Langerhans cells targeting HIV-1 vaccines

Abstract

Developing an effective HIV-1 vaccine is contingent on generating protective antibodies (Abs). Novel antigen delivery methods are needed to enhance immune responses. One promising approach involves directing antigens to dendritic cells (DC) through fused monoclonal antibodies (mAbs) to amplify both cellular and humoral responses. Here, the inventors aimed to refine Langerhans cells (EC) targeting by designing three Env monochains instead of 2 (EC3. Env3) mimicking natural Env conformation. The inventors demonstrated that EC3. Env3 construct (i) elicited a rapid and potent Env-IgG response, (ii) enhanced the avidity of anti-Env IgG that was accompanied by a marked expansion of Tfh and GC B cells and (iii) swiftly induced the formation of structured germinal centers in dLN, indicative of a robust immune response. Finally, significant Tier-1 NeutAb induction was observed in rabbits immunized with the construct. In conclusion, HIV Env antigen can be adaptively targeted to LC as a timer, intensifying both the magnitude and quality of humoral responses. The present invention thus relates to the use of such a construct as LC targeting HIV-1 vaccines.

Description

[0001] LANGERHANS CELLS TARGETING HIV-1 VACCINES

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine, in particular virology.

[0004] BACKGROUND OF THE INVENTION:

[0005] In recent years, the place of innovative vaccines based on the most recent knowledge of the induction / regulation and modulation of the immune response with the aim to elicit an integrated T and B cell immune responses against complex antigens has emerged beyond “classical” vaccine vectors (recombinant viruses, naked DNA or long peptides). Targeting antigens to endogenous DC appears as a promising strategy to reprogram the immune system (Steinman RM, Banchereau J. Taking dendritic cells into medicine. Nature. 2007;449:419-26). The current approach is to use a specific monoclonal antibody directed against a particular endocytic receptor to carry the antigen to the DC, resulting in processing and presentation of antigens but also in the activation of DC depending on the targeting. Targeting antigens to DC is a vaccine technology concept supported by more than a decade of animal model and human pre-clinical experimentation. In vitro targeting of HIV-1 antigens to DC receptors (e.g., DEC-205, CD40, DCIR, LOX-1) on human PBMCs, or in animal models, induces, even with minute amounts of antigens, strong and sustained T- and B cell responses, associated with control of HIV-1 infection. Despite extensive research over the past years, it remains difficult to define strategies that initiate T follicular helper (Tfh) cell differentiation, particularly in vaccination. Langerhans cells are arrayed at barrier sites of foreign antigen insult and were initially reported as preferentially selecting and expanding antigen-specific cytotoxic T lymphocytes (CTL). Thus Langerhans cells represent an important target to consider for the induction of antibody responses by DC targeting vaccine approaches. In said context, the inventors previously showed that anti-Langerin mAbs fused with the HIV-1 gp140z Envelope (aLC.Env) were efficient for inducing Tfh cell and B cell responses (Kervevan J. et al. Targeting human langerin promotes HIV-1 specific humoral immune responses. PLoS Pathog. 2021 Jul 29;17(7):el009749).

[0006] SUMMARY OF THE INVENTION:

[0007] The present invention is defined by the claims. In particular, the present invention relates to Langerhans cells targeting HIV-1 vaccines. DETAILED DESCRIPTION OF THE INVENTION:

[0008] Main definitions:

[0009] As used herein, the terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.

[0010] As used herein, the term “fusion protein” means a protein created by joining two or more polypeptide sequences together. The fusion polypeptides encompassed in this invention include translation products of a chimeric gene construct that joins the nucleic acid sequences encoding a first polypeptide, e.g., an RNA-binding domain, with the nucleic acid sequence encoding a second polypeptide, e.g., an effector domain, to form a single open-reading frame. In other words, a “fusion polypeptide” or “fusion protein” is a recombinant protein of two or more proteins which are joined by a peptide bond or via several peptides. The fusion protein may also comprise a peptide linker between the two domains. Within the fusion protein, the term "operably linked" is intended to indicate that the peptide of the present invention and the heterologous polypeptide are fused in- frame to each other.

[0011] As used herein, the term “linker” has its general meaning in the art and refers to an amino acid sequence of a length sufficient to ensure that the proteins form proper secondary and tertiary structures. Typically, linkers are those which allow the compound to adopt a proper conformation. The most suitable linker sequences (1) will adopt a flexible extended conformation, (2) will not exhibit a propensity for developing ordered secondary structure which could interact with the functional domains of fusion proteins, and (3) will have minimal hydrophobic or charged character which could promote interaction with the functional protein domains.

[0012] As used herein, the term “domain” refers to any portion of a polypeptide that adopts a tertiary structure.

[0013] As used herein, the terms “multimerization domain” and “multimerize” refer to the ability of a polypeptide, or domain of a polypeptide, to form dimers, trimers, tetramers, pentamers, or hexamers and / or to form heteromers with other multimerization domains. As used herein, the term “trimerization domain” refers to a multimerization domain that forms trimers.

[0014] The term “binding domain” as used herein refers to the one or more regions of a polypeptide that mediate specific binding with a target molecule (e.g., Langerin). Exemplary binding domains include an antibody variable domain, or a receptor binding domain of a ligand.

[0015] As used herein, the term “polynucleotide” as used herein refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”). It also includes modified, for example by alkylation, and / or by capping, and unmodified forms of the polynucleotide. More particularly, the term “polynucleotide” includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids “PNAs”) and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. In some embodiments, the polynucleotide comprises an mRNA. In other aspect, the mRNA is a synthetic mRNA. In some embodiments, the synthetic mRNA comprises at least one unnatural nucleobase. In some embodiments, all nucleobases of a certain class have been replaced with unnatural nucleobases (e.g., all uridines in a polynucleotide disclosed herein can be replaced with an unnatural nucleobase, e.g., 5 -methoxyuridine). In some embodiments, the polynucleotide (e.g., a synthetic RNA or a synthetic DNA) comprises only natural nucleobases, i.e., A, C, T and G in the case of a synthetic DNA, or A, C, T, and U in the case of a synthetic RNA.

[0016] As used herein, the term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as, for example, a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g. , rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "polynucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase “polynucleotide sequence that encodes a protein or a RNA” may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0017] As used herein, the expression “derived from” refers to a process whereby a first component (e.g., a first polypeptide), or information from that first component, is used to isolate, derive or make a different second component (e.g., a second polypeptide that is different from the first one).

[0018] As used herein, the “percent identity” between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443-53.). The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5. In general, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical irrespective of any chemical and / or biological modification. 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; 99 or 100% of identity with the second amino acid sequence.

[0019] As used herein, the term “targeting moiety” refers to any molecule that binds specifically to a target.

[0020] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to an antigen. In natural antibodies of rodents and primates, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains, lambda (1) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. In typical IgG antibodies, the light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans- placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FR) can participate in the antibody binding site, or influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences that together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L- CDR3 and H- CDR1, H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore, typically includes six CDRs, comprising the CDRs set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs. Accordingly, the variable regions of the light and heavy chains typically comprise 4 framework regions and 3 CDRs of the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (Kabat et al., 1992, hereafter “Kabat et al.”). The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues in SEQ ID sequences. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31- 35 (H-CDR1), residues 50-65 (H-CDR2) and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3) according to the Kabat numbering system. For the agonist antibodies described hereafter, the CDRs have been determined using CDR finding algorithms from www.bioinf.org.uk - see the section entitled « How to identify the CDRs by looking at a sequence » within the Antibodies pages.

[0021] As used herein, the term “immunoglobulin domain” refers to a globular region of an antibody chain (such as e.g., a chain of a conventional 4-chain antibody or of a heavy chain antibody or light chain), or to a polypeptide that essentially consists of such a globular region.

[0022] As used herein, the term "antibody fragment" refers to at least one portion of an intact antibody, preferably the antigen binding region or variable region of the intact antibody, that retains the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing / destabilizing, spatial distribution) an epitope of an antigen. “Fragments” comprise a portion of the intact antibody, generally the antigen binding site or variable region. Examples of antibody fragments include Fab, Fab', Fab’-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a “single-chain antibody fragment” or “single chain polypeptide”), including without limitation (1) single - chain Fv molecules (2) single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety and (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multispecific antibodies formed from antibody fragments. Fragments of the present antibodies can be obtained using standard methods.

[0023] As used herein, the term “single domain antibody”, “sdAb” or "VHH" 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 VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.

[0024] As used herein, the term “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0025] As used herein, the term “specificity” refers to the ability of an antibody to detectably bind target molecule (e.g., an epitope presented on an antigen) while having relatively little detectable reactivity with other target molecules. Specificity can be relatively determined by binding or competitive binding assays, using, e.g., Biacore instruments, as described elsewhere herein. Specificity can be exhibited by, e.g., an about 10:1, about 20:1, about 50:1, about 100:1, 10.000: 1 or greater ratio of affinity / avidity in binding to the specific antigen versus nonspecific binding to other irrelevant molecules. The term “binding” as used herein refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bond interactions, including interactions such as salt bridges and water bridges. In particular, as used herein, the term "binding" in the context of the binding of an antibody to a predetermined target molecule (e.g., an antigen or epitope) typically is a binding with an affinity corresponding to a KD of about 10-7M or less, such as about 10-8M or less, such as about 10-9M or less, about 10-10M or less, or about 10-11M or even less. The term “affinity”, as used herein, means the strength of the binding of an antibody to a target molecule (e.g., an epitope). The affinity of a binding protein is given by the dissociation constant Kd. For an antibody said Kd is defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen. The affinity constant Ka is defined by 1 / Kd. Preferred methods for determining the affinity of a binding protein can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference. One preferred and standard method well known in the art for determining the affinity of binding protein is the use of Biacore instruments.

[0026] As used herein, the term "HIV" refers to the human immunodeficiency virus. HIV includes, without limitation, HIV-1. HIV may be either of the two known types of HIV, i.e., HIV-1 or HIV-2. The HIV-1 virus may represent any of the known major subtypes or clades (e.g., Classes A, B, C, D, E, F, G, J, and H) or outlying subtype (Group 0). Also encompassed are other HIV- 1 subtypes or clades that may be isolated.

[0027] As used herein, the term “envelope glycoprotein”, “env glycoprotein” or “Env” refers to, but is not limited to, the glycoprotein that is expressed on the surface of the envelope of HIV virions and the surface of the plasma membrane of HIV infected cells, or a fragment thereof that can induce an immune response or produce an immunity against the HIV in a subject in need thereof. The env gene encodes gp160, which is proteolytically cleaved into gp120 and gp41. More specifically, gp160 trimerizes to (gp 160)3 and then undergoes cleavage into the two noncovalently associated fragments gp120 and gp41. Viral entry is subsequently mediated by a trimer of gp120 / gp41 heterodimers. gp120 is the receptor binding fragment, and binds to the CD4 receptor on a target cell that has such a receptor, such as, e.g., a T-helper cell. Gp41, which is non-covalently bound to gp 120, is the fusion fragment and provides the second step by which HIV enters the cell. Gp41 is originally buried within the viral envelope, but when gp120 binds to a CD4 receptor, gp120 changes its conformation causing gp41 to become exposed, where it can assist in fusion with the host cell. Gpl40 is the uncleaved ectodomain of trimeric gp160, i.e., (gp160)3, that has been used as a surrogate for the native state of the cleaved, viral spike. Thus, the term “HIV-1 gp140”, or “ gp140” or “ gp140 envelope protein” refers to a protein having two disulfide-linked polypeptide chains, the first chain comprising the amino acid sequence of the HIV gp120 glycoprotein and the second chain comprising the amino acid sequence of the water-soluble portion of HIV gp41 glycoprotein (“gp41 portion”).

[0028] As used herein, the term "subject", “host”, “individual” or “patient” refers to a mammal, preferably a human being, male or female at any age that is in-need of a therapy.

[0029] As used herein the term "antigen" refers to a molecule capable of being specifically bound by an antibody or by a T cell receptor (TCR) if processed and presented by MHC molecules. An antigen is additionally capable of being recognized by the immune system and / or being capable of inducing a humoral immune response and / or cellular immune response leading to the activation of B- and / or T-lymphocytes. An antigen can have one or more epitopes or antigenic sites (B- and T- epitopes).

[0030] As used herein, the term “immune response” refers to a reaction of the immune system to an antigen in the body of a host, which includes generation of an antigen-specific antibody and / or cellular cytotoxic response. The immune response to an initial antigenic exposure (primary immune response) is typically, detectable after a lag period of several days to two weeks; the immune response to subsequent stimulus (secondary immune response) by the same antigen is more rapid than in the case of the primary immune response. An immune response to a transgene product may include both humoral (e.g., antibody response) and cellular (e.g., cytolytic T cell response) immune responses that may be elicited to an immunogenic product encoded by the transgene. The level of the immune response can be measured by methods known in the art (e.g., by measuring antibody titre).

[0031] As used herein, the term "Langerhans cells" or “LC” includes hereafter, not only the Langerhans cells which are located in the epidermis, but also the emigrated Langerhans cells in the lymph nodes which have been activated and which are also called "Dendritic cells" and the non-lymphoid dendritic cells located in peripheral tissues, for instance in the pulmonary epithelium.

[0032] As used herein, the term “Langerin” has its general meaning in the art and refers to human C- type lectin domain family 4 member K polypeptide. In some embodiments, Langerin is the isoform of the human canonical sequence as reported by UniProtKB- Q9UJ71 (also referred as human CD207).

[0033] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients 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 patient 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 patient 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 patient 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 patient during treatment of an illness, e.g., to keep the patient 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 interval, 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 “pharmaceutical composition” refers to a composition described herein, or pharmaceutically acceptable salts thereof, with other agents such as carriers and / or excipients. The pharmaceutical compositions as provided herewith typically include a pharmaceutically acceptable carrier.

[0034] As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof.

[0035] As used herein, the term “vaccination” or “vaccinating” means, but is not limited to, a process to elicit an immune response in a subject against a particular antigen.

[0036] As used herein, the term "vaccine composition" is intended to mean a composition which can be administered to humans or to animals in order to induce an immune system response; this immune system response can result in the activation of certain cells, in particular APCs, T lymphocytes and B lymphocytes.

[0037] As used herein, the term “adjuvant” refers to a compound that can induce and / or enhance the immune response against an antigen when administered to a subject or an animal. It is also intended to mean a substance that acts generally to accelerate, prolong, or enhance the quality of specific immune responses to a specific antigen. In the context of the present invention, the term "adjuvant" means a compound, which enhances both innate immune response by affecting the transient reaction of the innate immune response and the more long-lived effects of the adaptive immune response by activation and maturation of the antigen-presenting cells (APCs) especially Dendritic cells (DCs).

[0038] As used herein, the expression "therapeutically effective amount" is meant a sufficient amount of the active ingredient of the present invention to induce an immune response at a reasonable benefit / risk ratio applicable to the medical treatment. Fusion proteins of the present invention:

[0039] The first object of the present invention relates to a fusion protein having the general formula of TM-MD-Ag wherein:

[0040] TM represents an anti-Langerin targeting moiety

[0041] MD represents a multimerization domain

[0042] Ag represents an HIV antigen.

[0043] Anti-Langerin targeting moiety:

[0044] According to the present invention, the targeting moiety is particularly suitable for targeting a population of Langerhans cells.

[0045] According to the present invention, the targeting moiety is a polypeptide having an anti- Langerin binding domain.

[0046] In some embodiments, the anti-Langerin binding domain comprises at least 1, 2, 3, 4, or 5 binding sites.

[0047] In some embodiments, the targeting moiety is an antibody-fragment such as an scFv or VHH or other functional fragment including an immunoglobulin devoid of light chains, Fab, Fab', F(ab*) 2, Fv, antibody fragment, diabody, scAB, single-domain heavy chain antibody, single- domain light chain antibody, Fd, CDR regions, or any portion or peptide sequence of the antibody that is capable of binding antigen or epitope. Typically the fragment derives from a mouse, chimeric, humanized or human antibody. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see e.g., Kohler and Milstein, Nature, 256:495, 1975).

[0048] In some embodiments, the targeting moiety is a heavy 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 is 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 0368 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.

[0049] In some embodiments, the targeting moiety is a single-chain antibody fragment (comprising a variable heavy chain region and / or a variable light chain region). In some embodiments the targeting moiety is selected from a Fab and a scFv. In some embodiments, the targeting moiety is a scFv. In some embodiments, the scFv can be derived from the variable heavy chain (VH) and variable light chain (VL) regions of an antigen-specific mAb linked by a flexible linker. The scFv retains the same specificity and a similar affinity as the full antibody from which it is derived. The peptide linker connecting scFv VH and VL domains joins the carboxyl terminus of one variable region domain to the amino terminus of the other variable domain without compromising the fidelity of the VH-VL paring and antigen-binding sites. Peptide linkers can vary from 10 to 30 amino acids in length. In some embodiments, the scFv peptide linker is a Gly / Ser linker and comprises one or more repeats of the amino acid sequence Gly-Gly-Gly-Ser or Gly-Gly-Gly-Gly-Ser.

[0050] In some embodiments, the scFv derives from the antibody 15B10 having ATCC Accession No. PTA-9852. In some embodiments, the scFv derives from the antibody 2G3 having ATCC Accession No. PTA-9853. In some embodiments, the scFv derives from the antibody 91E7, 37C1, or 4C7 as described in WO2011032161.

[0051] In some embodiments, the anti-Langerin scFv comprises a heavy chain comprising the complementarity determining regions CDR1H, CDR2H and CDR3H of the 15B10 antibody and a light chain comprising the complementarity determining regions CDR1L, CDR2L and CDR3L of the 15B10 antibody.

[0052] In some embodiments, the anti-Langerin scFv comprises a heavy chain comprising the complementarity determining regions CDR1H, CDR2H and CDR3H of the 2G3 antibody and a light chain comprising the complementarity determining regions CDR1L, CDR2L and CDR3L of the 2G3 antibody.

[0053] In some embodiments, the anti-Langerin scFv comprises a heavy chain comprising the complementarity determining regions CDR1H, CDR2H and CDR3H of the 4C7 antibody and a light chain comprising the complementarity determining regions CDR1L, CDR2L and CDR3L of the 4C7 antibody.

[0054] In some embodiments, the scFv comprises a VL and VH sequences as selected from Table A.

[0055] SEQ ID NO : 1 (Amino acid sequence of variable heavy chain region (VH) of 15B10 )

[0056] SEQ ID NO : 2 (Amino acid sequence of variable light chain (VL) 15B10 )

[0057] SEQ ID NO : 3 (Amino acid sequence of variable heavy chain region (VH) of 2G3 )

[0058] SEQ ID NO : 4 (Amino acid sequence of variable light chain (VL) 2G3 )

[0059] SEQ ID NO : 5 (Amino acid sequence of the variable heavy chain (VH) of 407 )

[0060] SEQ ID NO : 6 (Amino acid sequence of variable light chain (VL) of 4C7 )

[0061] In some embodiments, the anti-langerin scFv consists of the amino acid sequences as set forth in SEQ ID NO:7.

[0062] SEQ ID NO : 7 (Amino acid sequence of the 4C7 scFv)

[0063] Multimerization domains

[0064] According to the present invention, the multimerization domain is suitable to promote multimerization of the fusion protein. In particular, the multimerization domain is a trimerization domain.

[0065] Non-limiting examples of multimerization domains that promote stable trimers of soluble recombinant proteins include: the GCN4 leucine zipper, a T4 fibritin trimerization domain, and the trimerization motif from the lung surfactant protein (Hoppe et al. 1994 FEBS Lett 344:191- 195) or collagen (McAlinden et al. 2003 J Biol Chem 278:42200-42207). In some embodiments, the GCN4 trimerization domain comprises the amino acid sequence as set forth in SEQ ID NO:8 (IEDKIEEILSKIYHIENEIARIKKLIGEAP). In some embodiments, the trimerization domain is a fibritin trimerization domain, particularly a bacteriophage fibritin trimerization domain, more particularly a fibritin trimerization domain from bacteriophage T4. The T4 fibritin trimerization domain is, e.g., described in U.S. Pat. No. 6,911,205 or WO 01 / 19958, the contents of which is herein incorporated by reference. In some embodiments, the trimerization domain comprises the amino acid sequence as set forth in SEQ ID NO:9 or a sequence variant having an identity of at least 90% thereto.

[0066] HIV Antigens:

[0067] HIV antigens may be from any type (e.g., HIV-1 , HIV-2), group (e.g., group M, group N, group O, or group P), sub-type or clade (e.g., clade A, B, C, D, F, G, H, J, K) or circulating recombinant form of HIV. In particular, the HIV antigen elicits at least one of a humoral or a cellular immune response in a patient. In some embodiments, the HIV antigen is a HIV envelope glycoprotein.

[0068] In some embodiments, env glycoproteins are selected from the group consisting of gp160, gp140, gp120, or gp41. More particularly the env glycoprotein is gp140. In some embodiments, the HIV gp140 proteins include, but are not limited to, proteins wherein the gp41 portion comprises a point mutation such as I559P. I559P refers to a mutation introduced to change isoleucine at position 559 to proline.

[0069] In some embodiments, the HIV antigen comprises an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO: 10.

[0070] SEQ ID NO : 10 (Amino acid sequence of ZM96 gp140 )

[0071] Linker:

[0072] In some embodiments, the fusion protein of the present invention further comprises a linker. In some embodiments, the linker is used to link the targeting moiety to the multimerization domain.

[0073] Thus, in some embodiments, the fusion protein of the present invention has the general formula of TM-L-MD-Ag wherein:

[0074] TM represents an anti-Langerin targeting moiety,

[0075] L represents a linker,

[0076] MD represents a multimerization domain, and

[0077] Ag represents an HIV antigen.

[0078] In some embodiments, the linker is selected from the group consisting of FlexVl, fl, f2, f3, or f4 as described below. In some embodiments, the linker is FlexV 1.

[0079] Additional embodiments: In some embodiments, the fusion protein of the present invention further comprises one or more heterologous sequences such as purification tags, for example: β-galactosidase, glutathione-S- transferase, green fluorescent proteins (GFP), and epitope tags such as FLAG, myc tag, poly histidine (e.g., 6HIS). In some embodiments, the fusion protein of the present invention comprises an AviTag® sequence. The AviTag® sequence (U.S. Pat. Nos. 5,932,433, 5,874,239 & 5,723,584) is a unique peptide, just 15 residues long, that is recognized by biotin ligase (Schatz P. J., 1993). In the presence of ATP, the ligase specifically attaches biotin to the lysine residue in this sequence. Using vectors, the AviTag™ can be genetically fused to a much bigger polypeptide. This feature effectively allows any polypeptide that has been cloned to be tagged with a biotin molecule. The originality of the AviTag™ is that this peptide can be biotinylated by the E. Coli enzyme BirA. Thus, polypeptides containing this biotinylated peptide either at their NH2 or at their COOH terminus can interact with very strong affinity with streptavidine and can be multimerized or attached to streptavidine-coated surfaces (Altman et al, Science, 1996; Bodinier et al, Nature Medicine, 2000; Rabu et al, J. Biol. Chem., 2005).

[0080] In some embodiments, the amino acid sequences herein described comprise the sequence of a signal peptide. As used herein, the term "signal peptide" has its general meaning in the art and refers to a pre-peptide which is present as an N-terminal peptide on a precursor form of a protein. The function of the signal peptide is to facilitate translocation of the expressed polypeptide to which it is attached into the endoplasmic reticulum. The signal peptide is normally cleaved off in the course of this process. The signal peptide may be heterologous or homologous to the organism used to produce the polypeptide.

[0081] In some embodiments, the fusion protein of the present invention consists of the amino acid sequence as set forth in SEQ ID NO: 16.

[0082] Polynucleotides, vectors and host cells of the present invention:

[0083] A further object of the invention relates to a polynucleotide that encodes for the fusion protein of the present invention.

[0084] Typically, said polynucleotide is a DNA or RNA molecule, which may be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.

[0085] So, a further object of the invention relates to a vector comprising a polynucleotide of the present invention.

[0086] Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said antibody upon administration to a subject. Examples of promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40, LTR promoter and enhancer of Moloney mouse leukemia virus, promoter and enhancer of immunoglobulin H chain and the like. Any expression vector for animal cell can be used, so long as a gene encoding the human antibody C region can be inserted and expressed. Examples of suitable vectors include pAGE107, pAGE103, pHSG274, pKCR, pSGl beta d2-4 and the like. Other examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like. Other examples of viral vector include adenoviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication- defective recombinant viruses may be found for instance in WO 95 / 14785, WO 96 / 22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94 / 19478.

[0087] A further object of the present invention relates to a host cell which has been transfected, infected or transformed by a polynucleotide and / or a vector according to the present invention.

[0088] The polynucleotides of the invention may be used to produce the vaccine product of the present invention in a suitable expression system. Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E.coli, Kluyveromyces or Saccharomyces yeasts. Mammalian host cells include Chinese Hamster Ovary (CHO cells) including dhfr- CHO cells (described in Urlaub and Chasin, 1980) used with a DHFR selectable marker, CHOK1 dhfr+ cell lines, NSO myeloma cells, COS cells and SP2 cells, for example GS CHO cell lines together with GS Xceed™ gene expression system (Lonza), or HEK cells.

[0089] The present invention also relates to a method of producing a recombinant host cell expressing the fusion protein of the present invention, said method comprising the steps of: (i) introducing in vitro or ex vivo a recombinant polynucleotide or a vector as described above into a competent host cell, (ii) culturing in vitro or ex vivo the recombinant host cell obtained and (iii), optionally, selecting the cells which express and / or secrete said fusion protein.

[0090] The host cell as disclosed herein are thus particularly suitable for producing the vaccine product of the present invention. Indeed, when recombinant expression are introduced into mammalian host cells, the polypeptides are produced by culturing the host cells for a period of time sufficient in the host cells and, optionally, secreted into the culture medium in which the host cells are grown. The polypeptides can be recovered and purified for example from the culture medium after their secretion using standard protein purification methods.

[0091] Vaccines:

[0092] A further object of the present invention relates to a vaccine product that consists of a stabilized trimer of the fusion protein of the present invention. Said stabilized trimer thus comprising three env glycoproteins and three targeting moieties. The vaccine product can thus target Langerhans cells to elicit an immune response in a subject in need thereof.

[0093] The vaccine product as described herein may be administered as part of one or more pharmaceutical compositions. Except insofar as any conventional carrier medium is incompatible with the antibodies of the present invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatine; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil, sesame oil; olive oil; com oil and soybean oil; glycols; such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0094] The vaccine products as described herein are particularly suitable for preparing vaccine composition. Thus a further object of the present invention relates to a vaccine composition comprising a vaccine product that consists of a stabilized trimer of the fusion protein of the present invention.

[0095] In some embodiments, the vaccine composition of the present invention comprises an adjuvant. In some embodiments, the adjuvant is alum. In some embodiments, the adjuvant is Incomplete Freund’s adjuvant (IF A) or other oil based adjuvant that is present between 30-70%, preferably between 40-60%, more preferably between 45-55% proportion weight by weight (w / w). In some embodiments, the vaccine composition of the present invention comprises at least one Toll-Like Receptor (TLR) agonist which is selected from the group consisting of TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, and TLR8 agonists. In some embodiments, the adjuvant is a TLR3 agonist. In some embodiments, the adjuvant is Polyinosinic-polycytidylic acid (poly (I:C)) or Poly-ICLC that is a synthetic complex of carboxymethylcellulose, polyinosinic- polycytidylic acid, and poly-L-lysine double-stranded RNA.

[0096] Therapeutic methods:

[0097] The antibodies as well as the pharmaceutical or vaccine compositions as herein described are particularly suitable for inducing an immune response against HIV and thus can be used for vaccine purposes. In particular, the vaccine compositions of the present invention can be suitable to treat a subject (e.g., prevent an HIV infection or evoke a robust immune response to HIV) having, suspected of having, or at risk of developing an infection or related disease, particularly those related to HIV.

[0098] Therefore, a further object of the present invention relates to a method for vaccinating a subject in need thereof against HIV comprising administering a therapeutically effective amount of the vaccine product of the present invention.

[0099] In some embodiments, the subject is a human infant. In some embodiments, the subject is a human child. In some embodiments, the subject is a human adult. In some embodiments, the subject is an elderly human. In some embodiments, the subject is a premature human infant.

[0100] In some embodiments, the subject can be symptomatic or asymptomatic.

[0101] Typically, the active ingredient of the present invention (i.e., the antibodies and the pharmaceutical or vaccine compositions as herein described) is administered to the subject at a therapeutically effective amount. 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 disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed, 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 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. In particular, 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, in particular 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.

[0102] The antibodies and the pharmaceutical or vaccine compositions as herein described may be administered to the subject by any route of administration and in particular by oral, nasal, rectal, topical, buccal (e.g., sub-lingual), parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous) and transdermal administration, although the most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular active agent which is being used.

[0103] In some embodiments, the vaccine compositions of the present invention are particularly suitable for intradermal administration. In the context of the invention, intradermal is synonymous with intracutaneous. An intradermal application is typically given by injection. The intradermal administration is very attractive since the administration results in the local activation of the draining lymph node, resulting in a stronger local activation of the immune system. In particular, the intradermal immunization will cause antigen processing and activation of Langerhans cells

[0104] 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.

[0105] FIGURES:

[0106] Figure 1. Production and quality control of the LC3.Env3vaccine. (A) Diagram illustrating the LC3.Env3vaccine. The T4 fibritin trimerization domain associates with three Gpl40z HIV- 1 envelopes and three single-chain variable fragments (ScFv) of the 4C7 anti-langerin variable domain. Additional tags and linkers (FlexVl) are indicated. (B) The purified LC3.Env3vaccine was subjected to SDS-PAGE under reduced (r) and non-reduced (nr) conditions and stained with Coomassie blue for quality control. Arrows highlight trimeric forms (estimated at 531 kDa, non-reduced condition) versus the monomeric (177 kDa, reduced condition) chains of the vaccine. (C) Size- exclusion chromatography (SEC) diagram of LC3.Env3, depicting elution volume. Line corresponds to the elution of the vaccine, and position of standard molecular weights are mentioned. (D) ELISA binding assay to the human Langerin receptor of LC3.Env3(plain circles) and LC.Env (plain triangles) compared to non-targeting Env trimer (Env3, open circles, dotted line), using the Goat anti-HIV-Env mAb (clone 16H4) for detection. (E) Binding of the LC3.Env3versus LC.Env vaccine was assessed in a dose-dependent manner by FACS on human LC generated in vitro from CD34+ HSC (CD34-LC), compared to Env3and non- targeting hIgG4.Env (crosses). In D and E, data are mean (±SEM) of three independent experiments.

[0107] Figure 2. LC3.Env3elicits robust Germinal Center (GC) / Tfh responses in dLN. (A) C57BL / 6j mice were intradermally immunized without any adjuvant with 5μg of the HIV-1 envelope antigen, equivalent to 7.2qg of LC3.Env3, and 10μg of the monoclonal LC.Env vaccine. A prime-boost schedule was implemented, with a boost at day 21 , followed by sacrifice at day 28. (B) Tfh cell phenotyping. (Left) Representative dot plots of Tfh cells (PD-1+ CXCR5+) of one animal in each group. (Right) Percentage of Tfh cells induced post-boost in each group. (C) Percentages of total B cells, antibody-secreting cells (ASCs), and memory B cells (memB) were analyzed by flow cytometry. (D) Expansion of GC B cells (GL-7+ / FAS+) post-boost in each vaccinated group. Statistical analysis was conducted using Kruskal Wallis's multiple comparison test with Dunn’s correction (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001).

[0108] Figure 3. LC3.Env3improves LC targeting. (A) On the day of euthanasia, auricular dLN were collected, and cells were harvested for INF-γ T cell ELISpot analysis. Responses were quantified as the number of spots per million cells. Open circles represent unstimulated cells, while plain circles depict stimulation with Env peptide pools. (B) Representative dot plots of Env-specific GC B cells identified by biotinylated Env3detected with two anti-biotin PE / anti- biotin APC secondary antibodies. Representative dot plots are on the left, with the percentage of Env+ / +GC B cells in each mouse group on the right. Statistical analysis was conducted using Kruskal Wallis's multiple comparison test with Dunn’s correction (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, non-significant). Data represent the pooled results of three independent experiments. (C) Evaluation of Env- specific IgG in the blood sera of mice vaccinated with LC.Env and LC3.Env3using a Luminex assay with beads coated with the Env3at days 14 and 21 post-prime, and day 28 (one week post-boost). (Left) Data are presented as the mean fluorescent intensity (MFI), with the vertical dotted line indicating the boost at day 21. Geometric means (±SD) are provided. Circles represent the LC3.Env3group, triangles the LC.Env group, and crosses non-immunized mice (PBS). (Right) Overall intensity of the responses calculated by the area under the curve (AUC). Statistical analysis was conducted using Kruskal Wallis's multiple comparison test with Dunn’s correction (*, P<0.05; ***, P<0.001; ****, P<0.0001). (D) Similar to (C), calculation of the avidity index of Env IgG using the Luminex-beads Env3assay. Friedman test was performed for comparing avidity index of Env IgG induced by LC3.Env3at subsequent time points. Data represent the pooled results of three independent experiments.

[0109] Figure 4. Targeting the HIV Env trimer to Langerhans Cells enhances humoral antiviral responses. (A) Comparison of Env IgG titers induced by LC3.Env3and the non-targeting Env3HIV-1 trimer at different immunization doses using the Luminex-beads Env3assay. Geometric means (±SD) of MFI are provided, black circles, LC3.Env3; grey circles, Env3; open circles, lug of Env; plain circles, 250ng of Env. (B&C) The HIV-1 Env3trimer and LC3.Env3were labeled with the AF647 fluorochrome and administered intradermally in mice. The fluorochrome solution from the labeling kit was injected as a control (PBS group). After 24 hours, draining lymph nodes (dLNs) were collected for FACS analysis, evaluating absolute numbers of different resident versus migratory dendritic cell (DC) populations expressing or not expressing the Langerin receptor (B), and absolute numbers of AF647+ cells (considered as vaccine+) among the different DC populations (C). Statistical analysis was performed using the 2-way ANOVA test (*, P<0.05). The data presented are the pooled results of three different independent experiments.

[0110] Figure 5. LC3.Env3induces tier-1 neutralizing antibodies in rabbits. (A) New Zealand white rabbits were subcutaneously immunized with LC3.Env3at a 50μg dose without any adjuvant (plain circles). NaCl injection served as a control (open circles). The vaccine was administered at three time points (vertical dotted lines). Env-specific IgG amounts in sera were determined by ELISA 10-15 days post each injection. Titers are reported as the log (l / EC50). (B) Neutralizing assays were conducted to assess the sera's neutralizing capacity against tier-1 (MW965.26, SF162NW) HIV-1 viruses. Data are presented as Log IC50(mean±SEM). Dashed zone below a titer of 50 corresponds to low or non-neutralizing activity.

[0111] EXAMPLE:

[0112] Background:

[0113] Developing an effective HIV-1 vaccine is contingent on generating protective antibodies (Abs). Novel antigen delivery methods are needed to enhance immune responses. One promising approach involves directing antigens to dendritic cells (DC) through fused monoclonal antibodies (mAbs) to amplify both cellular and humoral responses. Previous studies have shown success in targeting skin Langerhans cells (LC) with anti -Lang erin mAbs fused to HIV-1 Envelope (LC.Env), inducing antigen-specific humoral responses in mice and human LC:T / B co-cultures (Kervevan J. et al. Targeting human langerin promotes HIV-1 specific humoral immune responses. PLoS Pathog. 2021 Jul 29; 17(7) :e 1009749). Here, we aimed to refine LC targeting by designing three Env monochains instead of 2 (LC3.Env3) mimicking natural Env conformation. We aim to investigate whether these new trimeric Env constructs could stimulate germinal center (GC) and Tfh cell reactions, ultimately leading to the production of Env- binding IgG and HIV neutralizing antibodies (NeutAb) in vivo.

[0114] Methods:

[0115] C57BL / 6j (B6) mice were intradermally immunized with LC3.Env3or control LC.Env mAb (5 mcg of Env antigen) at day (D) 0 and D21, without adjuvant. Antibody (magnitude and affinity) and cellular responses were assessed post-prime (PP) and post-boost (PB) using Luminex and FACS, respectively. GC / Tfh reactions in draining lymph nodes (dLN) were monitored through immunofluorescence at various times. Additionally, New Zealand white rabbits were subcutaneously immunized with 50 mcg LC3.Env3 without adjuvants at D0, 28 and 42, and serum NeutAb levels were evaluated.

[0116] Results:

[0117] Compared to LC.Env, LC3.Env3:

[0118] (i) elicited a rapid and potent Env-specific IgG response, with mean titers measuring 22- and 37-fold higher at D14 PP and D7 PB, respectively. (ii) enhanced the avidity of anti-Env IgG, resulting in an index increase of 12% and 36% at D14 PP and D7 PB, respectively. This was accompanied by a marked expansion of Tfh and GC B cells (GL-7+ / Fas+) at D7 PB.

[0119] (iii) swiftly induced the formation of structured germinal centers in dLN, indicative of a robust immune response.

[0120] Significant Tier-1 NeutAb induction was observed in rabbits immunized with LC3.Env3.

[0121] Conclusion:

[0122] This study underscores that HIV Env antigen can be adaptively targeted to LC as a trimer, intensifying both the magnitude and quality of humoral responses without using any adjuvant and after only two shots. In addition, the strategy of targeting SOSIP-like trimeric Env to LC holds substantial promise for eliciting broadly neutralizing antibodies (bNAb) against HIV.

[0123] 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.

Claims

CLAIMS:

1. A fusion protein having the general formula of TM-MD-Ag wherein:TM represents an anti-Langerin targeting moietyMD represents a multimerization domainAg represents an HIV antigen.

2. The fusion protein according to claim 1 wherein the targeting moiety is an antibody- fragment, more particularly a scFv having specificity for Langerin.

3. The fusion protein according to claim 3 wherein the scFv comprises a VL and VH sequences as selected from Table A.

4. The fusion protein according to claim 3 wherein the scFv consists of the amino acid sequences as set forth in SEQ ID NO:7.

5. The fusion protein according to any one of claims 1 to 4 wherein the multimerization domain is a trimerization domain that comprises an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:8 or 9.

6. The fusion protein according to any one of claims 1 to 5 wherein the HIV antigen is a HIV envelope glycoprotein selected from the group consisting of gp160, gp140, gp120, and gp41.

7. The fusion protein according to claim 6 wherein the HIV antigen comprises an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO: 10.

8. The fusion protein according to any one of claims 1 to 7 that has the general formula of TM-L-MD-Ag wherein:TM represents the anti-Langerin targeting moiety,L represents a linker,MD represents the multimerization domain, andAg represents the HIV antigen.

9. The fusion protein according to claim 8 wherein the linker is selected from the group consisting of SEQ ID NO: 11-15.

10. The fusion protein according to any one of claims 1 to 9 that consists of the amino acid sequence as set forth in SEQ ID NO:16.

11. A polynucleotide that encodes for the fusion protein according to any one of claims 1 to 10.

12. A vector comprising the polynucleotide according to claim 11.

13. A host cell which has been transfected, infected or transformed by the polynucleotide according to claim 11 and / or the vector according to claim 12.

14. A vaccine product that consists of a stabilized trimer of the fusion protein according to any one of claims 1 to 10.

15. A pharmaceutical composition that comprises the vaccine product according to claim 14.

16. A method for vaccinating a subject in need thereof against HIV comprising administering a therapeutically effective amount of the vaccine product according to claim 14.

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