DC-targeted vaccine against Nipah virus infection

Abstract

The present invention relates to antibodies directed against surface antigens of antigen-presenting cells whose heavy and / or light chains are conjugated or fused to Nipah virus antigenic polypeptides.

Description

[Technical Field]

[0001] The present invention relates to the field of medicine, in particular virology. [Background technology]

[0002] Nipah virus (NiV) is a recently emerged, highly pathogenic, zoonotic paramyxovirus that was first recognized in 1998-1999 due to outbreaks of severe febrile encephalitis in Malaysia and Singapore (Chua, KB et al., "Nipah virus: a recently emerged deadly paramyxovirus," Science 288.5470(2000): 1432-1435). The Bangladeshi strain (NiVB) has recurred in varying numbers in Bangladesh and northeastern India almost every year between 2001 and 2015. NiVB epidemics have a high mortality rate, averaging approximately 75% (Lo, Michael K. et al., "Characterization of Nipah virus from the 2008-2010 epidemic in Bangladesh," Emerging infectious diseases 18.2(2012):248.), and numerous cases of human-to-human transmission have been confirmed (Gurley, Emily S. et al., "Human-to-Human Transmission of Nipah Virus in the Bangladeshi Community," Emerging infectious diseases 13.7(2007):1031.). Several candidate vaccines exist, but all are in the preclinical stage. Although rVSV vectors expressing Nipah virus G (or F) are prime candidates for use as "emergency vaccines" in outbreak management (Foster, Stephanie L. et al., "Recombinant VSV-Vectored Vaccines Rapidly Protect Nonhuman Primates from Lethal Nipah Virus Disease," Proceedings of the National Academy of Sciences 119.12(2022):e2200065119), concerns remain about safety, development, and inappropriate transportation / storage in affected areas. Therefore, challenges remain to improve vaccines' ability to induce strong cellular and humoral immune responses, positioning them as new general vaccine platforms for preventive strategies as well as at the center of therapeutic clusters to rapidly and efficiently respond to Nipah. Summary of the Invention

[0003] The present invention is defined by the claims. In particular, the present invention relates to antibodies directed against surface antigens of antigen-presenting cells whose heavy and / or light chains are conjugated or fused to Nipah virus antigenic polypeptides. [Means for solving the problem]

[0004] definition As used herein, the term "subject" or "subject in need thereof" refers to a human or non-human mammal. Typically, the patient is infected with or susceptible to infection with Nipah virus.

[0005] As used herein, the term "Nipah virus" has its common meaning in the art and refers to a member of the Paramyxoviridae family and related to Hendra virus (formerly known as equine morbillivirus). Nipah virus was first isolated in 1999 from samples from outbreaks of encephalitis and respiratory disease among adult males in Malaysia and Singapore (see, e.g., Chua et al., Lancet. 1999 Oct. 9, 354(9186):1257-9 and Paton et al., Lancet. 1999 Oct. 9, 354(9186):1253-6). The host for Nipah virus is not yet known, but fruit bats (Pteropus bats) are suspected to be the natural host. Nipah virus contains a six-gene, 18.2 kb, negative-sense single-stranded RNA (ssRNA) genome that encodes nine proteins: nucleoprotein (N), phosphoprotein (P), interferon antagonists W and V, viral C protein, matrix protein (M), viral fusion and glycoproteins (F and G, respectively), and large polymerase (L).

[0006] 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 include modified amino acid polymers, such as those that undergo disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling moiety. Polypeptide, when discussed in the context of gene therapy, refers to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, that retains the desired biochemical function of the intact protein.

[0007] As used herein, the term "G protein" refers to Nipah virus glycoprotein G. The G protein has a globular head domain formed by a six-bladed beta-sheet propeller connected to a transmembrane anchor via a flexible stalk domain. The G protein binds to the cellular receptors ephrin B2 and ephrin B3, which mediate viral attachment. Following attachment, Nipah virus glycoprotein G undergoes a conformational change, thereby inducing glycoprotein F, which leads to membrane fusion. An exemplary amino acid sequence of the G protein is represented by SEQ ID NO: 1. SEQ ID NO: 1>sp / Q9IH62 / GLYCP_NIPAV glycoprotein G OS=Nipah virus, OX=121791, GN=G, PE=1, SV=1 MPAENKKVRFENTTSDKGKIPSKVIKSYYGTMDIKKINEGLLDSKILSAFNTVIALLGSIVIIVMNIMII QNYTRSTDNQAVIKDALQGIQQQIKGLADKIGTEIGPKVSLIDTSSTITIPANIGLLGSKISQSTASINENVNEKCKFTLPPLKIHECNISCPNPLPFREYRPQTEGVSNLVGLPNNICLQKTSNQILKPKLI SYTLPVVGQSGTCITDPLLAMDEGYFAYSHLERIGSCSRGVSKQRIIGVGEVLDRGDEVPSLFMTNVWTPPNPNTVYHCSAVYNNEFYYVLCAVSTVGDPILNSTYWSGSLMMTRLAVKPKSNGGGYNQHQLA LRSIEKGRYDKVMPYGPSGIKQGDTLYFPAVGFLVRTEFKYNDSNCPITKCQYSKPENCRLSMGIRPNSHYILRSGLLKYNLSDGENPKVVFIEISDQRLSIGPSKIYDSLGQPVFYQASFSWDTMIKFGDV LTVNPLVVNWRNNTVISRPGQSQCPRFNTCPEICWEGVYNDAFLIDRINWISAGVFLDSNQTAENPVFTVFKDNEILYRAQLASEDTNAQKTITNCFLLKNKIWCISLVEIYDTGDNVIRPKLFAVKIPEQCT

[0008] As used herein, the term "F protein" refers to Nipah virus glycoprotein F. Nipah virus glycoprotein F is a class I fusion protein with typical structural features, including heptad repeats and a hydrophobic fusion peptide that bind to each other to form a six-helix bundle that functions in the membrane fusion process. Nipah virus attaches to target cells via glycoprotein G, which then induces Nipah virus glycoprotein F, which undergoes a conformational change, thereby leading to membrane fusion. An exemplary amino acid sequence of the F protein is represented by SEQ ID NO:2. SEQ ID NO: 2>sp / Q9IH63 / FUS_NIPAV fusion glycoprotein F0 OS=Nipah virus, OX=121791, GN=F, PE=1, SV=1 MVVILDKRCYCNLLILILMISECSVGILHYEKLSKIGLVKGVTR KYKIKSNPLTKDIVIKMIPNVSNMSQCTGSVMENYKTRLNGILTPI KGALEIYKNNTHDLVGDVRLAGVIMAGVAIGIATAAQITAGVALYEAMKNADNINKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDKISCKQTELSLDLALS KYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYETLLRTLGYATEDFDDLLESDSITGQIIYVDLSSYYIIVRVYFPILTEIQQAYIQELLPVSFNNDNSEWISIVPNFILV RNTLISNIEIGFCLITKRSVICNQDYATPMTNNMRECLTGSTEKCPRELVVSSHVPRFALSNGVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCPTAVLGNVIISLGKY LGSVNYNSEGIAIGPPVFTDKVDISSQISSMNQSLQQSKDYIKEAQRLLDTVNPSLISMLSMIILYVLSIASLCIGLITFISFIIVEKKRNTYSRLEDRRVRPTSSGDLYYIGT

[0009] As used herein, the term "N protein" refers to the Nipah virus nucleoprotein that encapsidates the genome, protecting it from nucleases. The encapsidated genomic RNA is called the nucleocapsid (NC) and serves as a template for transcription and replication. An exemplary amino acid sequence of the N protein is represented by SEQ ID NO:3. SEQ ID NO: 3>sp / Q9IK92 / NCAP_NIPAV nucleoprotein OS=Nipah virus, OX=121791, GN=N, PE=1, SV=1 MSDIFEEAASFRSYQSKLGRDGRASAATATLTTKIRIFVATNSPELRWELTLFALDVIRSPSAAESMKVGAAFTLISMYSERPGALIRSLLNDPDIEAVIIDVGSMVNGIPVMERRGDKAQEEMEGLMRILKTARDSSKGKTPFVDSRAYGLRITDM STLVSAVITIEAQIWILIAKAVTAPDTAEESETRRWAKYVQQKRVNPFFALTQQWLTEMRNLLSQSLSVRKFMVEILIEVKKGGSAKGRAVEIISDIGNYVEETGMAGFATIRFGLETRYPALALNEFQSDLNTIKSLMLLYREIGPRAPYMVLLEES IQTKFAPGGYPLLWSFAMGVATTIDRSMGALNINRGYL EPMYFRLGQKSARHHAGGIDQNMANRLGLSSDQVAELAAAVQETSAGRQESNVQAREAKFAAGGVLIGGSDQDIDEGEEPIEQSGRQSVTFKREMSISSLANSVPSSSVSTSGGTRLTNSLLNLRSRLAAKAAKEAASSNATDDPAISNRTQGESEKKNNQDLKPAQNDLDFVRADV

[0010] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. As used herein, the term polynucleotide refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide includes both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute a double-stranded form.

[0011] As used herein, the phrase "derived from" refers to a process in which a first component (e.g., a first polypeptide), or information from that first component, is used to isolate, derive, or create a different second component (e.g., a second polypeptide that is different from the first polypeptide).

[0012] As used herein, the term "encode" refers to the essential property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand used as a template for transcription of the gene or cDNA can be said to encode the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The reference "nucleotide sequence encoding a protein or RNA" can also include introns, to the extent that a nucleotide sequence encoding a protein may contain intron(s) in some versions.

[0013] As used herein, the terms "vector," "cloning vector," and "expression vector" refer to a vehicle by which DNA or RNA sequences (e.g., foreign genes) can be introduced into a host cell, thereby transforming the host and promoting the expression (e.g., transcription and translation) of the introduced sequences.

[0014] As used herein, the term "promoter / regulatory sequence" refers to a polynucleotide sequence (e.g., a DNA sequence) recognized by the synthetic machinery of a cell or introduced synthetic machinery required to initiate specific transcription of a polynucleotide sequence, thereby enabling expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, this sequence may also include enhancer sequences and other control elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

[0015] As used herein, the term "operably linked" or "transcriptional control" refers to a functional linkage between a regulatory sequence and a heterologous polynucleotide sequence that results in expression of the latter. For example, a first polynucleotide sequence is operably linked to a second polynucleotide sequence when the first polynucleotide sequence is placed in a functional relationship with the second polynucleotide sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be adjacent to each other, and, for example, in the same reading frame as necessary to link two protein-coding regions.

[0016] As used herein, the term "transformation" refers to the introduction of a "foreign" (i.e., external or extracellular) gene, DNA, or RNA sequence into a host cell, so that the host cell expresses the introduced gene or sequence to produce a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. A host cell that receives and expresses the introduced DNA or RNA has been "transformed."

[0017] As used herein, the term "expression system" means a host cell and a compatible vector under appropriate conditions for expression of a protein encoded by, for example, foreign DNA carried by the vector and introduced into the host cell.

[0018] As used herein, the "percent identity" between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences (i.e., % identity = number of identical positions / total number of positions × 100). Sequence comparison 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. and Wunsch, Christian D. (1970). "A General Method Applicable to the Search for Similarity in the Amino Acid Sequences of Two Proteins." Journal of Molecular Biology. 48(3):443-53). The percent identity between two nucleotide or amino acid sequences can also be determined using algorithms such as EMBOSS Needle (pairwise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle can be used with a BLOSUM62 matrix, a "gap open penalty" of 10, a "gap extension penalty" of 0.5, a false "end gap penalty", a "end gap open penalty" of 10, and a "end gap extension penalty" of 0.5. Generally, "percent identity" is a function of the number of matched positions divided by the number of positions compared and multiplied by 100. For example, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, the identity is 60%. Percent identity is typically determined over the entire length of the query sequence over which the analysis is performed. Two molecules with identical primary amino acid or polynucleotide sequences are identical, regardless of chemical and / or biological modifications.According to the present invention, a first amino acid sequence having at least 80% identity to a second amino acid sequence means that the first sequence has 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the second amino acid sequence.

[0019] As used herein, the term "conjugate" or, interchangeably, "conjugated polypeptide" is intended to refer to a complex or chimeric molecule formed by the covalent linkage of one or more polypeptides. The terms "covalently linked" or "conjugated" mean that the polypeptide and non-peptide moiety are directly covalently linked to each other, or in other cases, are indirectly covalently linked to each other via intervening moiety(ies), such as a bridge, spacer, or linking moiety(ies). A particular conjugate is a fusion protein.

[0020] As used herein, the term "fusion protein" refers to a protein produced through the combination of two or more polypeptides derived from separate proteins. Certain fusion proteins can be produced by recombinant DNA technology and are typically used in biological research or therapy. Fusion proteins can also be produced through chemical covalent bonds with or without linkers between the polypeptide portions of the fusion protein. In a fusion protein, two or more polypeptides are fused directly or via a linker.

[0021] As used herein, the term "directly" means that the first amino acid at the N-terminus of a first polypeptide is fused to the last amino acid at the C-terminus of a second polypeptide. This direct fusion can occur naturally, as described in (Vigneron et al., Science 2004, PMID 15001714), (Warren et al., Science 2006, PMID 16960008), (Berkers et al., J. Immunol. 2015a, PMID 26401000), (Berkers et al., J. Immunol. 2015b, PMID 26401003), (Delong et al., Science 2016, PMID 26912858), (Liepe et al., Science 2016, PMID 27846572), (Babon et al., Nat. Med. 2016, PMID 27798614).

[0022] As used herein, the term "linker" has its common meaning in the art and refers to an amino acid sequence of sufficient length to ensure that a protein forms the appropriate secondary and tertiary structure. In some embodiments, the linker is a peptide linker containing at least one but fewer than 30 amino acids, e.g., 2-30 amino acids, preferably 10-30 amino acids, more preferably 15-30 amino acids, even more preferably 19-27 amino acids, and most preferably 20-26 amino acids. In some embodiments, the linker has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues. Typically, the linker allows the compound to adopt the appropriate structure. The most suitable linker sequences (1) adopt a flexible extended conformation, (2) do not tend to develop regular secondary structures that could interact with the functional domain of the fusion protein, and (3) have minimal hydrophobic or charged properties that could facilitate interaction with the functional protein domain.

[0023] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules containing an antigen-binding site that immunospecifically binds to an antigen. In natural antibodies from 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 (I) and kappa (K). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinct sequence domains. In a typical IgG antibody, the light chain contains two domains: a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains: one variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). Both the light chain variable region (VL) and heavy chain variable region (VH) determine binding recognition and specificity to antigens. The light chain constant region domain (CL) and heavy chain constant region domain (CH) confer important biological properties, such as antibody chain binding, secretion, transplacental mobility, complement binding, and Fc receptor (FcR) binding. The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of one light chain and one heavy chain variable region. Antibody specificity resides in the structural complementarity between the antibody binding site and an antigenic determinant. The antibody binding site is primarily composed of residues from hypervariable or complementarity-determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FRs) may participate in the antibody binding site or influence the overall domain structure and therefore the binding 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 an original immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3, respectively. Thus, an antigen-binding site typically contains six CDRs, including the CDRs set from each of the heavy and light chain V regions. The framework region (FR) refers to the amino acid sequence inserted between the CDRs.Thus, light and heavy chain variable regions typically contain four framework regions and three CDRs of the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Residues in antibody variable domains are conventionally numbered according to the system devised by Kabat et al. This system is described in Kabat et al., 1987, Immunological Significance of Protein Sequences, U.S. Department of Health and Human Services, NIH, USA (Kabat et al., 1992, hereinafter referred to as "Kabat et al."). Kabat residue designations do not necessarily correspond directly to the linear numbering of amino acid residues in the SEQ ID sequence. The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat numbering, corresponding to truncations of or insertions into components, whether framework or complementarity-determining regions (CDRs), of the basic variable domain structure. The correct Kabat numbering of residues can be determined for a given antibody by aligning the homologous residues in the antibody sequence with the "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 below, the CDRs are determined using the CDR algorithm taken from the section entitled "How to Identify CDRs by Looking at the Sequence" on the antibody page at www.bioinf.org.uk.

[0024] As used herein, the term "immunoglobulin domain" refers to a globular region of an antibody chain (such as a heavy chain or a light chain of an antibody), or a polypeptide consisting essentially of such a globular region.

[0025] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. The human IgG heavy chain Fc region is generally defined as comprising amino acid residues from position C226 or P230 to the carboxyl terminus of an IgG antibody. The numbering of residues in the Fc region is that of the EU index of Kabat. The C-terminal lysine of the Fc region (residue K447) can be removed, for example, during antibody production or purification. Thus, the antibody compositions of the present invention can include antibody populations in which all K447 residues have been removed, antibody populations in which the K447 residue has not been removed, and antibody populations having a mixture of antibodies containing the K447 residue and antibodies that do not contain the K447 residue.

[0026] As used herein, the term "chimeric antibody" refers to an antibody comprising the VH and VL domains of a non-human antibody and the CH and CL domains of a human antibody. In one embodiment, a "chimeric antibody" is an antibody molecule in which (a) the constant regions (i.e., heavy and / or light chains), or portions thereof, have been modified, substituted, or exchanged so that the antigen-binding site (variable region) is linked to constant regions of a different or modified class, effector function, and / or species, or to entirely different molecules that confer new properties to the chimeric antibody, such as CD40 ligand, hormones, growth factors, drugs, enzymes, toxins, agonist molecules, etc., or (b) the variable regions, or portions thereof, have been modified, substituted, or exchanged with variable regions having different or modified antigen specificities. Chimeric antibodies also include primatized, particularly humanized, antibodies. Furthermore, chimeric antibodies may contain residues not found in the recipient or donor antibody. These modifications are made to further refine antibody performance. For further details, see Jones et al., Nature 321, 522-525 (1986), Riechmann et al., Nature 332, 323-329 (1988), and Presta, Curr. Op. Struct. Biol. 2, 593-596 (1992). (See U.S. Patent No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA, 81, 6851-6855 (1984)).

[0027] As used herein, the term "humanized antibody" includes antibodies that have the six CDRs of a murine antibody but have humanized framework and constant regions. More specifically, the term "humanized antibody" as used herein can include antibodies in which CDR sequences derived from the germline of another mammal, such as a mouse, have been grafted onto human framework sequences.

[0028] As used herein, the term "human monoclonal antibody" is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by random or site-specific in vitro mutagenesis or by in vivo somatic mutation). However, in one embodiment, the term "human monoclonal antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammal, such as a mouse, have been grafted onto human framework sequences.

[0029] As used herein, the term "immune response" refers to the reaction of the immune system to an antigen in a host's body, including the production of antigen-specific antibodies and / or cytotoxic responses. The immune response to an initial antigen exposure (primary immune response) is typically detectable after a lag period of several days to two weeks, and the immune response to subsequent stimulation with the same antigen (secondary immune response) is more rapid than the primary immune response. The immune response to a transgene product can include both humoral (e.g., antibody response) and cellular (e.g., cytolytic T cell response) immune responses that can be elicited by the 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 titers).

[0030] As used herein, the term "APC" or "antigen-presenting cell" means a cell that can activate T cells, and includes, but is not limited to, certain macrophages, B cells, and dendritic cells.

[0031] As used herein, the term "dendritic cell" or "DC" refers to any member of a diverse population of morphologically similar cell types found in lymphoid or non-lymphoid tissues. These cells are characterized by their distinctive morphology and high levels of surface MHC class II expression (see Steinman et al., Ann. Rev. Immunol. 9, 271 (1991), incorporated herein by reference for a description of such cells).

[0032] As used herein, the term "CD40" has its general meaning in the art and refers to the human CD40 polypeptide receptor. In some embodiments, the CD40 is an isoform of the human canonical sequence as reported by UniProtKB-P 25942 (also referred to as human TNR5).

[0033] As used herein, the term "CD40L" has its general meaning in the art and refers to the human CD40L polypeptide as reported by UniProtKB-P25942, e.g., including its CD40-binding domain of SEQ ID NO: 4. CD40L can be expressed as a soluble polypeptide and is the natural ligand of the CD40 receptor. SEQ ID NO: 4>CD40L binding domain MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

[0034] As used herein, the term "CD40 agonist antibody" is intended to refer to an antibody that increases CD40-mediated signaling activity in the absence of CD40L in a cell-based assay, such as a B cell proliferation assay. In particular, a CD40 agonist antibody (i) induces B cell proliferation as measured in vitro by flow cytometric analysis or by analysis of replicate dilutions of CFSE-labeled cells, and / or (ii) induces secretion of cytokines such as IL-6, IL-12, or IL-15 as measured in vitro in a dendritic cell activation assay.

[0035] As used herein, the term "Langerin" has its general meaning in the art and refers to a human C-type lectin domain family 4 member K polypeptide. In some embodiments, Langerin is an isoform of the human canonical sequence as reported by UniProtKB-Q9UJ71 (also referred to as human CD207).

[0036] As used herein, the term "treatment" or "treating" refers to both preventative or prophylactic treatment and therapeutic or disease-modifying treatment, including treatment of patients at risk of or suspected of having a disease, as well as patients who are ill or diagnosed as suffering from a disease or medical condition, including the suppression of clinical recurrence. Treatment may be administered to patients with or who may ultimately suffer from a medical disorder to prevent, cure, delay the onset of, reduce the severity of, or ameliorate symptoms of a medical disorder or recurring disorder, or to extend the patient's survival beyond that expected in the absence of such treatment. A "therapeutic regimen" refers to a pattern of treatment for a disease, such as, for example, the pattern of medication used during treatment. A therapeutic regimen can include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or portion of a therapeutic regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide high levels of a drug to the patient during the initial period of the regimen. An induction plan may employ (in part or in whole) a "loading plan," which may involve administering a higher dose of a drug than a physician would employ during a maintenance plan, administering a drug more frequently than a physician administers during a maintenance plan, or both. The phrase "maintenance plan" or "maintenance period" refers to a treatment plan (or portion of a treatment plan) used to maintain a patient during treatment of a disease, e.g., keeping a patient in remission for an extended period of time (months or years). A maintenance plan may employ continuous treatment (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent treatment (e.g., discontinued treatment, intermittent treatment, treatment upon relapse, or treatment upon reaching certain predetermined criteria (e.g., pain, disease manifestation, etc.)).

[0037] As used herein, the term "pharmaceutical composition" refers to a composition described herein, or a pharmaceutically acceptable salt thereof, together with other agents, such as carriers and / or excipients. The pharmaceutical compositions provided herein typically include a pharmaceutically acceptable carrier.

[0038] As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, adapted to the particular dosage form desired. Remington's Pharmaceutical-Sciences, 16th Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), discloses various carriers used in formulating pharmaceutical compositions and known techniques for their preparation.

[0039] As used herein, the term "vaccination" or "vaccinating" refers to, but is not limited to, a process for eliciting an immune response in a subject against a particular antigen.

[0040] As used herein, the term "vaccine composition" is intended to mean a composition that can be administered to a human or animal to induce an immune system response, which can result in the activation of certain cells, particularly APCs, T lymphocytes, and B lymphocytes.

[0041] As used herein, the term "antigen" refers to a molecule that, when processed and presented by MHC molecules, can be specifically bound by an antibody or a T cell receptor (TCR). An antigen is further capable of being recognized by the immune system and / or of inducing a humoral and / or cellular immune response that leads to the activation of B and / or T lymphocytes. An antigen can have one or more epitopes or antigenic sites (B epitopes and T epitopes).

[0042] As used herein, the term "adjuvant" refers to a compound that, when administered to a subject or animal, can induce and / or enhance an immune response to an antigen. It is also intended to mean a substance that acts to generally accelerate, prolong, or enhance the quality of a specific immune response to a particular antigen. In the context of the present invention, the term "adjuvant" refers to a compound that enhances both the innate immune response by affecting the transient response of the innate immune response and the longer-term effects of the adaptive immune response by activating and maturing antigen-presenting cells (APCs), particularly dendritic cells (DCs).

[0043] As used herein, the phrase "therapeutically effective amount" means a sufficient amount of the active ingredients of the invention to induce an immune response at a reasonable benefit / risk ratio applicable to any medical treatment.

[0044] Antibodies of the Invention A first object of the present invention is an antibody directed against a surface antigen of an antigen-presenting cell, the heavy and / or light chain of which is selected from the group consisting of SEQ ID NO: 1 (Niv(G) B The present invention relates to an antibody conjugated or fused to a polypeptide having at least 80% identity with the amino acid sequence ranging from the amino acid residue at position 71 (Q) to the amino acid residue at position 602 (T) in the ectodomain of the antibody.

[0045] In some embodiments, the light chain of the antibody is SEQ ID NO: 1 (Niv(G) B The polypeptide is conjugated or fused to a polypeptide having at least 80% identity with the amino acid sequence ranging from the amino acid residue at position 71 (Q) to the amino acid residue at position 602 (T) in the ectodomain.

[0046] In some embodiments, the heavy chain of the antibody is SEQ ID NO: 1 (Niv(G) B The polypeptide is conjugated or fused to a polypeptide having at least 80% identity with the amino acid sequence ranging from the amino acid residue at position 71 (Q) to the amino acid residue at position 602 (T) in the ectodomain.

[0047] The heavy and / or light chains of the antibody are linked via their C-terminus to Niv(G) B In some embodiments, the heavy and / or light chains of the antibody are conjugated or fused to an ectodomain of Niv(G). B It is fused to the N-terminus of the ectodomain.

[0048] In some embodiments, the heavy and / or light chains of the antibody are linked to Niv(G) by using chemical linkage. BIt is conjugated to the ectodomain.Several methods for binding or conjugating an antibody to its conjugated moiety are known in the art.Examples of linker types that have been used to conjugate a moiety to an antibody include, but are not limited to, hydrazone, thioether, ester, disulfide, and peptide-containing linkers, such as valine-citrulline linker.Linkers can be selected that are susceptible to cleavage by the low pH in lysosomal compartments, or susceptible to cleavage by proteases, such as proteases preferentially expressed in tumor tissues, such as cathepsins (e.g., cathepsins B, C, D). Techniques for conjugating polypeptides, in particular, are well known in the art (see, e.g., Arnon et al., "Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al., eds. Alan R. Liss, Inc., 1985); "Antibodies for Drug Delivery" by Hellstorm et al., in Controlled Drug Delivery (Robinson et al., eds., Marcel Deiker Inc., 2nd ed. 1987); "Antibody Carriers for Cytotoxic Agents in Cancer Therapy: A Review" by Thorpe, in Monoclonal Antibodies '84: Biological And Clinical Application (Pinchera et al., eds. 1985); Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al., eds. Academic See, "Analysis, Results, and Future Prospects of the Therapeutic Use of Radiolabeled Antibodies in Cancer Treatment," in J.D. McGraw-Hill Press, 1985; and Thorpe et al., 1982, Immunol. Rev. 62:119-58. See also, e.g., WO 89 / 12624.) Typically, peptides are covalently attached to lysine or cysteine ​​residues on the antibody via N-hydroxysuccinimide ester or maleimide functionalities, respectively.Conjugation methods using engineered cysteines or the incorporation of unnatural amino acids have been reported to improve conjugation homogeneity (Axup, JY, Bajjuri, KM, Ritland, M., Hutchins, BM, Kim, CH, Kazane, SA, Halder, R., Forsyth, JS, Santidrian, AF, Staffin, K. et al. (2012), "Synthesis of site-specific antibody-drug conjugates using unnatural amino acids," Proc. Natl. Acad. Sci. USA 109, 16101-16106, Junutula, JR, Flagella, KM, Graham, RA, Parsons, KL, Ha, E., Raab, H., Bhakta, S., Nguyen, T., Dugger, DL, Li, G. et al. (2010), "Engineered Thio-Trastuzumab-DM1 Conjugate with Improved Therapeutic Index Targeting Human Epidermal Growth Factor Receptor 2-Positive Breast Cancer," Clin. Cancer Res. 16, 4769-4778. Junutula et al. (Nat. Biotechnol. 2008, 26:925-32) developed a cysteine-based site-specific conjugate called "THIOMAB" (TDC), which is claimed to exhibit an improved therapeutic index compared to traditional conjugation methods. Conjugation to unnatural amino acids incorporated into antibodies has also been explored for ADCs, although the generality of this approach has not yet been established (Axup et al., 2012). In particular, one skilled in the art can envision engineered Fc-containing polypeptides with acyl donor glutamine-containing tags (e.g., nucleotide-containing peptide tags or Q-tags) or endogenous glutamines that have been made reactive by polypeptide engineering (e.g., via amino acid deletion, insertion, substitution, or mutation on the polypeptide). Furthermore, transglutaminase can be covalently crosslinked with amine donating agents (e.g., small molecules containing or bound to reactive amines) to form stable and homogeneous populations of engineered Fc-containing polypeptides conjugated with acyl donor glutamine-containing tags or amine donating agents site-specifically conjugated to Fc-containing polypeptides via accessible / exposed / reactive endogenous glutamines (WO 2012 / 059882).

[0049] In some embodiments, the heavy and / or light chains of the antibody are linked to Niv(G) by a dockerin domain or domains, as described in U.S. Patent Application Publication Nos. 2016 / 0031988 and 2012 / 0039916. B It is conjugated to an ectodomain and non-covalently binds to the cohesin fusion protein.

[0050] In some embodiments, the heavy and / or light chains of the antibody are Niv(G) B It is fused to an ectodomain to form a fusion protein.

[0051] In some embodiments, Niv(G) B The ectodomains are fused directly or via a linker to the heavy and / or light chains. As used herein, the term "directly" refers to the Niv(G) B This means that the first amino acid at the N-terminus of the ectodomain is fused to the last amino acid at the C-terminus of the heavy or light chain. This direct fusion can occur naturally, as described in (Vigneron et al., Science 2004, PMID 15001714), (Warren et al., Science 2006, PMID 16960008), (Berkers et al., J. Immunol. 2015a, PMID 26401000), (Berkers et al., J. Immunol. 2015b, PMID 26401003), (Delong et al., Science 2016, PMID 26912858), (Liepe et al., Science 2016, PMID 27846572), and (Babon et al., Nat. Med. 2016, PMID 27798614).

[0052] In some embodiments, Niv(G) B The N-terminus of the ectodomain is fused directly or via a linker to the C-terminus of the heavy chain.

[0053] In some embodiments, the linker is selected from the group consisting of SEQ ID NO:5 (FlexV1), SEQ ID NO:6 (f1), SEQ ID NO:7 (f2), SEQ ID NO:8 (f3), or SEQ ID NO:9 (f4), as described below. QTPTNTISVTPTNNSTPTNNSNPKPNP (FlexV1, SEQ ID NO: 5) SSVSPTTSVHPTPTSVPPTPTKSSP (f1, SEQ ID NO: 6) PTSTPADSSTITPTATPTATPTIKG (f2, SEQ ID NO: 7) TVTPTATATPSAIVTTITPTATTKP (f3, SEQ ID NO: 8) TNGSITVAATAPTVTPTVNATPSAA (f4, SEQ ID NO: 9)

[0054] In some embodiments, the antibodies of the invention SEQ ID NO: 1 (Niv(G) B the heavy chain of an antibody conjugated or fused to a polypeptide having at least 80% identity to an amino acid sequence ranging from the amino acid residue at position 71 (Q) to the amino acid residue at position 602 (T) in the ectodomain of the antibody; The light chain is SEQ ID NO: 2 (predicted epitope-rich peptide Niv(F) B ) is conjugated or fused to a polypeptide having at least 80% identity with the amino acid sequence ranging from the amino acid residue at position 45 (K) to the amino acid residue at position 90 (I).

[0055] In some embodiments, the C-terminus of the light chain of the antibody is SEQ ID NO: 2 (predicted epitope-rich peptide Niv(F) B and the C-terminus of the heavy chain of the antibody is conjugated or fused to the N-terminus of a polypeptide having at least 80% identity to the amino acid sequence ranging from amino acid residue 45 (K) to amino acid residue 90 (I) of SEQ ID NO: 1 (Niv(G) BIt is conjugated or fused to the N-terminus of a polypeptide having at least 80% identity with the amino acid sequence ranging from the amino acid residue at position 71 (Q) to the amino acid residue at position 602 (T) in the ectodomain.

[0056] In some embodiments, the antibodies of the invention SEQ ID NO: 1 (Niv(G) B the heavy chain of an antibody conjugated or fused to a polypeptide having at least 80% identity to an amino acid sequence ranging from the amino acid residue at position 71 (Q) to the amino acid residue at position 602 (T) in the ectodomain of the antibody; The light chain is SEQ ID NO: 3 (predicted epitope-rich peptide Niv(N) B ) is conjugated or fused to a polypeptide having at least 80% identity with the amino acid sequence ranging from amino acid residue (I) at position 318 to amino acid residue (L) at position 355 in the sequence.

[0057] In some embodiments, the C-terminus of the light chain of the antibody is SEQ ID NO: 3 (predicted epitope-rich peptide Niv(N) B and the C-terminus of the heavy chain of the antibody is conjugated or fused to the N-terminus of a polypeptide having at least 80% identity to the amino acid sequence ranging from amino acid residue (I) at position 318 to amino acid residue (L) at position 355 in SEQ ID NO: 1 (Niv(G) B It is conjugated or fused to the N-terminus of a polypeptide having at least 80% identity with the amino acid sequence ranging from the amino acid residue at position 71 (Q) to the amino acid residue at position 602 (T) in the ectodomain.

[0058] In some embodiments, the antibodies of the invention SEQ ID NO: 1 (Niv(G) B the heavy chain of an antibody conjugated or fused to a polypeptide having at least 80% identity to an amino acid sequence ranging from the amino acid residue at position 71 (Q) to the amino acid residue at position 602 (T) in the ectodomain of the antibody; The light chain is: i) SEQ ID NO: 2 (predicted epitope-rich peptide Niv(F) B ii) a polypeptide having at least 80% identity with the amino acid sequence ranging from the amino acid residue at position 45 (K) to the amino acid residue at position 90 (I) in SEQ ID NO: 3 (a predicted epitope-rich peptide Niv(N) B ) and a polypeptide having at least 80% identity with the amino acid sequence ranging from amino acid residue (I) at position 318 to amino acid residue (L) at position 355 in the sequence.

[0059] In some embodiments, the antibodies of the invention SEQ ID NO: 1 (Niv(G) B the heavy chain of said antibody conjugated or fused to a polypeptide having at least 80% identity to an amino acid sequence ranging from the amino acid residue (Q) at position 71 to the amino acid residue (T) at position 602 in the ectodomain of said antibody, The light chain is conjugated or fused to both fusion proteins, i) SEQ ID NO: 3 (predicted epitope-rich peptide Niv(N) B ii) a polypeptide having at least 80% identity with the amino acid sequence ranging from amino acid residue (I) at position 318 to amino acid residue (L) at position 355 in SEQ ID NO: 2 (predicted epitope-rich peptide Niv(F) B ) to a polypeptide having at least 80% identity with the amino acid sequence ranging from amino acid residue 45 (K) to amino acid residue 90 (I) in the sequence.

[0060] In some embodiments, the light chain comprises SEQ ID NO: 2 (predicted epitope-rich peptide Niv(F) B ) directly or via a linker to a fusion protein comprising a polypeptide having at least 80% identity with the amino acid sequence ranging from the amino acid residue at position 45 (K) to the amino acid residue at position 90 (I) of SEQ ID NO: 3 (a predicted epitope-rich peptide Niv(N) BThe amino acid sequence of the polypeptide having at least 80% identity with the amino acid sequence ranging from the amino acid residue at position 318 (I) to the amino acid residue at position 355 (L) in the polypeptide of the present invention is conjugated or fused to the N-terminus of the polypeptide having at least 80% identity with the amino acid sequence ranging from the amino acid residue at position 318 (I) to the amino acid residue at position 355 (L) in the polypeptide of the present invention.

[0061] In some embodiments, the light chain of the antibody is conjugated or fused to the fusion protein via its C-terminus.

[0062] In some embodiments, SEQ ID NO: 3 (predicted epitope-rich peptide Niv(N) B A polypeptide having at least 80% identity with the amino acid sequence ranging from amino acid residue (I) at position 318 to amino acid residue (L) at position 355 in SEQ ID NO: 2 (predicted epitope-rich peptide Niv(F)) is linked via a linker. B The polypeptide is fused to a polypeptide having at least 80% identity with the amino acid sequence ranging from the amino acid residue at position 45 (K) to the amino acid residue at position 90 (I) in the sequence of the fragment 11 of the present invention.

[0063] In some embodiments, the linker consists of the amino acid sequence set forth in SEQ ID NO:10. SEQ ID NO: 10 AEAAAKEAAAKA

[0064] In some embodiments, the antibodies of the invention SEQ ID NO: 1 (Niv(G) B the heavy chain of an antibody conjugated or fused to a polypeptide having at least 80% identity to an amino acid sequence ranging from the amino acid residue at position 71 (Q) to the amino acid residue at position 602 (T) in the ectodomain of the antibody; The light chain conjugated or fused to both fusion proteins has at least 80% identity to the amino acid sequence set forth in SEQ ID NO:11. SEQ ID NO: 11 KYKIKSNPLTKDIVIKMIPNVSNMSQCTGSVMENYKTRLNGILTPI AEAAAKEAAAKA IQTKFAPGGYPLLWSFAMGVATTIDRSMGALNINRGYL

[0065] In some embodiments, the antibody is an IgG antibody, preferably an IgG1 or IgG4 antibody, or even more preferably an IgG4 antibody.

[0066] In some embodiments, the antibody is a chimeric antibody, particularly a chimeric mouse / human antibody.

[0067] In some embodiments, the antibody is a humanized antibody.

[0068] Chimeric or humanized antibodies can be prepared based on the sequence of the mouse monoclonal antibody prepared as described above. DNA encoding heavy and light chain immunoglobulins can be obtained from the hybrid cells of the target mouse and can be engineered to contain non-mouse (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to generate chimeric antibodies, mouse variable regions can be linked to human constant regions using methods known in the art (see, for example, U.S. Patent No. 4,816,567 by Cabilly et al.). To generate humanized antibodies, mouse CDR regions can be inserted into a human framework using methods known in the art. See, for example, Winter, U.S. Patent No. 5,225,539, and Queen et al., U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370.

[0069] In some embodiments, the antibody is a human antibody. In some embodiments, human antibodies can be identified using transgenic or transchromosomic mice carrying parts of the human immune system rather than the mouse immune system. These transgenic and transchromosomic mice include those referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "human Ig mice." HuMAb mice® (Medarex, Inc.) contain human immunoglobulin gene minilocuses encoding unrearranged human heavy chain (μ and γ) and K light chain immunoglobulin sequences, along with targeted mutations that inactivate the endogenous μ and K chain loci (see, e.g., Lonberg et al., 1994 Nature 368(6474):856-859). In another embodiment, human antibodies can be raised using mice carrying human immunoglobulin sequences on transgenes and transchromosomes, such as mice carrying a human heavy chain transgene and a human light chain transchromosome. Such mice, referred to herein as "KM mice," are described in detail in Ishida et al., WO 02 / 43478.

[0070] In some embodiments, the antibody is specific for a cell surface marker of a professional APC. The antibody may be specific for a cell surface marker of another professional APC, such as a B cell or macrophage.

[0071] In some embodiments, the antibody is selected from antibodies that specifically bind to a DC immunoreceptor (DCIR), MHC class I, MHC class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD19, CD20, CD29, CD31, CD40, CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2, MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2, IFN-γ receptor and IL-2 receptor, ICAM-1, Fey receptor, LOX-1, and ASPGR.

[0072] In some embodiments, the antibody is specific for CD40.

[0073] In some embodiments, the anti-CD40 antibody is derived from the 12E12 antibody: a heavy chain comprising complementarity determining regions CDR1H, CDR2H and CDR3H, wherein CDR1H has the amino acid sequence GFTFSDYYMY (SEQ ID NO: 12), CDR2H has the amino acid sequence YINSGGGSTYYPDTVKG (SEQ ID NO: 13), and CDR3H has the amino acid sequence RGLPFHAMDY (SEQ ID NO: 14); a light chain comprising complementarity determining regions CDR1L, CDR2L and CDR3L, wherein CDR1L has the amino acid sequence SASQGISNYLN (SEQ ID NO: 15), CDR2L has the amino acid sequence YTSILHS (SEQ ID NO: 16), and CDR3L has the amino acid sequence QQFNKLPPT (SEQ ID NO: 17).

[0074] In some embodiments, the anti-CD40 antibody is derived from the 11B6 antibody: a heavy chain comprising complementarity determining regions CDR1H, CDR2H and CDR3H, wherein CDR1H has the amino acid sequence GYSFTGYYMH (SEQ ID NO: 18), CDR2H has the amino acid sequence RINPYNGATSYNQNFKD (SEQ ID NO: 19), and CDR3H has the amino acid sequence EDYVY (SEQ ID NO: 20); a light chain comprising complementarity determining regions CDR1L, CDR2L and CDR3L, wherein CDR1L has the amino acid sequence RSSQSLVHSNGNTYLH (SEQ ID NO: 21), CDR2L has the amino acid sequence KVSNRFS (SEQ ID NO: 22), and CDR3L has the amino acid sequence SQSTHVPWT (SEQ ID NO: 23).

[0075] In some embodiments, the anti-CD40 antibody is derived from the 12B4 antibody: a heavy chain comprising complementarity determining regions CDR1H, CDR2H and CDR3H, wherein CDR1H has the amino acid sequence GYTFTDYVLH (SEQ ID NO: 24), CDR2H has the amino acid sequence YINPYNDGTKYNEKFKG (SEQ ID NO: 25), and CDR3H has the amino acid sequence GYPAYSGYAMDY (SEQ ID NO: 26); a light chain comprising complementarity determining regions CDR1L, CDR2L and CDR3L, wherein CDR1L has the amino acid sequence RASQDISNYLN (SEQ ID NO: 27), CDR2L has the amino acid sequence YTSRLHS (SEQ ID NO: 28), and CDR3L has the amino acid sequence HHGNTLPWT (SEQ ID NO: 29).

[0076] In some embodiments, the anti-CD40 antibody is selected from the group consisting of selected mAb1, mAb2, mAb3, mAb4, mAb5, and mAb6 listed in Table A. [Table 1]

[0077] SEQ ID NO: 30 (amino acid sequence of the variable heavy chain region (VH) (v2) of humanized 11B6) EVQLVQSGAEVKKPGASVKISKASGYSFTGYYMHWVKQAHGQGLEWIGRINPYNGATSYNQNFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCAREDYVYWGQGTTVTVSSAS SEQ ID NO: 31 (amino acid sequence of the variable heavy chain region (VL) Vk(v2) of humanized 11B6 VL) DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPWTFGGGTK SEQ ID NO: 32 (amino acid sequence of the variable heavy chain region VH(v3) of humanized 11B6) EVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYMHWVRQAPGQGLEWIGRINPYNGATSYNQNFKDRVTLTVDKSTSTAYMELSSLRSEDTAVYYCAREDYVYWGQGTTVTVSSAS SEQ ID NO: 33 (VH amino acid sequence of mAb3 (12B4)) EVQLQQSGPELVKPGASVKMSCKASGYTFTDYVLHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGYPAYSGYAMDYWGQGTSVTVSSAS SEQ ID NO: 34 (VL amino acid sequence of mAb3 (12B4)) DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCHHGNTLPWTFGGGTK SEQ ID NO: 35 (VH amino acid sequence of mAb4 (24A3 HC)) DVQLQESGPDLVKPSQSLSLTCTVTGYSITSDYSWHWIRQFPGNKLEWMGYIYYSGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDSATYFCARFYYGYSFFDYWGQGTTLTVSSAS SEQ ID NO: 36 (VL amino acid sequence of mAb4 (24A3 KC)) QIVLTQSPAFMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTK SEQ ID NO: 37 (VH amino acid sequence of mAb5) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSAS SEQ ID NO: 38 (VL amino acid sequence of mAb5) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTK SEQ ID NO: 39 (VH amino acid sequence of mAb6 (12E12 H3 humanized HC)) EVQLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQAPGKGLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARRGLPHAMDYWGQGTLVTVSSAS SEQ ID NO: 40 (VL amino acid sequence of mAb6 (humanized K2 12E12)) DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQFNKLPPTFGGGTK

[0078] In some embodiments, the anti-CD40 antibody comprises a heavy chain whose variable domain has the amino acid sequence set forth as SEQ ID NO:30 and a light chain whose variable domain has the sequence set forth as SEQ ID NO:31.

[0079] In some embodiments, the anti-CD40 antibody comprises a heavy chain whose variable domain has the amino acid sequence set forth as SEQ ID NO: 32 and a light chain whose variable domain has the sequence set forth as SEQ ID NO: 31.

[0080] In some embodiments, the anti-CD40 antibody comprises a heavy chain whose variable domain has the amino acid sequence set forth as SEQ ID NO:33 and a light chain whose variable domain has the sequence set forth as SEQ ID NO:34.

[0081] In some embodiments, the anti-CD40 antibody comprises a heavy chain whose variable domain has the amino acid sequence set forth as SEQ ID NO:35 and a light chain whose variable domain has the sequence set forth as SEQ ID NO:36.

[0082] In some embodiments, the anti-CD40 antibody comprises a heavy chain whose variable domain has the amino acid sequence set forth as SEQ ID NO:37 and a light chain whose variable domain has the sequence set forth as SEQ ID NO:38.

[0083] In some embodiments, the anti-CD40 antibody comprises a heavy chain whose variable domain has the amino acid sequence set forth as SEQ ID NO:39 and a light chain whose variable domain has the sequence set forth as SEQ ID NO:40.

[0084] In some embodiments, the anti-CD40 antibody is a CD40 agonist antibody. CD40 agonist antibodies are described in International Publication Nos. 2010 / 009346, 2010 / 104747, and 2010 / 104749. Other anti-CD40 agonist antibodies in development include CP-870,893, a fully human IgG2 CD40 agonist antibody developed by Pfizer. It binds CD40 with a KD of 3.48×10-10 M but does not block the binding of CD40L (see, e.g., U.S. Pat. No. 7,338,660) to SGN-40, a humanized IgG1 antibody developed by Seattle Genetics from the murine antibody clone S2C6 generated using a human bladder cancer cell line as an immunogen. It binds to CD40 with a KD of 1.0 x 10-9 M and functions by enhancing the interaction between CD40 and CD40L, thereby exhibiting partial agonism (Francisco JA et al., Cancer Res, 60:3225-31, 2000). More specifically, the CD40 agonist antibody is selected from the group consisting of selected mAb1, mAb2, mAb3, mAb4, mAb5, and mAb6, as set forth in Table A.

[0085] In some embodiments, the antibody is specific to Langerin. In some embodiments, the antibody is derived from antibody 15B10, which has ATCC accession number PTA-9852. In some embodiments, the antibody is derived from antibody 2G3, which has ATCC accession number PTA-9853. In some embodiments, the antibody is derived from antibody 91E7, 37C1, or 4C7, as described in WO 2011 / 032161.

[0086] In some embodiments, the anti-Langerin antibody 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.

[0087] In some embodiments, the anti-Langerin antibody 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.

[0088] In some embodiments, the anti-Langerin antibody 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.

[0089] In some embodiments, the antibody is selected from the group consisting of selected mAb7, mAb8, and mAb9, as set forth in Table B. [Table 2]

[0090] SEQ ID NO: 41 (amino acid sequence of the variable heavy chain region (VH) of 15B10) SVKMSCKASGYTFTDYVISWVKQRTGQGLEWIGDIYPGSGYSFYNENFKGKATLTADKSSTTAYMQLSSLTSEDSAVYFCA SEQ ID NO: 42 (amino acid sequence of the variable light chain (VL) of 15B10) ASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTNFTLKISRVEAEDLGLYFCS SEQ ID NO: 43 (amino acid sequence of the variable heavy chain region (VH) of 2G3) SSVKMSCKASGYTFTDYVISWVKQRTGQGLEWIGDIYPGSGYSFYNENFKGKATLTADKSSTTAYMQLSSLTSEDSAVYFCA SEQ ID NO: 44 (amino acid sequence of the variable light chain (VL) of 2G3) VTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRVSGVPARFSGSLIGDKAALTITGAQTEDEAIYFCA SEQ ID NO: 45 (amino acid sequence of the heavy chain of 4C7) QVQLQQSGAELVRPGASVTLSCKASGYTFIDHDMHWVQQTPVYGLEWIGAIDPETGDTGYNQKFKGKAILTADKSSRTAYMELRSLTSEDSAVYYCTIPFYYSNYSPFAYWGQG ALVTVSAAKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNP CPPLKECPPCADLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDAQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKT ISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGKAS SEQ ID NO: 46 (amino acid sequence of the light chain of 4C7) QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQRKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPLTFGAGTKLELK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0091] In some embodiments, the anti-Langerin antibody comprises a heavy chain whose variable domain has the amino acid sequence set forth as SEQ ID NO:41 and a light chain whose variable domain has the sequence set forth as SEQ ID NO:42.

[0092] In some embodiments, the anti-Langerin antibody comprises a heavy chain whose variable domain has the amino acid sequence set forth as SEQ ID NO:43 and a light chain whose variable domain has the sequence set forth as SEQ ID NO:44.

[0093] In some embodiments, the anti-Langerin antibody comprises a heavy chain having the amino acid sequence set forth as SEQ ID NO:45 and a light chain having the sequence set forth as SEQ ID NO:46.

[0094] Antibodies of the present invention can be produced by any technique known per se in the art, including, but not limited to, any chemical, biological, genetic, or enzymatic technique, alone or in combination. Knowing the amino acid sequence of a desired sequence, one skilled in the art can easily produce said polypeptide using standard techniques for producing polypeptides. For example, antibodies of the present invention can be synthesized by recombinant DNA technology, as is now well known in the art. For example, these fragments can be obtained as DNA expression products after incorporating a DNA sequence encoding the desired (poly)peptide into an expression vector and introducing such a vector into a suitable eukaryotic or prokaryotic host that expresses the desired polypeptide, which can then be isolated using well-known techniques.

[0095] In some embodiments, the amino acid sequences described herein comprise one or more sequences derived from restriction cloning site(s) present in the polynucleotide encoding said amino acid sequence. Typically, said sequences may consist of two amino acid residues and typically include AP, AS, AR, PR, SA, TR, and TS sequences.

[0096] In some embodiments, the amino acid sequences described herein include signal peptide sequences. As used herein, the term "signal peptide" has its general meaning in the art and refers to a prepeptide present as an N-terminal peptide on the precursor form of a protein. The function of a signal peptide is to facilitate the translocation of the expressed polypeptide to which it is attached into the endoplasmic reticulum. The signal peptide is usually cleaved during this process. The signal peptide may be heterologous or homologous to the organism used to produce the polypeptide.

[0097] In some embodiments, the antibodies of the invention a heavy chain having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 47; and a light chain having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:48. SEQ ID NO: 47 MGWSLILLFLVAVATRVHSEVQLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQAPGKGLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCA RRGLPFHAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVD KRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS QTPTNTISVTPTNNSTPTNNSNPKPNPASQNYTRSTDNQAMIKDALQSIQQQIKGLADKIGTEIGPKVSLIDTSSTITIPANIGLLGSKISQSTASINENVNEKCKFTLPPLKIHECNISCPNPLPFREYKPQTEGVSNLVGLPNNICLQKTSNQILKPKL ISYTLPVVGQSGTCITDPLLAMDEGYFAYSHLEKIGSCSRGVSKQRIIGVGEVLDRGDEVPSLFMTNVWTPSNPNTVYHCSAVYNNEFYYVLCAVSVVGDPILNSTYWSGSLMMTRLAVKPKNNGESYNQHQFA LRNIEKGKYDKVMPYGPSGIKQGDTLYFPAVGFLVRTEFKYNDSNCPIAECQYSKPENCRLSMGIRPNSHYILRSGLLKYNLSDEENSKIVFIEISDQRLSIGSPSKIYDSLGQPVFYQASFSWDTMIKFGDVQ TVNPLVVNWRDNTVISRPGQSQCPRFNKCPEVCWEGVYNDAFLIDRINWISAGVFLDSNQTAENPVFTVFKDNEVLYRAQLASEDTNAQKTITNCFLLKNKIWCISLVEIYDTGDNVIRPKLFAVKIPEQCTLK SEQ ID NO: 48 DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQFNKLPPTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0098] In some embodiments, the antibodies of the invention a heavy chain having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 47; and a light chain having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:49. SEQ ID NO: 49 DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQFNKLPPTFGGGTKLEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECAS KYKIKSNPLTKDIVIKMIPNVSNMSQCTGSVMENYKTRLNGILTPIAEAAAKEAAAKAIQTKFAPGGYPLLWSFAMGVATTIDRSMGALNINRGYL

[0099] Polynucleotides, Vectors, and Host Cells of the Invention A further object of the present invention relates to polynucleotides encoding the heavy and / or light chains of the antibodies of the invention.

[0100] Typically, the polynucleotide is a DNA or RNA molecule that may be contained in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.

[0101] Therefore, a further object of the present invention relates to a vector comprising a polynucleotide of the invention.

[0102] Such vectors may contain regulatory elements, such as promoters, enhancers, and terminators, to induce or directly express the antibody upon administration to a subject. Examples of promoters and enhancers used in animal cell expression vectors include the SV40 early promoter and enhancer, the Moloney murine leukemia virus long terminal repeat promoter and enhancer, and the immunoglobulin heavy chain promoter and enhancer. Any animal cell expression vector can be used as long as it can insert and express a gene encoding a human antibody C region. Examples of suitable vectors include pAGE107, pAGE103, pHSG274, pKCR, and pSG1 beta d2-4. Other examples of plasmids include replicative plasmids containing an origin of replication, or integrative plasmids such as pUC, pcDNA, and pBR. Other examples of viral vectors include adenovirus, retrovirus, herpesvirus, and AAV vectors. Such recombinant viruses can 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 viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in WO 95 / 14785, WO 96 / 22378, U.S. Pat. No. 5,882,877, U.S. Pat. No. 6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056, and WO 94 / 19478.

[0103] A further object of the present invention relates to host cells transfected, infected or transformed with the polynucleotides and / or vectors according to the invention.

[0104] Polynucleotides of the present invention can be used to produce antibodies 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, but are not limited to, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, and plant cells). Specific examples include E. coli, Kluyveromyces, or Saccharomyces yeast. Mammalian host cells include Chinese hamster ovary (CHO) cells, including dhfr- CHO cells (described in Urlaub and Chasin, 1980) used with a DHFR selection marker, the CHOK1 dhfr+ cell line, NSO myeloma cells, COS cells, and SP2 cells, e.g., the GS CHO cell line supplemented with the GS Xceed™ Gene Expression System (Lonza), or HEK cells.

[0105] The present invention also relates to a method for producing a recombinant host cell expressing an antibody according to the invention, said method comprising the steps of (i) introducing a recombinant polynucleotide or vector into competent host cells in vitro or ex vivo as described above, (ii) culturing the resulting recombinant host cells in vitro or ex vivo, and (iii) optionally selecting cells that express and / or secrete the antibody.

[0106] Such recombinant host cells can be used for the production of the antibodies of the invention.

[0107] Thus, the host cells disclosed herein are particularly suitable for producing the antibodies of the present invention. In practice, when recombinant expression is introduced into mammalian host cells, the polypeptide is produced by culturing the host cells for a period of time sufficient for expression of the antibody in the host cells, and optionally secreting the antibody into the medium in which the host cells grow. The antibodies can be recovered and purified from the medium after their secretion, for example, using standard protein purification methods.

[0108] Pharmaceutical and vaccine compositions The antibodies described herein can be administered as part of one or more pharmaceutical compositions. Except insofar as any conventional carrier medium is incompatible with the antibodies of the invention, such as by producing any undesirable biological effects or otherwise causing adverse interactions with any other component(s) of the pharmaceutical composition, its use is considered within the scope of the invention. Some examples of materials which may 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, for example, sodium carboxymethylcellulose, ethylcellulose, and acetylcellulose; excipients such as tragacanth powder, malt, gelatin, talc, cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar-agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and phosphate buffer; and other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate. Coloring agents, release agents, coating agents, sweetening agents, flavorings, and perfuming agents, preservatives, and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0109] The antibodies described herein are particularly suitable for preparing vaccine compositions. A further object of the present invention therefore relates to a vaccine composition comprising an antibody of the present invention.

[0110] In some embodiments, the vaccine compositions of the invention comprise an adjuvant. In some embodiments, the adjuvant is alum. In some embodiments, the adjuvant is Freund's incomplete adjuvant (IFA) or other oil-based adjuvant, present at a weight / weight ratio (W / W) of 30-70%, preferably 40-60%, and more preferably 45-55%. In some embodiments, the adjuvant is polyinosinic / polycytidylic acid (poly(I:C)) or polyinosinic / polycytidylic acid-poly-L-lysine / carboxymethylcellulose (poly-ICLC). In some embodiments, the vaccine compositions of the invention comprise at least one Toll-like receptor (TLR) agonist selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, and TLR8 agonists.

[0111] Treatment method The antibodies and pharmaceutical or vaccine compositions described herein are particularly suitable for inducing an immune response against Nipah virus and can therefore be used for vaccine purposes.

[0112] Therefore, a further object of the present invention relates to a method for vaccinating a subject in need thereof against Nipah virus, comprising administering a therapeutically effective amount of an antibody of the present invention.

[0113] In some embodiments, the antibodies and pharmaceutical or vaccine compositions described herein are particularly suitable for the treatment of Nipah virus infection.

[0114] In some embodiments, the subject may be a human or any other animal (e.g., bird and mammal) susceptible to Nipah virus infection (e.g., domestic animals such as cats and dogs, livestock such as horses, cows, pigs, chickens, etc.). Typically, the subject is a mammal, including non-primates (e.g., camels, donkeys, zebras, cows, pigs, horses, goats, sheep, cats, dogs, rats, and mice) and primates (e.g., monkeys, chimpanzees, and humans). In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a livestock or pet. In some embodiments, the subject is a human. 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.

[0115] In some embodiments, the subject may be symptomatic or asymptomatic.

[0116] Typically, the active ingredients of the present invention (i.e., antibodies and pharmaceutical or vaccine compositions described herein) are administered to a subject in a therapeutically effective amount. It will be understood that the total daily dosage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dosage level for any particular subject will depend on a variety of factors, including factors well known in the medical field, such as the disorder being treated and its severity, the activity of the specific compound used, the specific composition used, the subject's age, weight, general health, sex, and diet, the time of administration, route of administration, and excretion rate of the specific compound used, the duration of treatment, and drugs used in combination with or concomitantly with the specific polypeptide used. For example, it is well known to those skilled in the art to initiate doses of a compound at levels lower than those required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. However, the daily dosage of a product can vary 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 administration to the subject for symptomatic control. The medicament typically contains about 0.01 mg to about 500 mg of the active ingredient, particularly 1 mg to about 100 mg of the active ingredient. An effective amount of the medicament is usually provided at 0.0002 mg / kg to about 20 mg / kg of body weight per day, particularly about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0117] The antibody and pharmaceutical or vaccine compositions described herein may be administered to a subject by any route of administration, particularly oral, nasal, rectal, topical, buccal (e.g., sublingual), 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 the nature of the particular active agent being used.

[0118] The present invention is further illustrated by the following figures and examples, which should not, however, be construed as limiting the scope of the present invention. [Brief explanation of the drawings]

[0119] [Figure 1] Figure 1 shows epitope mapping of the NiV G antigen in relation to CD40mAb. Within the NiV genome, ORFs encoding the G, F, and N proteins are highlighted in blue, orange, and red, respectively. The full-length aa sequence was examined for predicted HLA-I (NetMHC 4.0) and -II (NetMHC II 2.3) and linear B cell epitopes (BepiPred 2.0). For HLA-I and -II, the color intensity represents the density of predicted epitopes with at least 30% global HLA coverage. (Top row) The ectodomain of the G protein associated with the Gen-1 and Gen-2 vaccines. [Figure 2] Figure 1 shows epitope mapping of NiV F and N antigens in relation to CD40mAb. The refined domains of F and N in relation to the Gen-2 vaccine are depicted. [Figure 3] FIG. 1 is a schematic diagram of the Generation 1 (Gen-1) and Generation 2 (Gen-2) vaccines. [Figure 4] FIG. 1 shows a summary of Gen-1, Gen-2, and IgG4 control vaccine SDS PAGE in non-reduced (NR) vs. reduced (R) conditions. [Figure 5] Figure 1 shows that the G antigen fused to the Gen-1 vaccine was confirmed by Western blot using serum from NiV(G)-immunized mice, with in-house produced G protein tested as a positive control. Molecular weight (kDa) markers are shown in the left column. Arrows indicate the heavy chain (HC) and light chain (LC) of the vaccine structure at their expected sizes. [Figure 6]Figure 1 shows size exclusion chromatography (SEC) of Gen-1 and Gen-2 vaccines with standard protein size markers. Recombinant mAbs are indicated by arrows. [Figure 7] Figure 1 shows a binding assay of CD40.NiV vaccine to mouse cells expressing the human CD40 receptor. Mouse splenocytes from hCD40My / HuTg mice were cultured with 10 nM of CD40.NiV Gen-1 mAb and then labeled with T-, B-, and DC cell markers, respectively. Untreated and non-targeting IgG4.NiV-treated splenocytes were used as negative controls. [Figure 8] Figure 1 shows a binding assay of CD40.NiV vaccine to AGM PBMCs. Same method as in Figure 7, using PBMCs from untreated AGMs. Two in-house produced anti-CD40 mAbs that were not related to the G protein (clones VH3 and 11B6) were used as positive controls. Vaccine binding was demonstrated on CD40-expressing B cells, with less extension to the monocyte / macrophage population. [Figure 9] Figure 1 shows that Gen-1 and -2 CD40.NiV vaccines induced humoral immune responses in hCD40Hy / Mu transgenic mice. G-specific IgG titers were determined by ELISA on days 0, 21, and 28 post-immunization in mice immunized twice with the molar equivalent of 10 μg CD40.NiV (Gen-1), Gen-2, or Niv (G) protein alone, with (solid circles) and without (open circles) poly-ICLC. Median AUC [Min-Max] values ​​are presented and compared with control animals (poly-ICLC only). [Figure 10] 9 shows the neutralizing activity of post-vaccinated mouse sera detected by Luminex-based pseudoneutralization assay on days 0, 21, and 28 post-immunization. Statistical analysis as in FIG. 9. [Figure 11]Figure 1 shows an in vitro neutralization assay was performed on NiV-B infected cells using serially diluted sera collected on day 28 post-immunization. IC50 was calculated and reported as titer. Statistical analysis is the same as in Figure 9. [Figure 12] Biotinylated NiV(G) protein was used to stain Niv(G)-specific B cells in the dLN. (Left) Representative dot plot. Niv(G)-specific B cells are considered double-positive for the two anti-biotin mAbs. (Right) Percentage (%) of GC B cells (FAS+GL-7+) and the percentage of Niv(G)-specific B cells within this population. Animals were immunized with 30 μg of CD40.NiV(Gen-2) poly-ICLC and compared with a poly-ICLC-negative group. Mann-Whitney unpaired t-test, **, p<0.01; ***, p<0.0001. [Figure 13] Figure 1 shows that Gen-1 and -2 CD40.NiV vaccines induced cellular immune responses in hCD40Hy / Mu transgenic mice. IFN-g T cell responses to G, F, and N overlapping pools of peptides were assessed by ELISPOT one week after boost. (Left) Specific IFN-g responses to the G antigen in mice immunized with Gen-1 and Gen-2, respectively. The number of spots per million splenocytes is reported. (Right) Total specific IFN-g responses to the three antigens of Gen-2 in mice immunized with 10 and 30 μg of vaccine, respectively. (Bottom right) Percentage of G, F, and N-specific responses in 10 and 30 μg of vaccine, respectively. Solid circles indicate the presence of poly-ICLC; open circles indicate the absence of poly-ICLC. [Figure 14]Figure 1 shows the immune response of AGMs vaccinated with CD40.NiV Gen-2. Graphical representation of the study design. Diamonds represent sampling. Nine AGMs were immunized with CD40.NiV Gen-2 with poly-ICLC at 3-week intervals from the primary boost. Niv(G)-specific IgG (left) and IgA (right) titers in the poly-ICLC group (n=3, left) versus the vaccinated group (n=9, right) at days 0, 10, 21, 35, and 56 postprime. Individual Ig titers (log(1 / EC50)) are shown, and whiskers indicate the median [Min-Max] for each group and time point. Priming and boost are indicated by arrows. Dotted line, threshold of Ig titer detection. [Figure 15] As shown in FIG. 10, the neutralizing activity of AGM sera was measured by an indirect inhibition assay. [Figure 16] FIG. 1 shows in vitro neutralization of Niv-B by sera from vaccinated AGMs. [Figure 17] Figure 13 shows that total specific IFN-γ responses to G, F, and N antigens were measured in AGM circulating PBMCs collected on days 21 and 35 post-immunization. The number of spots per million PBMCs is reported. The dotted line represents the threshold of positivity defined by the mean response (+3SD) of the non-vaccinated AGM group. (Right) Percentage (%) of specific T cell responses to each vaccine antigen (mean ± SD). [Figure 18] Figure 1 shows in vitro Niv-B neutralizing activity of AGM sera after immunization and challenge (80π days): poly-ICLC group (n=3, filled circles), CD40.NiV(Gen-2) with poly-ICLC (n=9, open circles), thin black line, modeling of decline of neutralizing Abs after peak of response (35 days). [Figure 19]Figure 1. Protection assay in AGM. Survival curve. Nine AGM were immunized twice with CD40.NiV(Gen-2) and challenged with 102 Pfu Niv-B (intratracheal route). Highly untreated animals served as controls. Gehan-Breslow-Wilcoxon test, ****, p<0.0001. [Figure 20] 1 shows clinical scores and temperatures of naive (filled circles) and vaccinated (open circles) AGMs. The dotted line indicates the score threshold for ethical standards (left panel), and the dotted line indicates the median temperature on day 0 (right panel). [Figure 21] Virus dissemination after challenge. (A) Amount of viral NiV RNA quantified by RT-PCR (N gene) in PBLs (left). Values ​​were normalized to those of the GADPH housekeeping gene. Genomic RNA was measured by RT-qPCR in nasal (center) and pharyngeal (right) swabs. Mean values ​​(±SEM) are presented for unvaccinated (filled circles, n=7) vs. CD40.NiV(Gen-2) vaccinated (open circles, n=9). (B) Same as A for fluids collected at necropsy. [Example]

[0120] method: Molecular cloning and production: Generation 1: The ectodomain of the NiV-B G protein (Figure 1, Bangladesh strain (NiVB) (GenBank: AY988601.1) (Harcourt BH et al., Emerging Infect. Dis. 2005) ectodomain highlighted) was cloned into the UCOE vector at the C-terminus of the heavy chain of an anti-CD40 mAb (clone 12E12 / VH3) using a flexible linker (see Figure 3).

[0121] Generation 2: The AA sequences of the F and N proteins (Bangladesh strain) were screened for predicted MHC-I and -II epitope density and HLA diversity using online software (NetMHC) (Figure 2). The sequences were further broken down by analyzing the presence of predicted linear B cell epitopes and screening for non-hydrophobic stable protein domains. One peptide in F and one in N were linked to the C-terminus of the anti-CD40 light chain (clone 12E12 / VH3) via a linker domain (see Figure 3, right).

[0122] Production in CHO cell lines: CHO cells were transfected with plasmids expressing the heavy and light chains (see Figure 3). Culture supernatants were collected, and protein A was captured and purified on an FPLC. Purified vaccine batches were controlled for their endotoxin levels (<0.5 ng / mg protein).

[0123] Biochemical quality control: The quality of the vaccine batches was analyzed by i) SDS-PAGE analysis (under reducing and non-reducing conditions) followed by Coomassie blue staining (Figure 4), and ii) SEC analysis using standard proteins (Figure 6). The Gen1 vaccine was evaluated by Western blot (Figure 5) using serum from C57BL / 6 mice immunized with in-house produced G protein (+CpG). The H chain fused to the G protein was revealed by mouse serum.

[0124] Binding assay: The Gen-1 antibody and the same 12E12 / VH3 clone without antigen were marked with fluorescent dye. Clone 11B6 was also tested in parallel as a positive control. Mouse splenocytes from hCD40Tg mice (Figure 7) and PBMCs from AGM (Figure 8) were cultured in vitro with the fluorescent vaccine. The cell phenotypes were determined and analyzed by FACS.

[0125] Immunological responses in hCD40Tg mice: hCD40 Hy / Mu Tg mice (CO-1704 hTnfrsf5-OST5(CD40) knock-in HOM) were subcutaneously immunized twice with CD40.NiV(G) (Gen-1 or -2) or NiV(G) protein alone, with or without any adjuvant (see Table 1). Serum was collected at weeks 0, 3, and 4. Splenocytes were collected at week 4. Serum was tested for the amount of G-specific IgG by ELISA. Serial dilutions were evaluated to determine antibody titers (Figure 9). The neutralizing activity of serum Abs was measured by an in-house Luminex assay (Figure 10) or an in vitro NiV-B neutralization assay (Figure 11). Serial dilutions were performed to determine the neutralizing effect of serum against NiV infection. The formation of germinal center B cells and G-specific B cells within this population was estimated by FACS (Figure 12).

[0126] Splenocytes were stimulated in vitro with either a pool of overlapping peptides (OVLP at 1 μg / mL; Figure 13) encompassing domains selected from the NiV G, F, and N proteins contained within the vaccine (Figures 1 and 2). IFN-γ production was assessed by ELISpot.

[0127] Immunological responses in AGM Nine AGMs were vaccinated twice with the CD40.NiV(Gen-2) vaccine, three weeks apart. Three untreated animals served as controls. Blood samples were collected periodically to measure serum G-specific IgG and IgA by ELISA (Figure 14). The neutralizing activity of serum was determined as in the mouse study (Figures 15, 16, and 18). IFNg-ELISPOT was performed on PBMCs collected on days 21 and 35 post-immunization (Figure 17).

[0128] Challenge assay Vaccinated animals (n=9) vs. naive AGM (n=8) were treated with Niv-B for 10 min. 2 pfu. Survival was monitored over a 3-week period (Figure 19).

[0129] Clinical and blood biochemistry follow-up Clinical examinations were performed daily after challenge and the following parameters were scored: temperature, weight, dehydration, respiration, responsiveness, fecal examination and neurological symptoms. For ethical reasons, animals were euthanized when they reached a total score of >15 (Figure 20).

[0130] Virology RNA extraction from infected AGM PBLs: Blood samples from infected AGMs collected in EDTA tubes were diluted with Pharmalyse buffer, vortexed, and incubated in the dark for 15 minutes. They were then centrifuged at 200 × g for 5 minutes, and the pellets were washed with 2 mL of PBS 1% FBS, 2 mM EDTA. Finally, the pellets were resuspended in 600 μL of RLT lysis buffer and purified according to the manufacturer's instructions (Macherey-Nagel). Samples were stored at -80°C until use.

[0131] RT-qPCR: Viral RNA was extracted using the Qiamp viral RNA kit (Qiagen) for serum, swab, urine, BAL, vitreous, and breast effusion samples, and the Nucleospin kit (Macherey-Nagel) for PBMCs and organs. Viral load was assessed by one-step RT-qPCR using NiV-N-specific and GAPDH primers, as appropriate (Figure 21).

[0132] result: NiV predicted immunogenic peptides and CD40. In silico refinement of NiV vaccine design. We have successfully produced two CD40.NiV vaccine candidates. Gen-1 and -2 contain the gold-standard NiV G (glycoprotein) surface antigen, while the GEN-2 vaccine also binds peptides in silico selected from the F and N proteins and predicted to be enriched in T- and B-cell epitopes (Figures 1-8). The NiV surface glycoprotein (G) is the gold-standard antigen for inducing protective humoral responses. Other cellular effectors, such as helper and effector T-cells, may also participate in host defense. We screened the NiV G ectodomain (ECD) to identify vaccine epitopes using NetMHC 4.0 and NetMHCII 2.3 software, which predict T-cell epitopes that bind to a large panel of class I and class II HLA molecules, respectively. Linear B-cell epitopes were predicted using BepiPred 2.0. The NiV G ECD vaccine region was predicted to contain 3,522 and 15 T- and B-cell epitopes, respectively. Due to their conservation across NiV strains, we further identified vaccine epitopes from the fusion (F) and nucleocapsid (N) proteins. Regions with strong binding epitopes and the highest HLA coverage were highlighted, as were linear B-cell epitopes. The refined peptides of NiV F (aa 45-90) and NiV N (aa 318-355) contain 356 and 266 predicted T-cell epitopes, respectively, and three linear B-cell epitopes. Overall, these amino acid sequences were screened for homology with other henipaviruses, yielding 100% homology between different Nipah strains for the F and N peptides and over 98% homology for the NiV G ECD (Figures 1-8). We next engineered vectors expressing the NiV-B G ECD fused to the C-terminus of the heavy chain (HC) of the anti-human CD40 humanized 12E12 IgG4 monoclonal antibody, with further selected peptides of NiV F and NiV N fused to the C-terminus of the light chain (LC) (designated CD40.NiV) (Fig. 3). Vaccines were produced in CHO cells and controlled for their quality.Here, we confirm the binding of CD40.NiV vaccine to human CD40 and AGM CD40 receptors in vitro using splenocytes from mice transgenic for the human CD40 receptor (CD40Hy / Mu transgenic mice, hCD40Tg) and PBMCs from AGM, respectively ( Figures 7–8 ).

[0133] CD40.NiV induces specific T- and B-cell responses in hCD40Tg mice We first identified hCD40 My / Hu We evaluated immunogenicity in Tg mice. hCD40Tg mice were immunized with two subcutaneous (SC) injections of CD40.NiV (Gen-1 or Gen-2) vaccine (10 μg) or the same amount of NiV G protein, both with poly-ICLC (50 μg), on days 0 and 21. Antibody-mediated immune responses were first assessed by Luminex assay. Our results showed significant G-specific IgG titers with both vaccines (Figure 9). Three weeks after the first injection, mice immunized with CD40-targeted NiV G protein showed extremely high anti-NiV G IgG levels (P < 0.01), highlighting the benefit conferred by the DC targeting system. These antibody levels were significantly improved when i) the G protein was targeted to APCs and ii) mice were immunized with adjuvant. We next compared the avidity of NiV G-specific IgG after boosting using a multiplex immunoassay approach. Strikingly, the avidity index was significantly improved when NiV G targeted the CD40 receptor (P < 0.05), suggesting the benefit of such targeting for the induction of B-cell affinity maturation (data not shown). Remarkably, we set up an in-house Luminex-based assay to evaluate the inhibition of G binding to the ephrin B2 receptor by circulating IgG in serum. Our results revealed the potent capacity of immunized serum to mimic this protein G / receptor interaction (Figure 10). Remarkably, the neutralizing capacity of vaccine-induced IgG was confirmed by a neutralization assay performed at BSL-4 (Figure 11).

[0134] G-specific B cells were detected by FACS within the germinal center B cell population of immunized mice (Figure 12). We further confirmed the induced B-cell response in CD40.NiV (+ poly-ICLC) vaccinated mice by detecting germinal center (GC) B-cells in the draining lymph nodes (dLN) (P < 0.001 compared with the poly-ICLC control group) (Figure 12). Furthermore, FACS staining demonstrated a significant NiV G-specific GC B-cell population in the dLN of vaccinated animals (P < 0.01). To demonstrate specific T-cell responses to various antigens from CD40.NiV, we performed IFN-g ELISpot assays on splenocytes using pools of overlapping peptides (Figure 13). We detected dose-dependent IFN-g-producing cells specific for the NiV G ECD and NiV F and N peptides. Finally, we demonstrated by IFN-g ELISpot performed on mouse splenocytes that Gen-2 vaccine-induced cells specifically responded to the G, F, and N peptides, respectively, which were compromised in the vaccine (Figure 13). Overall, these results demonstrate the immunogenicity of the CD40.NiV vaccine candidate.

[0135] CD40.NiV vaccine induces early and robust humoral and T-cell responses in AGM We then evaluated the cellular and humoral responses of the Gen-2 vaccine in the African green monkey (AGM) model, which is the most relevant model for testing infection with the Bangladeshi NiV strain. Twelve animals were imported and housed at the Bioprim Animal Center (Toulouse). Two groups of animals were immunized with the homologous regimen in the first boost, followed by three AGMs receiving poly-ICLC alone and nine AGMs receiving 200 μg of the Gen-2 vaccine plus poly-ICLC. Serial collection of blood samples was performed as follows: (i) serum for G-specific Ig titers and neutralization, and (ii) PBMCs for IFN-g ELISpot (Figure 14). We demonstrated that our vaccine candidate induced a potent and sustained humoral response, with circulating IgG neutralizing the virus in vitro (assessed at BSL-4) (Figures 14-16). All vaccinated animals demonstrated specific and significant IgG and IgA titers 10 days after the first vaccination (Figure 14). IgG titers increased over time and then remained stable until day 56, while serum IgA levels declined. Vaccination elicited the neutralizing potential of NiV G-specific IgG by day 10 after the first vaccination, reaching significance 2 weeks after the boost (mean neutralizing titer of 3.2 (±0.2), P<0.001) and remaining high through day 56 (Figures 15, 16, and 18). We estimated a significant mean decrease in neutralizing titer of 0.016 logs per day, leading to a prediction of maintenance of neutralizing titers above the detectable threshold (mean log titer of 2.2 (±0.1)) up to 100 days after the boost. A Lumix-based surrogate inhibition assay was used to confirm the neutralizing capacity of specific antibodies after the first and second vaccinations (P<0.001) (data not shown). We used sera from five vaccinated AGMs to evaluate cross-neutralization against NiV-B, -M (Malaysia), and -C (Cambodia) and HeV by in vitro infection of VeroE6 cells with recombinant (F / G) VSV particles (data not shown). Two weeks after the boost, all animals demonstrated comparable cross-reactive humoral responses to NiV (F / G) proteins and positive cross-reactive responses to Hendra virus (data not shown).Using pools of peptides against each vaccine antigen, IFN-g ELISpot assays were performed on PBMCs collected 21 days (before the boost) and 35 days after the prime (Figure 17). The overall response was significant 2 weeks after the boost (P<0.05) and positive in 5 / 8 test animals. Our results highlight polyclonal IFN-g T cell responses to all vaccine antigens (Figure 17).

[0136] Overall, CD40.NiV(Gen-2) induces cellular responses associated with a potent, rapid, and durable humoral response in the AGM model, with high titers of neutralizing antibodies.

[0137] Clinical outcome and survival for challenged animals We then tested vaccine-induced protection against NiV infection in African green monkeys (AGM). Control and vaccinated AGM were transferred to BSL-4 where NiV (10 2pfu intratracheal challenge (Bangladesh strain). The challenge dose was selected based on a previous experiment involving five non-vaccinated animals infected under the same conditions. Remarkably, we observed complete protection in vaccinated animals (day 22 postchallenge, n = 8), whereas all non-vaccinated animals died between D7 and D11 (n = 8) (Figure 19). Clinically, only mild and transient clinical signs were evident in vaccinated animals, with an average clinical score of less than 6, primarily attributable to lack of responsiveness (Figure 20). The control group showed a high clinical score, with lethargy, tachypnea, dyspnea, and gastrointestinal symptoms, accompanied by fever (>38.9°C in all animals). Animals showed various hematological and serum biochemical changes during the challenge phase (data not shown). Enzyme activity levels reflecting liver damage (aspartate aminotransferase, AST, creatine kinase, CK) exceeded normal values ​​in control animals but not in vaccinated animals during the critical disease period. We also observed disturbances in white blood cell and red blood cell counts, indicating lymphopenia and thrombocytopenia, in control animals, whereas no significant abnormalities were observed in vaccinated animals during the post-immunization and challenge phases. Necropsy revealed the pathological and pathophysiological processes of NiV-B infection in control animals (data not shown). Examination of lung tissue revealed interstitial pneumonia, edema, and vasculitis accompanied by inflammatory cell infiltration in all control animals, but was rarely observed in vaccinated animals (observed in 2 of 9 animals, AGM #S1134 and #O1376, data not shown).

[0138] Non-vaccinated animals showed follicular depletion in the spleen, whereas eight CD40.NiV-vaccinated AGMs showed follicular hyperplasia, suggesting that a strong adaptive immune response was induced after challenge. Notably, we did not observe any lesions associated with the frontal cortex in any of the animals. From a clinical perspective, CD40.NiV(Gen-2) appears to confer complete protection against disease progression and death.

[0139] Plasma and tissue viral loads in challenged AGM We assessed NiV viremia by qRT-PCR in PBLs from vaccinated and control animals for organs, fluids, and swabs at routine time points and at the end of the study (euthanasia or 28 dpc). All control animals (n = 3 receiving poly-ICLC + n = 4 untreated AGM) showed detectable high levels of NiV-B (range 4.5-5.5 log10 copies / mL) in PBLs at 7-8 dpc, whereas we did not detect virus in vaccinated subjects until 22 dpc (Figure 21A). With the exception of one animal (AGM#S1134) in which NiV-B transcripts were detected by RT-PCR in the lung, NiV-B replication was undetectable in nasal and nasopharyngeal swabs, bronchoalveolar lavage (BAL), serum, urine, and fluids, including chest exudates, as well as in organs (lungs, spleen, and nervous tissue) in vaccinated AGMs, whereas high levels were detected in control animals (Figure 21B). This strong antiviral effect was confirmed by the absence of NiV N protein expression in lung and spleen tissues, as assessed by tissue immunofluorescence (data not shown). Viral syncytia were detected in the lungs of non-immunized animals. Notably, NiV-B infection of the brain could not be confirmed by tissue immunofluorescence. Overall, CD40.NiV(Gen-2) vaccination resulted in sterilizing immunity that limited both virus transmission and virus shedding in the AGM.

[0140] Immunological and cytokine characteristics of challenged animals We further characterized the postchallenge immune response by immunological phenotype of cell populations from 0 to 22 dpc. Animals that died showed profound and significant defects in lymphoid populations (CD20+ B- and CD3+ T cells) (data not shown). Among T cells, the percentage of CD8+ T cells was markedly reduced during NiV infection (data not shown). These changes were transient and not significant in the vaccinated group. Innate immunity was also affected by NiV-B infection, with circulating monocytes almost completely disappearing in animals that died, whereas in vaccinated animals, the numbers of inflammatory, intermediate, and classical monocytes remained stable after a critical period of infection (7–9 dpc) (data not shown). Overall, these data indicate that exposure to NiV challenge induces dramatic perturbations of innate and adaptive cellular immunity, which are attenuated and / or nonsignificant in CD40.NiV-immunized animals.

[0141] Changes in the expression of hemocyte genes in vaccinated AGM To decipher early changes in gene expression associated with vaccination, we performed RNA-seq analysis of peripheral whole blood samples from animals receiving adjuvanted CD40.NiV(Gen-2) at D0 (preprime), 1 day after the prime (PP, D1), immediately before the boost (D21), and 1 day after the boost (PB, D22). For a small number of animals in the adjuvant control group, we analyzed differentially expressed genes (DEGs) at different time points relative to the baseline of vaccinated animals. Principal component analysis (PCA) demonstrated changes in gene abundance at PP and PB relative to baseline (D0 before injection) (data not shown). A total of 773 DEGs were significantly modulated at D22 (1 day after boost). Interestingly, 437 of these DEGs were recorded at D1 and D22 (1 day after PP and PB). An additional 236 DEGs were common between 1 day after PP and PB (data not shown). The most highly upregulated genes from D0 to D22 included those playing key roles in antiviral innate and pro-inflammatory responses (e.g., ISG15, MX1, IFI44, CXCL10, and IL-27) and adaptive immunity (e.g., antigen uptake by SIGLEC-1, T cell chemotaxis by CCL8, and CXCL11) (data not shown). The majority of these transcripts were found to be upregulated PP and PB (data not shown). Panels of the most highly downregulated genes were also associated with signatures of primary or secondary immune responses to the vaccine (e.g., CD1c, CD79A, CCR6, and CXCR4). Globally, the most highly upregulated and downregulated genes in vaccinated animals were primarily associated with pathways involved in immune system processes and defense against viruses and other organisms (data not shown). Humoral immune responses mediated by circulating immunoglobulins were particularly promoted in PB, highlighting a prominent antibody-mediated response after the second injection of CD40.NiV(Gen-2) (data not shown).

[0142] Overall, the antiviral defense module of genes was most significantly upregulated at D22 (data not shown). Detailed dynamics of the genes involved in this module demonstrated rapid and significant activation in all PPs and PBs (data not shown). Ten transcripts, including interferon-inducible antiviral proteins (RSAD2 and RIG1), were significantly upregulated in PPs. Other gene network modules were also highlighted in PPs and PBs (e.g., regulation of virus response, cytokine signaling, neutrophil degranulation, and leukocyte activation). Although we were unable to distinguish between vaccine and adjuvant effects in vaccine recipients, our transcriptome analysis highlights a vaccine-induced gene signature.

[0143] conclusion Here, we demonstrate the efficacy of an innovative DC-targeted vaccine candidate to prevent NiV-B infection in a challenge experiment in the AGM model. More than 30 years after the discovery of the immunological properties of DCs, we demonstrate for the first time that targeting viral antigens to professional APCs can be used effectively as a preventative measure against lethal viral challenge. In contrast to most other NiV vaccine platforms, the DC-targeting strategy enabled us to engineer subunit constructs containing immunogenic and cross-reactive epitopes from the NiV G ECD as well as the F and N NiV proteins (Bossart et al., 2012; Foster et al., 2022; Geisbert et al., 2021; Loomis et al., 2020; Mohammed et al., 2020; Woolsey et al., 2023; Yoneda et al., 2013).

[0144] The CD40.NiV vaccine induced both IgG and IgA antibodies in the AGM as early as 10 days postpriming. We also show that neutralizing responses can be maintained 100 days after the peak of the Ab response, with a stable estimated mean log titer of approximately 2.2 (±0.1).

[0145] The responses were shown to cross-neutralize multiple strains of NiV, but also HeV. We found that the CD40.NiV vaccine elicited T cell responses to NiV G ECD and targeted F and N peptides in two preclinical models, although detectable at lower levels. Transcriptome analysis revealed a common set of DEGs at day 1 (D1 and D22) after the prime and boost, including those for antiviral innate (ISG15, MX1, IFI44) and adaptive immunity. Differentially expressed genes in vaccinated animals were primarily associated with pathways involved in immune system processes and defense against viruses with innate immunity at early time points (D1, PP), as described in other vaccine strategies (Hagan et al., 2022). Interestingly, changes in gene pathways related to humoral responses were significant at day 22 after the boost.

[0146] Overall, these results highlight a vaccine signature associated with the protective efficacy of the vaccine.

[0147] quotation Throughout this application, various references describe the state of the art to which this invention pertains, the disclosures of which are hereby incorporated by reference into the present disclosure.

Claims

1. An antibody directed against a surface antigen of an antigen-presenting cell, The heavy and / or light chain is selected from SEQ ID NO: 1 (Niv(G) Ｂ An antibody conjugated or fused to a polypeptide having at least 80% identity with the amino acid sequence ranging from the amino acid residue at position 71 (Q) to the amino acid residue at position 602 (T) in the ectodomain of the antibody.

2. 2. The antibody of claim 1, wherein the light chain of the antibody is conjugated or fused to a polypeptide having at least 80% identity to the amino acid sequence ranging from the amino acid residue (Q) at position 71 to the amino acid residue (T) at position 602 in SEQ ID NO: 1 (Niv(G)B ectodomain).

3. 2. The method of claim 1, wherein the heavy chain of the antibody is conjugated or fused to the polypeptide having at least 80% identity to the amino acid sequence ranging from the amino acid residue (Q) at position 71 to the amino acid residue (T) at position 602 in SEQ ID NO: 1 (Niv(G)B ectodomain).

4. The antibody of any one of claims 1 to 3, wherein the heavy chain and / or the light chain of the antibody is conjugated or fused to the Niv(G)B ectodomain via its C-terminus.

5. The antibody of any one of claims 1 to 3, wherein the heavy chain and / or the light chain of the antibody is fused to the N-terminus of the Niv(G)B ectodomain.

6. The antibody of any one of claims 1 to 5, wherein the heavy chain and / or the light chain of the antibody is fused to the Niv(G)B ectodomain to form a fusion protein.

7. The antibody of any one of claims 1 to 6, wherein the Niv(G)B ectodomain is fused to the heavy and / or light chain directly or via a linker.

8. 2. The method of claim 1, wherein the linker is selected from the group consisting of SEQ ID NO:5 (FlexV1), SEQ ID NO:6 (f1), SEQ ID NO:7 (f2), SEQ ID NO:8 (f3), or SEQ ID NO:9 (f4).

9. The antibody SEQ ID NO: 1 (Niv(G) Ｂ the heavy chain of said antibody conjugated or fused to said polypeptide having at least 80% identity to said amino acid sequence ranging from said amino acid residue (Q) at position 71 to said amino acid residue (T) at position 602 in the ectodomain of said antibody, The light chain is SEQ ID NO: 2 (predicted epitope-rich peptide Niv(F) Ｂ ) is conjugated or fused to a polypeptide having at least 80% identity with the amino acid sequence ranging from the amino acid residue (K) at position 45 to the amino acid residue (I) at position 90 in The antibody according to any one of claims 1 to 8.

10. The antibody SEQ ID NO: 1 (Niv(G) Ｂ the heavy chain of said antibody conjugated or fused to said polypeptide having at least 80% identity to said amino acid sequence ranging from said amino acid residue (Q) at position 71 to said amino acid residue (T) at position 602 in the ectodomain of said antibody, The light chain is SEQ ID NO: 3 (predicted epitope-rich peptide Niv(N) Ｂ ) is conjugated or fused to a polypeptide having at least 80% identity with the amino acid sequence ranging from the amino acid residue (I) at position 318 to the amino acid residue (L) at position 355 in The antibody according to any one of claims 1 to 8.

11. The antibody SEQ ID NO: 1 (Niv(G) Ｂ the heavy chain of said antibody conjugated or fused to said polypeptide having at least 80% identity to said amino acid sequence ranging from said amino acid residue (Q) at position 71 to said amino acid residue (T) at position 602 in the ectodomain of said antibody, The light chain is selected from the group consisting of: i) SEQ ID NO: 2 (predicted epitope-rich peptide Niv(F) Ｂ ii) a polypeptide having at least 80% identity to the amino acid sequence ranging from the amino acid residue (K) at position 45 to the amino acid residue (I) at position 90 in SEQ ID NO: 3 (a predicted epitope-rich peptide Niv(N) Ｂ and a polypeptide having at least 80% identity with the amino acid sequence ranging from the amino acid residue (I) at position 318 to the amino acid residue (L) at position 355 in The antibody according to any one of claims 1 to 8.

12. The antibody SEQ ID NO: 1 (Niv(G) Ｂ the heavy chain of said antibody conjugated or fused to said polypeptide having at least 80% identity to said amino acid sequence ranging from said amino acid residue (Q) at position 71 to said amino acid residue (T) at position 602 in the ectodomain of said antibody, The light chain is conjugated or fused to both fusion proteins, i) SEQ ID NO: 3 (predicted epitope-rich peptide Niv(N) Ｂ ii) a polypeptide having at least 80% identity to the amino acid sequence ranging from the amino acid residue (I) at position 318 to the amino acid residue (L) at position 355 of SEQ ID NO: 2 (predicted epitope-rich peptide Niv(F) Ｂ ) to the polypeptide having at least 80% identity with the amino acid sequence ranging from the amino acid residue (K) at position 45 to the amino acid residue (I) at position 90 in The antibody according to any one of claims 1 to 8.

13. SEQ ID NO: 3 (predicted epitope-rich peptide Niv(N) Ｂ ) from the amino acid residue (I) at position 318 to the amino acid residue (L) at position 355 in SEQ ID NO: 2 (predicted epitope-rich peptide Niv(F) Ｂ ) is fused via a linker to the polypeptide having at least 80% identity with the amino acid sequence ranging from the amino acid residue (K) at position 45 to the amino acid residue (I) at position 90 in The antibody described in claim 12.

14. The antibody of claim 13, wherein the linker consists of the amino acid sequence defined in SEQ ID NO:

10.

15. The antibody SEQ ID NO: 1 (Niv(G) Ｂ the heavy chain of said antibody conjugated or fused to said polypeptide having at least 80% identity to said amino acid sequence ranging from said amino acid residue (Q) at position 71 to said amino acid residue (T) at position 602 in the ectodomain of said antibody, a light chain conjugated or fused to both of said fusion proteins having at least 80% identity with the amino acid sequence set forth in SEQ ID NO: 11; The antibody described in claim 14.

16. The antibody of any one of claims 1 to 15, wherein the antibody is an IgG4 antibody.

17. The antibody of any one of claims 1 to 16, wherein the antibody is specific for CD40.

18. The anti-CD40 antibody is derived from the 12E12 antibody, a heavy chain comprising complementarity determining regions CDR1H, CDR2H and CDR3H, wherein the CDR1H has the amino acid sequence GFTFSDYYMY (SEQ ID NO: 12), the CDR2H has the amino acid sequence YINSGGGGSTYYPDTVKG (SEQ ID NO: 13), and the CDR3H has the amino acid sequence RGLPFHAMDY (SEQ ID NO: 14); a light chain comprising complementarity determining regions CDR1L, CDR2L and CDR3L, wherein the CDR1L has the amino acid sequence SASQGISNYLN (SEQ ID NO: 15), the CDR2L has the amino acid sequence YTSILHS (SEQ ID NO: 16), and the CDR3L has the amino acid sequence QQFNKLPPT (SEQ ID NO: 17); The anti-CD40 antibody is derived from the 11B6 antibody, a heavy chain comprising complementarity determining regions CDR1H, CDR2H and CDR3H, wherein the CDR1H has the amino acid sequence GYSFTGYYMH (SEQ ID NO: 18), the CDR2H has the amino acid sequence RINPYNGATSYNQNFKD (SEQ ID NO: 19), and the CDR3H has the amino acid sequence EDYVY (SEQ ID NO: 20); a light chain comprising complementarity determining regions CDR1L, CDR2L and CDR3L, wherein the CDR1L has the amino acid sequence RSSQSLVHSNGNTYLH (SEQ ID NO: 21), the CDR2L has the amino acid sequence KVSNRFS (SEQ ID NO: 22), and the CDR3L has the amino acid sequence SQSTHVPWT (SEQ ID NO: 23); The anti-CD40 antibody is derived from the 12B4 antibody, a heavy chain comprising complementarity determining regions CDR1H, CDR2H and CDR3H, wherein the CDR1H has the amino acid sequence GYTFTDYVLH (SEQ ID NO: 24), the CDR2H has the amino acid sequence YINPYNDGTKYNEKFKG (SEQ ID NO: 25), and the CDR3H has the amino acid sequence GYPAYSGYAMDY (SEQ ID NO: 26); a light chain comprising complementarity determining regions CDR1L, CDR2L and CDR3L, wherein the CDR1L has the amino acid sequence RASQDISNYLN (SEQ ID NO: 27), the CDR2L has the amino acid sequence YTSRLHS (SEQ ID NO: 28), and the CDR3L has the amino acid sequence HHGNTLPWT (SEQ ID NO: 29); The antibody described in claim 17.

19. 18. The antibody of claim 17, wherein the anti-CD40 antibody is selected from the group consisting of selected mAb1, mAb2, mAb3, mAb4, mAb5 and mAb6 listed in Table A.

20. a heavy chain having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 47; a light chain having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 48; The antibody of claim 1, comprising:

21. a heavy chain having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 47; a light chain having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 49; The antibody of claim 1, comprising:

22. A polynucleotide encoding the heavy and / or light chain of the antibody according to any one of claims 1 to 21.

23. A vector comprising the polynucleotide of claim 22.

24. A host cell transfected, infected or transformed with the polynucleotide of claim 22 and / or the vector of claim 23.

25. A vaccine composition comprising the antibody of any one of claims 1 to 21.

26. 22. A method of vaccinating a subject in need thereof against Nipah virus, comprising administering a therapeutically effective amount of the antibody of any one of claims 1 to 21.

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