human cytomegalovirus which are target of neutralizing antibodies. PLoS Pathog 7:el002172.

54. Razonable, R. R., and A. Humar. 2013. Cytomegalovirus in solid organ transplantation. Am J Transplant 13 Suppl 4:93-106.

55. Roche, S., S. Bressanelli, F. A. Rey, and Y. Gaudin. 2006. Crystal structure of the low-pH form of the vesicular stomatitis virus glycoprotein G. Science 313: 187-91.

56. Roche, S., F. A. Rey, Y. Gaudin, and S. Bressanelli. 2007. Structure of the prefusion form of the vesicular stomatitis virus glycoprotein G. Science 315:843-8.

57. Sakuntabhai, A., C. Turbpaiboon, I. Casademont, A. Chuansumrit, T. Lowhnoo, A. Kajaste-Rudnitski, S. M. Kalayanarooj, K. Tangnararatchakit, N. Tangthawornchaikul, S. Vasanawathana, W. Chaiyaratana, P. T. Yenchitsomanus, P. Suriyaphol, P. Avirutnan, K. Chokephaibulkit, F. Matsuda, S. Yoksan, Y. Jacob, G. M. Lathrop, P. Malasit, P. Despres, and C. Julier. 2005. A variant in the CD209 promoter is associated with severity of dengue disease. Nat Genet 37:507-13.

58. Sallusto, F., and A. Lanzavecchia. 1994. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony- stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med 179: 1109-18.

59. Schaefer, M., N. Reiling, C. Fessler, J. Stephani, I. Taniuchi, F. Hatam, A. O. Yildirim, H. Fehrenbach, K. Walter, J. Ruland, H. Wagner, S. Ehlers, and T. Sparwasser. 2008. Decreased pathology and prolonged survival of human DC-SIGN transgenic mice during mycobacterial infection. J Immunol 180:6836-45. 60. Schlick, K., M. Grundbichler, J. Auberger, J. M. Kern, M. Hell, F. Hohla, G. Hopfinger, and R. Greil. 2015. Cytomegalovirus reactivation and its clinical impact in patients with solid tumors. Infect Agent Cancer 10:45.

61. Sharma, S., T. W. Wisner, D. C. Johnson, and E. E. Heldwein. 2013. HCMV gB shares structural and functional properties with gB proteins from other herpesviruses. Virology

435:239-49.

62. Sinzger, C, G. Hahn, M. Digel, R. Katona, K. L. Sampaio, M. Messerle, H. Hen gel, U. Koszinowski, W. Brune, and B. Adler. 2008. Cloning and sequencing of a highly productive, endotheliotropic virus strain derived from human cytomegalovirus TB40/E. J Gen Virol 89:359-68.

63. Soilleux, E. J., L. S. Morris, G. Leslie, J. Chehimi, Q. Luo, E. Levroney, J. Trowsdale, L. J. Montaner, R. W. Doms, D. Weissman, N. Coleman, and B. Lee. 2002. Constitutive and induced expression of DC-SIGN on dendritic cell and macrophage subpopulations in situ and in vitro. J Leukoc Biol 71:445-57.

64. Sonnabend, J., S. S. Witkin, and D. T. Purtilo. 1983. Acquired immunodeficiency syndrome, opportunistic infections, and malignancies in male homosexuals. A hypothesis of etiologic factors in pathogenesis. JAMA 249:2370-4.

65. Soroceanu, L., A. Akhavan, and C. S. Cobbs. 2008. Platelet-derived growth factor-alpha receptor activation is required for human cytomegalovirus infection. Nature 455:391-5.

66. Spindler, N., U. Diestel, J. D. Stump, A. K. Wiegers, T. H. Winkler, H. Sticht, M. Mach, and Y. A. Muller. 2014. Structural basis for the recognition of human cytomegalovirus glycoprotein B by a neutralizing human antibody. PLoS Pathog 10:el004377.

67. Spindler, N., P. Rucker, S. Potzsch, U. Diestel, H. Sticht, L. Martin-Parras, T. H. Winkler, and M. Mach. 2013. Characterization of a discontinuous neutralizing epitope on glycoprotein B of human cytomegalovirus. J Virol 87:8927-39.

68. Su, S. V., P. Hong, S. Baik, O. A. Negrete, K. B. Gurney, and B. Lee. 2004. DC- SIGN binds to HIV-1 glycoprotein 120 in a distinct but overlapping fashion compared with ICAM-2 and ICAM-3. J Biol Chem 279: 19122-32.

69. Tacken, P. J., W. Ginter, L. Berod, L. J. Cruz, B. Joosten, T. Sparwasser, C. G.

Figdor, and A. Cambi. 2011. Targeting DC-SIGN via its neck region leads to prolonged antigen residence in early endosomes, delayed lysosomal degradation, and cross-presentation. Blood 118:4111-9. 70. Tassaneetrithep, B., T. H. Burgess, A. Granelli-Piperno, C. Trumpfheller, J. Finke, W. Sun, M. A. Eller, K. Pattanapanyasat, S. Sarasombath, D. L. Birx, R. M. Steinman, S. Schlesinger, and M. A. Marovich. 2003. DC-SIGN (CD209) mediates dengue virus infection of human dendritic cells. J Exp Med 197:823-9.

71. van Liempt, E., C. M. Bank, P. Mehta, J. J. Garcia- Vallejo, Z. S. Kawar, R.

Geyer, R. A. Alvarez, R. D. Cummings, Y. Kooyk, and I. van Die. 2006. Specificity of DC- SIGN for mannose- and fucose-containing glycans. FEBS Lett 580:6123-31.

72. Vanarsdall, A. L., B. J. Ryckman, M. C. Chase, and D. C. Johnson. 2008. Human cytomegalovirus glycoproteins gB and gH/gL mediate epithelial cell-cell fusion when expressed either in cis or in trans. J Virol 82: 11837-50.

73. Vigerust, D. J., S. Vick, and V. L. Shepherd. 2012. Characterization of functional mannose receptor in a continuous hybridoma cell line. BMC Immunol 13:51.

74. Wagner, B., B. Kropff, H. Kalbacher, W. Britt, V. A. Sundqvist, L. Ostberg, and M. Mach. 1992. A continuous sequence of more than 70 amino acids is essential for antibody binding to the dominant antigenic site of glycoprotein gp58 of human cytomegalovirus. J Virol 66:5290-7.

75. Wang, X., S. M. Huong, M. L. Chiu, N. Raab-Traub, and E. S. Huang. 2003. Epidermal growth factor receptor is a cellular receptor for human cytomegalovirus. Nature 424:456-61.

76. Wiegers, A. K., H. Sticht, T. H. Winkler, W. J. Britt, and M. Mach. 2015.

Identification of a neutralizing epitope within antigenic domain 5 of glycoprotein B of human cytomegalovirus. J Virol 89:361-72.

77. Zeev-Ben-Mordehai, T., D. Vasishtan, A. Hernandez Duran, B. Vollmer, P.

White, A. Prasad Pandurangan, C. A. Siebert, M. Topf, and K. Grunewald. 2016. Two distinct trimeric conformations of natively membrane- anchored full-length herpes simplex virus 1 glycoprotein B. Proc Natl Acad Sci U S A 113:4176-81.

Claims

CLAIMS:

A method for treating an infection in a subject in need thereof comprising a step of administering to said subject an agent that blocks the interaction between DC-SIGN and an infectious ligand.

The method according to claim 1 wherein the infection is caused by HCMV.

The method according to claim 1 wherein the infectious ligand is HCMV envelope glycoprotein B (HCMV gB).

The method according to claim 1 wherein, the agent that blocks the interaction between DC-SIGN and the infectious ligand is a monoclonal antibody.

The method according to claim 4, wherein, the monoclonal antibody having the specificity for DC-SIGN comprises: a) a heavy chain having at least 70% of identity with the sequence set forth as SEQ ID NO:7; and a light chain having at least 70% of identity with the sequence set forth as SEQ ID NO: 8; b) a heavy chain having at least 70% of identity with the sequence set forth as SEQ ID NO: 15; and a light chain having at least 70% of identity with the sequence set forth as SEQ ID NO: 16; c) a heavy chain having at least 70% of identity with the sequence set forth as SEQ ID NO:23 and a light chain having at least 70% of identity with the sequence set forth as SEQ ID NO:24; d) a heavy chain having at least 70% of identity with the sequence set forth as SEQ ID NO:34 and a light chain having at least 70% of identity with the sequence set forth as SEQ ID NO:36; or e) i) a heavy chain having at least 70% of identity with the sequence set forth as SEQ ID NO:35 and a light chain having at least 70% of identity with the sequence set forth as SEQ ID NO:36. The method according to claim 5, wherein, the monoclonal antibody having the specificity for DC-SIGN comprises: a) i) a heavy chain wherein the variable domain comprises: a H-CDR1 having the sequence set forth as SEQ ID NO: 1; a H-CDR2 having the sequence set forth as SEQ ID NO: 2; a H-CDR3 having the sequence set forth as SEQ ID NO: 3; ii) a light chain wherein the variable domain comprises L-CDR1 having the sequence set forth as SEQ ID NO: 4; a L-CDR2 having the sequence set forth as SEQ ID NO:5; a L-CDR3 having the sequence set forth as SEQ ID NO:6; b) i) a heavy chain wherein the variable domain comprises: a H-CDR1 having the sequence set forth as SEQ ID NO: 9; a H-CDR2 having the sequence set forth as SEQ ID NO: 10; a H-CDR3 having the sequence set forth as SEQ ID NO: 11; ii) a light chain wherein the variable domain comprises L-CDR1 having the sequence set forth as SEQ ID NO: 12; a L-CDR2 having the sequence set forth as SEQ ID NO: 13; a L-CDR3 having the sequence set forth as SEQ ID NO: 14; c) i) a heavy chain wherein the variable domain comprises: a H-CDR1 having the sequence set forth as SEQ ID NO: 17; a H-CDR2 having the sequence set forth as SEQ ID NO: 18; a H-CDR3 having the sequence set forth as SEQ ID NO: 19; ii) a light chain wherein the variable domain comprises L-CDR1 having the sequence set forth as SEQ ID NO: 20; a L-CDR2 having the sequence set forth as SEQ ID NO:21; a L-CDR3 having the sequence set forth as SEQ ID NO:22; d) i) a heavy chain wherein the variable domain comprises: a H-CDR1 having the sequence set forth as SEQ ID NO: 25; a H-CDR2 having the sequence set forth as SEQ ID NO: 26; a H-CDR3 having the sequence set forth as SEQ ID NO: 27; ii) a light chain wherein the variable domain comprises L-CDR1 having the sequence set forth as SEQ ID NO: 31; a L-CDR2 having the sequence set forth as SEQ ID NO:32; a L-CDR3 having the sequence set forth as SEQ ID NO:33; or e) i) a heavy chain wherein the variable domain comprises: a H-CDR1 having the sequence set forth as SEQ ID NO: 28; a H-CDR2 having the sequence set forth as SEQ ID NO: 29; a H-CDR3 having the sequence set forth as SEQ ID NO: 30; ii) a light chain wherein the variable domain comprises L-CDR1 having the sequence set forth as SEQ ID NO: 31; a L-CDR2 having the sequence set forth as SEQ ID NO:32; a L-CDR3 having the sequence set forth as SEQ ID NO:33.

7. The method according to claims 5 to 6 wherein, the monoclonal antibody is an antibody which competes for binding to DC-SIGN with at least one antibody according to claim 4.

8. A nucleic acid molecule encoding the monoclonal antibody according to claims 5 to 6. 9. A nucleic acid molecule which encodes for the heavy chain or the light chain of the monoclonal antibody of claims 5 to 6.

10. A vector comprising the nucleic acid molecule according to claims 8 to 9.

11. A host cell which has been transfected, infected or transformed by the nucleic acid according to claims 8 to 9 and/or the vector according to claim 10.

12. A pharmaceutical composition comprising the monoclonal antibody of claims 5 to 6.