Vaccine delivery of HIV-1 ENV trimers to langerhans cells

Abstract

Developing an effective HIV-1 vaccineis 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, vaccine candidates such as LC3.SOSIP(w) and LC3.SOSIP(s) showed promising results in New Zealand white rabbits. These candidates elicited significantly higher Env-specific IgG levels than controls, demonstrated high affinity for SOSIP antigens in ELISA binding assays, and achieved broad neutralizing capabilities against multiple HIV-1 pseudoviruses in TZM-bl neutralization assays. In conclusion, HIV Env antigen can be adaptively targeted to LC as a trimer, intensifying both the magnitude and quality of humoral responses. The present invention thus relates to the use of such constructs as LC targeting HIV-1 vaccines.

Description

[0001] VACCINE DELIVERY OF HIV-1 ENV TRIMERS TO LANGERHANS CELLS

[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). Langerhans cells also 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 gpl40z 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. As used herein, 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.

[0013] 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.

[0014] 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).

[0015] 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).

[0016] 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.

[0017] As used herein, the term “targeting moiety” refers to any molecule that binds specifically to a target.

[0018] 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, transplacental 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.

[0019] 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.

[0020] 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.

[0021] 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 lama sdAb.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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 gpl60, which is proteolytically cleaved into gpl20 and gp41. More specifically, gpl60 trimerizes to (gp 160)3 and then undergoes cleavage into the two noncovalently associated fragments gpl20 and gp41. Viral entry is subsequently mediated by a trimer of gpl20 / gp41 heterodimers. gpl20 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 gpl20, 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 gpl20 binds to a CD4 receptor, gpl20 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 gpl60, i.e., (gpl60)3, that has been used as a surrogate for the native state of the cleaved, viral spike. Thus, the term “HIV-1 gpl40”, or “gp!40” or “gpl40 envelope protein” refers to a protein having two disulfide-linked polypeptide domains, the first domain comprising the amino acid sequence of the HIV gpl20 glycoprotein (“gpl20 domain”) and the second chain comprising the amino acid sequence of the water-soluble portion of HIV gp41 glycoprotein (“gp41 domain”).

[0027] As used herein, the term "modified gp!40 envelope glycoprotein" refers to the gpl40 envelope glycoprotein that has been altered to improve its properties for use in vaccines. This includes modifications such as the A501C and T605C mutations to form a disulfide bond between the gpl20 and gp41 domains, the I559P mutation to facilitate trimerization, the deletion of the gp41 transmembrane and cytoplasmic tail domains to create a soluble protein, and the addition of other mutations to enhance immunogenicity and stability. Furthermore, these modifications may include the T332N substitution to create epitopes for broadly neutralizing antibodies (bNAbs) and the removal of the membrane-proximal external region (MPER) to reduce aggregate formation. The modified gpl40 envelope glycoprotein leads to the formation of stabilized trimers that retain the epitopes for several neutralizing antibodies and related agents (CD4-IgG2, bl 2, 2G12, 2F5 and 4E10) and the CD4-IgG2 molecule, so that the overall antigenic structure of the gpl40 protein has not been adversely impaired by the trimer-stabilizing substitutions.

[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.]).

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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. 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.

[0039] Fusion proteins of the present invention:

[0040] The first object of the present invention relates to a fusion protein wherein an anti-Langerin targeting moiety is fused to a modified gpl40 envelope glycoprotein (SOSIP) that comprises a) the A501C and T605C mutations to form a disulfide bond between gpl20 and gp41 domains (SOS) and b) the mutation I559P to facilitate trimerization (IP).

[0041] Modified gp!40 envelope glycoproteins:

[0042] In some embodiments, the modified gpl40 envelope glycoprotein of the present invention derives from a gpl40 envelope glycoprotein that is isolated from HIV-1 Clade A virus, HIV-1 Clade B virus, HIV-1 Clade C virus, a HIV-1 Clade A pseudo-virus, HIV-1 Clade B pseudovirus or a HIV-1 Clade C pseudo-virus. The modified gpl40 envelope glycoprotein of the present invention derives from an gpl40 envelope glycoprotein that is isolated from the 6535 virus, the 13095 virus, the 16055 virus, the 25710 virus, the 25925 virus, the CAAN virus or the ZmlO9F virus. In some embodiments, the modified gpl40 envelope glycoprotein of the present invention derives from an gpl40 envelope glycoprotein that is isolated from the HIV-1 strain KNH1144 as described in US Patent No. 7,939,083. In some embodiments, the modified gpl40 envelope glycoprotein of the present invention derives from an gpl40 envelope glycoprotein that uses the tier 2, HIV-1 BG505, subtype A gpl40 as its base sequence Sanders, Rogier W., et al. "A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP. 664 gpl40, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies. " PLoS pathogens 9.9 (2013): e 1003618).

[0043] In some embodiments, the modified gpl40 envelope glycoprotein of the present invention does not comprise the gp41 transmembrane (TM) and cytoplasmic tail (CT) domains so as to create a soluble gpl40 envelope glycoprotein.

[0044] In some embodiments, the membrane-proximal external region (MPER) is also deleted to improve trimer solubility and reduce aggregate formation. As used herein, the term "membrane-proximal external region" or “MPER” refers to a segment of the gp41 subunit of the HIV-1 envelope glycoprotein complex that is located adjacent to the viral membrane and plays a crucial role in the process of viral fusion and entry into the host cell.

[0045] In some embodiments, the modified gpl40 envelope glycoprotein of the present invention also comprises the T332N substitution to create the epitopes for several bNAbs that depend on the presence of a glycan moiety.

[0046] In some embodiments, the modified gpI40 envelope glycoprotein of the present invention comprises a gpl20 domain that consists of an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO: 1.

[0047] SEQ ID NO: 1> gp!20 domain NLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMV EQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYR LDWQINENQGNRSNNSNKEYRLINCNTSACTQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPC PSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIR IGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKWKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNC GGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQCMYAPPIQGVIRCVSN ITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKWKIEPLGVAPTRCKRRWG

[0048] In some embodiments, the modified gpl40 envelope glycoprotein of the present invention comprises a gp41 domain that consists of an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:2.

[0049] SEQ ID NO: 2> gp41 domain AVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARV LAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEES QNQQEKNEQDLLALD In some embodiments, the cleavable sequence (i.e., REKR) (SEQ ID NO:3) that is located between the gp120 and gp41 domains is replaced by a furin cleavable sequence. Furin is an intracellular protease that recognizes and cleaves the specific amino acid sequence R-X-K / R-R (SEQ ID NO:4), where X can be any amino acid (Thomas, Gary. "Furin at the cutting edge: from protein traffic to embryogenesis and disease. "Nature reviews Molecular cell biology 3.10 (2002): 753-766). In particular, the furin cleavable sequence consists of the amino acid sequence as set forth in SEQ ID NO:5 (i.e., RRRRRR). By incorporating a furin cleavable site into the gpl40 envelope glycoprotein, the cleavage process can be more efficiently controlled within the host cells, leading to the production of a more stable and correctly processed protein. This modification can enhance the overall functionality and immunogenicity of the gpl40 trimer, ensuring it retains the necessary epitopes for effective immune recognition.

[0050] In some embodiments, the modified gp140 envelope glycoprotein of the present invention consists of the BG505 SOSIP.664(w) sequence that is disclosed in Sanders, Rogier W., et al. "A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP. 664 gpl40, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies. " PLoS pathogens 9.9 (2013): e!003618.

[0051] In some embodiments, the modified gp140 envelope glycoprotein of the present invention consists of the amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:6.

[0052] SEQ ID NO: 6> BG505 SOSIP.664 (w) sequence NLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMV EQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYR LDWQINENQGNRSNNSNKEYRLINCNTSACTQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPC PSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIR IGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKWKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNC GGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQCMYAPPIQGVIRCVSN ITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKWKIEPLGVAPTRCKRRWGRRRRRRAVGI GAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVE RYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQ EKNEQDLLALD

[0053] In some embodiments, the cleavable sequence (i.e REKR) that is located between the gp120 and gp41 domains is replaced by a linker. Typically the linker plays a crucial role in ensuring the proper folding and functionality of the resulting protein. In some embodiments, the flexible linker sequence that is composed of glycine and serine residues has proven particularly effective. These amino acids are known for their flexibility and ability to maintain the spatial arrangement necessary for proper protein folding and functionality. In some embodiments, the linker consists of the amino acid sequence as set forth in SEQ ID NO:7 (i.e., GGGSGGGGSGGGGSGG). This particular linker sequence provides sufficient length and flexibility to allow for the necessary conformational changes, facilitating the creation of a stable and effective gpl40 trimer.

[0054] In some embodiments, the modified gp140 envelope glycoprotein of the present invention consists of the SOSIP BG505.664(s) sequence that is disclosed in Ivelin S. Georgiev et al. Single-Chain Soluble BG505. SOSIP gp!40 Trimers as Structural and Antigenic Mimics of Mature Closed HIV- 1 Env. Journal of Virology 89(10). 2015.

[0055] In some embodiments, the modified gpI40 envelope glycoprotein of the present invention consists of the amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO: 8.

[0056] SEQ ID NO: 6> SOSIP BG505.664 ( s ) sequence NLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMV EQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYR LDWQINENQGNRSNNSNKEYRLINCNTSAITQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPC PSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIR IGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKWKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNC GGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQAMYAPPIQGVIRCVSN ITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKWKIEPLGVAPTRCKRRWGGGSGGGGSGG GGSGGAVGIGAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQS LLRAPEAQQHLLKLTVWGIKQ LQARVLAVERYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYG LLEESQNQQEKNEQDLLALD

[0057] Anti-Langerin targeting moiety:

[0058] According to the present invention, the targeting moiety is particularly suitable for targeting a population of Langerhans cells.

[0059] According to the present invention, the targeting moiety is a polypeptide having an anti-Langerin binding domain.

[0060] In some embodiments, the anti-Langerin binding domain comprises at least 1, 2, 3, 4, or 5 binding sites. 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, singledomain 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).

[0061] In some embodiments, the targeting moiety is a 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 0368684, 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.

[0062] 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.

[0063] 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. 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.

[0064] 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.

[0065] 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.

[0066] In some embodiments, the scFv comprises a VL and VH sequences as selected from Table A

[0067]

[0068] SEQ ID NO: 9 (Amino acid sequence of variable heavy chain region (VH) of 15B10) SVKMSCKASGYTFTDYVISWVKQRTGQGLEWIGDIYPGSGYSFYNENFKGKATLTADKSSTTAYMQLSS LTSEDSAVYFCA SEQ ID NO: 10 (Amino acid sequence of variable light chain (VL) 15B10) ASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTNFTLKISRVEAED LGLYFCS SEQ ID NO: 11 (Amino acid sequence of variable heavy chain region (VH) of 2G3) SLKLSCAASGLTFNIYAMNWVRQAPGKGLEWVARIRNKSNNYATYYADSVKDRFTISRDDSQSLLYLQM NNLKTEDTAMYYC SEQ ID NO: 12 (Amino acid sequence of variable light chain (VL) 2G3 ) VTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRVSGVPARFSGSLIGDKAALTITGAQTEDEA IYFCA SEQ ID NO: 13 (Amino acid sequence of the variable heavy chain (VH) of 4C7 ) QVQLQQSGAELVRPGASVTLSCKASGYTFIDHDMHWVQQTPVYGLEWIGAIDPETGDTGYNQKFKGKAI LT AD K S S RT AYME LRSLTSED S AVY YCT IPFYYSNYSP FAY GQ GT LVT VS SEQ ID NO: 14 (Amino acid sequence of variable light chain (VL) of 407) QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQRKPGSSPKPWIYATSNLASGVPARFSGSGSGTS YSLTISRVEAEDAATYYCQQWSSNPLTFGAGTKLEIK

[0069] In some embodiments, the anti-langerin scFv consists of the amino acid sequences as set forth in SEQ ID NO: 15.

[0070] SEQ ID NO: 15 (Amino acid sequence of the 407 scFv) QVQLQQSGAELVRPGASVTLSCKASGYTFIDHDMHWVQQTPVYGLEWIGAIDPETGDTGYNQKFKGKAI LT AD K S S RT AYME LRSLTSED S AVY YCT IPFYYSNYSP FAYWGQ GT LVT VS SGGGGSGGGGSGGGGS QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQRKPGSSPKPWIYATSNLASGVPARFSGSGSGTS YSLTISRVEAEDAATYYCQQWSSNPLTFGAGTKLEIK

[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 modified gp140 envelope glycoprotein.

[0073] Thus, in some embodiments, the fusion protein of the present invention has the general formula of TM-L-gpl40 wherein:

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

[0075] L represents a linker, and,

[0076] gpl40 represents the modified gpl40 envelope glycoprotein.

[0077] 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 FlexVl.

[0078] QTPTNTISVTPTNNSTPTNNSNPKPNP ( flexVl, SEQ ID NO: 16) SSVSPTTSVHPTPTSVPPTPTKSSP ( fl, SEQ ID NO: 17 ) PTSTPADSSTITPTATPTATPTIKG ( f2, SEQ ID NO: 18 ) TVTPTATATPSAIVTTITPTATTKP ( f 3, SEQ ID NO: 19)

[0079] TNGS I TVAATAPTVTPTVNAT P SAA ( f4, SEQ ID NO: 20)

[0080] 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: P-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. Colt 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 streptavidin-coated surfaces (Altman et al, Science, 1996; Bodinier et al, Nature Medicine, 2000; Rabu et al, J. Biol. Chem., 2005)

[0081] 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.

[0082] Best embodiments:

[0083] In some embodiments, the fusion protein of the present invention consists of an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:21 or SEQ IDNO:22.

[0084] SEQ ID NO: 21 > LC3. SOSIP (w) (vP81) NLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMV EQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYR LDWQINENQGNRSNNSNKEYRLINCNTSACTQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPC PSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIR IGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKWKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNC GGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQCMYAPPIQGVIRCVSN ITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKWKIEPLGVAPTRCKRRWGRRRRRRAVGI GAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVE RYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQ EKNEQDLLALD / TPTNTTSVTPTNNSTPTNNSNPKPNP / GLNDIFEAQKTEWHE / HHHHHH / QVQLQQS GAELVRPGASVTLSCKASGYTFIDHDMHWVQQTPVYGLEWIGAIDPETGDTGYNQKFKGKAILTADKSS RTAYMELRSLTSEDSAVYYCTIPFYYSNYSPFAYWGQGTLVTVS / SGGGGSGGGGSGGGGS / QIVLSQS PAILSASPGEKVTMTCRASSSVSYMHWYQRKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISR VEAEDAATYYCQQWSSNPLTFGAGTKLEIK SEQ ID NO: 22 > LC3. SCSI P ( s ) (vP66) NLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMV EQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYR LDWQINENQGNRSNNSNKEYRLINCNTSACTQACPKVSFEPIPIHYCAPAGFAILKCKDKKFNGTGPC PSVSTVQCTHGIKPWSTQLLLNGSLAEEEVMIRSENITNNAKNILVQFNTPVQINCTRPNNNTRKSIR IGPGQAFYATGDIIGDIRQAHCNVSKATWNETLGKWKQLRKHFGNNTIIRFANSSGGDLEVTTHSFNC GGEFFYCNTSGLFNSTWISNTSVQGSNSTGSNDSITLPCRIKQIINMWQRIGQCMYAPPIQGVIRCVSN ITGLILTRDGGSTNSTTETFRPGGGDMRDNWRSELYKYKWKIEPLGVAPTRCKRRWGRRRRRRAVGI GAVFLGFLGAAGSTMGAASMTLTVQARNLLSGIVQQQSNLLRAPEAQQHLLKLTVWGIKQLQARVLAVE RYLRDQQLLGIWGCSGKLICCTNVPWNSSWSNRNLSEIWDNMTWLQWDKEISNYTQIIYGLLEESQNQQ EKNEQDLLALD / LK / TPTNTI SVTPTNNSTPTNNSNPKPNP / GLNDIFEAQKIEWHE / HHHHHH / QVQL QQSGAELVRPGASVTLSCKASGYTFIDHDMHWVQQTPVYGLEWIGAIDPETGDTGYNQKFKGKAILTAD KSSRTAYMELRSLTSEDSAVYYCTI PFYYSNYSPFAYWGQGTLVTVS / SGGGGSGGGGSGGGGS / QIVL SQSPAILSASPGEKVTMTCRASSSVSYMHWYQRKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLT ISRVEAEDAATYYCQQWSSNPLTFGAGTKLEIK

[0085] Polynucleotides, vectors and host cells of the present invention:

[0086] A further object of the invention relates to a polynucleotide that encodes for the fusion protein of the present invention.

[0087] 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.

[0088] So, a further object of the invention relates to a vector comprising a polynucleotide of the present invention.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] Vaccines:

[0095] 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.

[0096] According to the present invention, the 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.

[0097] 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 corn 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.

[0098] 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.

[0099] 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.

[0100] Therapeutic methods:

[0101] 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.

[0102] 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.

[0103] 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.

[0104] In some embodiments, the subject can be symptomatic or asymptomatic. 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.

[0105] 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.

[0106] 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

[0107] 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.

[0108] FIGURES:

[0109] Figure 1. Production and Quality Control of LC3. SOSIP(w). (A) This diagram illustrates the LC3. SOSIP(w) vaccine. The BG505.664 Env sequence is linked via a flexible linker (Flexvl) to three single-chain variable fragments (ScFvs) derived from the 4C7 anti-Langerin variable domain. The gpl40 Env sequence is cleaved by furin into the gp41 ectodomain and the gp120 domain, resulting in the formation of trimers. (B) A size-exclusion chromatography (SEC) diagram shows the purification of LC3. SOSIP(w) from CHO supernatants using a HIS-tag column and FPLC elution. The elution profile over time indicates the position of the eluted vaccine fraction (Fl, indicated by a dotted square), along with markers for standard molecular weights. (C) The purified LC3. SOSIP(w) vaccine from fraction Fl was analyzed using SDS-PAGE and stained with Coomassie blue for quality control. Arrows indicate trimeric forms (shown under non-reducing conditions, nr) compared to the monomeric chains of the vaccine (shown under reducing conditions, r). (D) Electron microscopy (EM) images of LC3. SOSIP(w) (left panel) reveal the structure of HIV-1 envelope trimers, highlighted with white blurred squares in the right panel.

[0110] Figure 2. Production and Quality Control of LC3. SOSIP(s). (A) This diagram illustrates the LC3. SOSIP(s) vaccine. As shown in Fig-1, the BG505.664 Env sequence features a GS linker connecting the gp41 and gp120 domains. This sequence is attached via a flexible linker (Flexvl) to three single-chain variable fragments (ScFvs) derived from the 4C7 anti-langerin variable domain. The gp41 ectodomain and gp120 domains are covalently linked by a repeated GS motif to stabilize the structure, leading to the formation of trimers. (B) The size-exclusion chromatography (SEC) diagram demonstrates the purification of LC3. SOSIP(s) from CHO supernatants using a HIS-tag column and FPLC elution. The elution profile indicates the presence of two eluted vaccine fractions (Fl and F2, marked by a dotted square) alongside standard molecular weight markers. (C) The purified LC3. SOSIP(w) vaccine from fractions Fl and F2 was analyzed using SDS-PAGE and stained with Coomassie blue for quality control. Arrows indicate trimeric forms (shown under non-reducing conditions, nr) compared to the monomeric chains of the vaccine (shown under reducing conditions, r). Fraction Fl was enriched in Env trimers and was used in preclinical animal models (see Fig 3 and 4). (D) Electron microscopy (EM) images of LC3. SOSIP(s) (left panel) reveal the structure of HIV- 1 envelope trimers, highlighted by white blurred squares in the right panel. Notably, a 3D trimeric structure of LC3. SOSIP(s) was predicted from the EM analysis (right panel).

[0111] Figure 3. LC3. SOSIP(w) and LC3. SOSIP(s) induce SOSIP binding IgG and tier-1 neutralizing antibodies in rabbits. (A) New Zealand white rabbits (n=5 per group) were subcutaneously immunized either with LC3. SOSIP(w) (plain circles) or LC3. SOSIP(s) (open circles), at a 30pg dose without any adjuvant (equivalent to 20 pg dose of Env antigen). NaCl injection served as a control (cross symbols). The vaccine was administered at three time points (DO, D28, D42, dotted lines). Env-specific IgG amounts in sera were determined by Luminex, using beads coated with either SOSIP(w) (left panel) or SOSIP(s) (right panel). Titers are reported as the log (1 / EC50). (B) Neutralizing assays were conducted to assess the sera's neutralizing capacity against tier-1 (MW965.26, open circles; SF162NW, open triangles) HIV-1 viruses. Data are presented as Log IC50(mean±SEM). Dashed zone below a titer of 20 corresponds to low or non-neutralizing activity. (C) Env-specific IgG titers were compared in rabbits immunized with a low dose (4 pg) of BG505 SOSIP.664 either targeted to the Langerin receptor (LC3. SOSIP(w), plain circles) or non-targeted (open circles). Mean MFI values ± SEM are reported for animals immunized without adjuvant (left) or co-administered with MPLA (right). Statistical analysis was performed using the Kruskal-Wallis test with Dunn’s multiple comparison correction, comparing vaccinated groups (**p < 0.01). (D) As in (B), NeutAb responses were assessed against the MW965.26 strain in rabbits immunized with a low dose of either LC3. SOSIP(W) or BG505 SOSIP.664(w). Sequential IRD50values for individual animals are shown, with or without MPLA adjuvant (solid vs dotted lines).

[0112] Figure 4. LC3. SOSIP(s) elicits Env-specific antibody responses in mice. (A) C57BL / 6j mice were intradermally immunized without any adjuvant using 1.44 pg of LC3. SOSIP(s), which is equivalent to 1 pg of the HIV-1 envelope antigen. A prime-boost schedule was followed, with a boost administered on day 21, and the mice were sacrificed on day 28. (B) Env IgG titers were measured on day 28 using ELISA, comparing serial dilutions of sera from vaccinated mice (n=4) to those from mock-immunized mice (PBS, n=4), using SOSIP(s) as the target Env antigen. The titers were reported as the logarithm of the dilution factor. Statistical analyses were conducted using non-parametric Mann-Whitney t-tests were used (*; P < 0.05). LOQ, limit of quantification.

[0113] EXAMPLE:

[0114] Methods:

[0115] Mice

[0116] Male and female C57BL / 6j mice aged seven to eight weeks, were obtained from Charles River. Mice were kept in a pathogen-free environment with human care in the infrastructural facility of the Mondor Institute of Biomedical Research (U955 INSERM-Paris Est Creteil University, France). The mice were kept at 20-24 °C, 50% ± 15% humidity, and on a 12-hour light / 12-hour dark cycle.

[0117] Animal housing and experimental procedures were conducted according to French and European Regulations (Parlement Europeen et du Conseil du 22 septembre 2010, Decret n° 2013-118 du ler fevrier 2013 relatifa la protection des animaux utilises a des fins scientifiques) and the National Research Council Guide for the Care and Use of Laboratory Animals (National Research Council (U. S.), Institute for Laboratory Animal Research (U. S.), and National Academies Press (U. S.), Eds., Guide for the care and use of laboratory animals, 8th ed. Washington, D. C: National Academies Press, 2011). The French authorities accepted all procedures presented to the Institutional Animal Care and Use Committee (APAFIS#25329-2020051119073072 v4).

[0118] Rabbits

[0119] New Zealand white rabbits were housed and immunized at Charles River Laboratories (France).

[0120] Immunogens & reagents

[0121] Production and quality controls of vaccines and antigens

[0122] The HIV-1 SOSIP Envelop (BG505.664) sequence was inserted at the N-terminal codon of the anti-Langerin variable domains (in-house developed mAb, clone 4C7, cross-reacting with the human, rabbit and mouse Langerin[l]), which were associated as ScFv using a GS(4) linker. The BG505.664(w) trimeric Env structure, provided by Weissenhorn’s lab, forms after furin cleavage, while the BG505.664(s) trimeric Env structure, obtained from the NIH, contains a GS linker between the gp41 and gpl20 domains. Both sequences share 97.35% amino acid homology (Table-1). These novel Langerin-targeting vaccine constructs lack a human IgG4 backbone but include 6HIS-tag and AviTag sequences for FPLC purification and biotinylation, respectively. Previously published methods were followed for cloning vectors, generating stably transfected Expi-CHO-S cells, purifying antibodies via his-tag collection, and filtering using FPLC (ÄKTA Pure, Cytiva). The recombinant constructs were stored in 125 mM cavitron cyclodextrin buffer or 1M arginine buffer (100 mM Tris, pH 7) at -80°C until use. Quality control assessments included: i) SDS-PAGE with Coomassie staining, ii) endotoxin levels below 0.5 ng / mg of protein, and iii) Cytiva SEC 200 26 / 600 G analysis as per manufacturer instructions. Trimeric structures of vaccines were further validated by electron microscopy (EM). The LC3. SOSIP(w) and LC3. SOSIP(s) vaccines had molecular weights of 307 kDa and 315 kDa, respectively, in trimeric form, and 102 kDa and 105 kDa in monomeric form. Glycosylation of Env led to higher molecular weight bands than expected.

[0123] Animal immunization and groups

[0124] Mice and immunization schemes

[0125] Mice were primed and boosted intradermally (i.d.) with 10μL / ear of the vaccine without any adjuvant. Doses corresponding to 1 pg of Env bound to an anti-Langerin construct (LC3SOSIP(s)) were applied i d. in the ears of the mice. Animals injected i d. with PBS were used as a control group. Every week from prime until the sacrifice on day 28, blood was drawn, and serum was examined by ELISA for anti-Env IgG antibodies.

[0126] Rabbits ’ immunization schemes

[0127] New Zealand white rabbits (at Charles River Laboratories) were immunized subcutaneously (s.c.) with the LC3. SOSIP(w) or LC3. SOSIP(s) at 30pg dose without any adjuvant. NaCL injection was used as a control. The vaccine was injected at 3 -time points (DO, D28, D42) and blood samples were collected 10-15 days after each injection. Blood Sera were used for Env-specific IgG quantification by Luminex, and for HIV neutralizing assay.

[0128] Vaccine-induced Immune responses

[0129] Antibodies

[0130] Env IgG titration by Luminex assay

[0131] The quantity of Env-specific IgG from rabbit serum was measured using Luminex bead-based immunoassay. Luminex beads were made by employing the Bio-Plex Amine Coupling Kit (Bio-Rad, France) to covalently couple the Trimeric HIV-1 SOSIP(s) or SOSIP(w) Env protein that was manufactured in-house with MagPlex beads. A freshly made solution of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC, 2.5 mg) and N-hydroxysulfosuccinimide (S-NHS, 2.5 mg) (Thermo Fischer, USA) was added to an activation buffer to resuspend 10×106MagPlex-COOH Microspheres (Bio-Rad). The activated beads were stirred at room temperature for 20 minutes using a Thermo Fischer Hula-Mixer. Following the PBS washing of the beads, 100μg of the HIV-1 Env antigen was added to 1 mL of PBS. Two hours at RT coupling reaction were conducted with beads agitation. After washing the beads, they were again suspended in 500 pL of blocking buffer and allowed to agitate at room temperature for 30 minutes. Beads were lastly rinsed with storage buffer, resuspended in 1mL of buffer, counted with an Auto-2000 cell counter (Nexcelom), and kept at 4°C in the dark until needed. Trimeric HIV-1 Env linked beads have been added (50μL) to a Bio-Plex Flat Bottom Pro 96-well Plate (Bio-Rad) after being diluted at 40,000 beads / mL in PBS. After two cycles of PBS (0.05% tween) rinsing in a Bio-Rad magnetic plate washer, 50 pL of separate serum samples, each diluted in PBS (1 / 100), were transferred to the plate wells. For one hour, sealed plates that were shielded from light were agitated at 750 rpm. The beads were then subjected to three rounds of washing on the magnetic plate washer before adding 0.5μg / mL of the secondary antibody, antimouse IgG-PE (cat# 12-4010-87, ThermoFisher) for 45 minutes at 750 rpm. Following three rounds of washing, the beads were again reconstituted with 80 μL of Sheath fluid (Bio-Rad), mixed for two minutes, and then immediately read using a Bioplex-200 plate reader (Bio-Rad) set to DD gate 5000–25000 with 50 μL of acquiring volume. The program Bioplex Manager 6.1 was used to determine the median fluorescence intensity (MFI)

[0132] ELISA binding assay

[0133] Sera from mice vaccinated with LC3. SOSIP(s) or LC3. SOSIP(w), LC. Env were subjected to serial dilution to assess their binding to the in-house-produced SOSIP(s) or SOSIP(w) trimeric Env antigen. In brief, SOSIP were immobilized at 2 ug / mL in carbonate buffer (pH 9.6) overnight at 4°C, followed by washing in TBS-Tween20 0.05% and saturation using Superblock (Thermo). The samples were then applied for 1 hour at room temperature and detected, using a goat anti-mouse IgG polyclonal antibody coupled with HRP (Jackson ImmunoResearch) (1:20,000). After washing, HRP substrate was added and the reaction was stopped with H2SO4 IM. Reaction in each well was measured at 450 nm (with a reference at 670 nm) using a Tristar2 reader (Berthold Technologies).

[0134] TZM-bl neutralization assays

[0135] Neutralization assays using TZM-bl cells and rabbit sera were conducted. Pseudoviruses were generated as previously [2] by co-transfecting 293T cells with the HIV-1 env expression plasmid MW965.26, along with SF162NW and 96ZM651, along with an env-deficient HIV-1 backbone plasmid (pSG3ΔEnv). These viral strains were obtained from the NIH AIDS reagent program. The ability of the sera to neutralize HIV-1 was assessed through TZM-bl neutralization, following previously established methods [3], Sera from non-immunized rabbits served as negative controls. The 50% inhibitory reciprocal dilution (IRD50) was defined as the reciprocal dilution of the sample causing a 50% reduction in relative luminescence units (RLU).

[0136] Results:

[0137] The results depicted in the figures indicate that immunization with LC3. SOSIP(w) or LC3. SOSIP(s) in New Zealand white rabbits elicited a robust immune response. Specifically, the Env-specific IgG levels, measured via Luminex bead-based immunoassay, were significantly higher in vaccinated rabbits compared to controls. This demonstrates the vaccine's ability to induce strong humoral responses. Additionally, ELISA binding assays confirmed high affinity of the sera from vaccinated mice for the SOSIP antigens. Furthermore, TZM-bl neutralization assays showed that the sera from immunized rabbits achieved a 50% inhibitory reciprocal dilution (IRD50) against multiple HIV-1 pseudoviruses, suggesting broad neutralizing capabilities. Collectively, these results underline the potential efficacy of the LC3. SOSIP vaccine candidates in inducing targeted and potent immune responses.

[0138] REFERENCES:

[0139] 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.

[0140] 1. Kervevan J, Bouteau A, Lanza JS, Hammoudi A, Zurawski SM, Surenaud M, et al. Targeting human langerin promotes HIV-1 specific humoral immune responses. PLoS Pathogens. 2021;17. doi:10.1371 / journal.ppat.1009749

[0141] 2. Rouers A, Klingler J, Su B, Samri A, Laumond G, Even S, et al. HIV-Specific B Cell Frequency Correlates with Neutralization Breadth in Patients Naturally Controlling HIV-Infection. EBioMedicine. 2017;21: 158-169. doi:10.1016 / j.ebiom.2017.05.029

[0142] 3. Li M, Gao F, Mascola JR, Stamatatos L, Polonis VR, Koutsoukos M, et al. Human Immunodeficiency Virus Type 1 env Clones from Acute and Early Subtype B Infections for Standardized Assessments of Vaccine-Elicited Neutralizing Antibodies. J Virol. 2005;79: 10108-10125. doi: 10.1128 / JVI.79.16.10108-10125.2005

Claims

CLAIMS:

1. A fusion protein wherein an anti-Langerin targeting moiety is fused to a modified gp140 envelope glycoprotein (SOSIP) that comprises a) the A501C and T605C mutations to form a disulfide bond between the gp120 and gp41 domains (SOS) and b) the mutation I559P to facilitate trimerization (IP).

2. The fusion protein according to claim 1 wherein the modified gp140 envelope glycoprotein comprises a gp120 domain that consists of an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:

13. The fusion protein according to claim 1 or 2 wherein the modified gp140 envelope glycoprotein comprises a gp41 domain thar consists of an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:2.

4. The fusion protein according to any one of claims 1 to 3 wherein the cleavable sequence that is located between the gp120 and gp41 domains is replaced by a furin cleavable sequence.

5. The fusion protein according to claim 4 wherein the furin cleavable consist of the amino acid sequence as set forth in SEQ ID NO:5.

6. The fusion protein according to claim 5 wherein the modified gp140 envelope glycoprotein of the present invention consists of the amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:6.

7. The fusion protein according to any one of claims 1 to 3 wherein the cleavable sequence that is located between the gp120 and gp41 domains is replaced by a linker.

8. The fusion protein according to claim 4 wherein the linker consists of the amino acid sequence as set forth in SEQ ID NO:7.

9. The fusion protein according to claim 8 wherein the modified gp140 envelope glycoprotein consists of the amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:8.

10. The fusion protein according to any one of claims 1 to 9 wherein the targeting moiety is an antibody-fragment, more particularly a scFv having specificity for Langerin.

11. The fusion protein according to claim 10 wherein the scFv comprises a VL and VH sequences as selected from Table A.

12. The fusion protein according to claim 3 wherein the scFv consists of the amino acid sequences as set forth in SEQ ID NO: 15.

13. The fusion protein according to any one of claims 1 to 12 that comprises a linker that links the targeting moiety to the modified gp140 envelope glycoprotein.

14. The fusion protein according to claim 13 wherein the linker consists of the amino acid sequence as set forth in SEQ ID NO: 16.

15. The fusion protein according to any one of claims 1 to 14 that consists of an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:21 or SEQ ID NO:22.

16. A polynucleotide that encodes for the fusion protein according to any one of claims 1 to 15.

17. A host cell which has been transfected, infected or transformed by the polynucleotide according to claim 16.

18. A pharmaceutical composition that comprises fusion protein according to any one of claims 1 to 15.

19. A method for vaccinating a subject in need thereof against HIV comprising administering a therapeutically effective amount of the fusion protein according to any one of claims 1 to 15.

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