DC-targeting anti-Nipah virus infection vaccine

Abstract

Nipah virus (NiV) is a paramyxovirus which appears recently, is high in pathogenicity and is affected by both people and livestock. The inventor designs an anti-CD40 monoclonal antibody which is associated with a NiV G protein extracellular domain (a first generation vaccine), or associated with the NiV G protein extracellular domain and epitopes screened from NiV F protein and N protein (a second generation vaccine). And carrying out quality control on the vaccine batch. The immunogenicity of the two vaccines has been tested in an hCD40Tg mouse. And the dose-dependent reaction of the antigen on IFNg T cells is observed. And after three weeks of boosted immunization, specific IgG can be detected in a group inoculated with 10 micrograms of vaccine for immunization. And one week after boosted immunization, the B cell reaction is obviously improved. All samples show a neutralization effect in the fourth week, and the average titer is 1: 500. The inventor also proves the efficacy of an innovative DC-targeting vaccine candidate for preventing NiV-B infection in a challenge experiment of an AGM model. The immune reaction caused by the vaccine not only can crossly neutralize various NiV strains, but also can neutralize HeV strains. The nipah virus antigen targeting professional APCs can be used as an effective means for preventing nipah virus lethal dose attack. Thus, the present invention relates to antibodies directed against antigens on the surface of an antigen presenting cell wherein the heavy chain and / or light chain is bound or fused to a Nipah virus antigenic polypeptide.

Description

Technical Field

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

[0002] Nipah virus (NiV) is a recently emerged, highly pathogenic, zoonotic paramyxovirus first discovered in a severe febrile encephalitis outbreak in Malaysia and Singapore in 1998-1999 (Chua, KB, et al. "Nipah virus: a recently emergent deadly paramyxovirus." Science 288.5470(2000):1432-1435.). The Bangladesh strain (NiVB) has been reported in Bangladesh and northeastern India in multiple outbreaks of varying magnitude, with outbreaks occurring almost every year from 2001 to 2015. NiVB outbreaks have a high case fatality rate, averaging about 75% (Lo, Michael K., et al. "Characterization of Nipah virus from outbreaks in Bangladesh, 2008–2010." Emerging infectious diseases 18.2 (2012): 248.), and human-to-human transmission is often observed (Gurley, Emily S., et al. "Person-to-person transmission of Nipah virus in a Bangladeshi community." Emerging infectious diseases 13.7 (2007): 1031.). There are currently a variety of candidate vaccines, but all are in the preclinical stage. Although rVSV vectors expressing Nipah virus G (or F) are the leading candidates for new "emergency vaccines" for epidemic management (Foster, Stephanie L., et al. "A recombinant VSV-vectored vaccine rapidly protects nonhuman primates against lethal Nipah virus disease." Proceedings of the National Academy of Sciences 119.12(2022):e2200065119.), issues such as safety, development, and insufficient transportation / storage in epidemic areas remain concerns. Therefore, improving the ability of vaccines to induce strong cellular and humoral immune responses and using them as a new universal vaccine platform for preventive strategies, as well as at the core of therapeutic means, remains a challenge for rapidly and efficiently responding to Nipah virus. Summary of the invention Summary of the invention:

[0004] The present invention is defined by the claims. In particular, the present invention relates to antibodies against antigens on the surface of antigen presenting cells, wherein the heavy chain and / or light chain is coupled or fused to a Nipah virus antigen polypeptide. Detailed description of the invention:

[0006] definition:

[0007] As used herein, the term "subject" or "subject in need thereof" is intended to refer to a human or non-human mammal. Typically, the patient is infected or at risk of being infected with Nipah virus.

[0008] As used herein, the term "Nipah virus" has a general meaning in the art and refers to a virus belonging to the family Paramyxoviridae, and is related to Hendra virus (formerly known as equine measles virus). Nipah virus was initially isolated in 1999 when examining samples of an outbreak of encephalitis and respiratory disease in adult males in Malaysia and Singapore (see, e.g., Chua et al., Lancet. October 9, 1999; 354(9186): 1257-9 and Paton et al., Lancet. October 9, 1999; 354(9186): 1253-6). The host of Nipah virus is not yet clear, but flying foxes (Pteropus bats) are suspected to be natural hosts. Nipah virus contains a negative-sense single-stranded RNA (ssRNA) genome of 18.2 kb consisting of six genes, which encode nine proteins: nucleoprotein (N), phosphoprotein (P), interferon antagonist factors W and V, viral C protein, matrix protein (M), viral fusion protein and glycoprotein (F and G, respectively), and large polymerase (L).

[0009] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to amino acid polymers of any length. These terms also include amino acid polymers that have been modified; for example, by disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. When in the context of gene therapy, a polypeptide refers to the corresponding intact polypeptide or any fragment or genetically engineered derivative thereof that retains the desired biochemical function of the intact protein.

[0010] As used herein, the term "G protein" refers to Nipah virus glycoprotein G. The G protein has a globular head domain formed by six β-sheet propellers connected to a transmembrane anchor region by a flexible stem domain. The G protein binds to the cellular receptors ephrinB2 and ephrin B3, mediating viral attachment. After attachment, Nipah virus glycoprotein G undergoes a conformational change, leading to the triggering of glycoprotein F, resulting in membrane fusion. An exemplary amino acid sequence of the G protein is shown in SEQ ID NO: 1.

[0011] SEQ ID NO:1>sp|Q9IH62|GLYCP_NIPAV glycoprotein G OS=Nipah virus OX=121791 GN=GPE=1 SV=1

[0012] MPAENKKVRFENTTSDKGKIPSKVIKSYYGTMDIKKINEGLLDSKILSAFNTVIALLGSIVIIVMNIMII QNYTRSTDNQAVIKDALQGIQQQIKGLADKIGTEIGPKVSLIDTSSTITIPANIGLLGSKISQSTASINENVNEKC KFTLPPLKIHECNISCPNPLPFREYRPQTEGVSNLVGLPNNICLQKTSNQILKPKLISYTLPVVGQSGTCITDPLLA MDEGYFAYSHLERIGSCSRGVSKQRIIGVGEVLDRGDEVPSLFMTNVWTPPNPNTVYHCSAVYNNEFYYVLCAVSTV GDPILNSTYWSGSLMMTRLAVKPKSNGGGYNQHQLALRSIEKGRYDKVMPYGPSGIKQGDTLYFPAVGFLVRTEFKY NDSNCPITKCQYSKPENCRLSMGIRPNSHYILRSGLLKYNLSDGENPKVVFIEISDQRLSIGSPSKIYDSLGQPVFY QASFSWDTMIKFGDVLTVNPLVVNWRNNTVISRPGQSQCPRFNTCPEICWEGVYNDAFLIDRINWISAGVFLDSNQT AENPVFTVFKDNEILYRAQLASEDTNAQKTITNCFLLKNKIWCISLVEIYDTGDNVIRPKLFAVKIPEQCT

[0013] 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. These features include a heptad repeat sequence and a hydrophobic fusion peptide, which bind to each other to form a six-helix bundle and play a role in membrane fusion. Nipah virus attaches to target cells through glycoprotein G, which then undergoes conformational changes, triggering Nipah virus glycoprotein F, leading to membrane fusion. An exemplary amino acid sequence of the F protein is shown in SEQ ID NO: 2.

[0014] SEQ ID NO:2>sp|Q9IH63|FUS_NIPAV fusion glycoprotein F0 OS=Nipah virus OX=121791 GN=F PE=1 SV=1

[0015] MVVILDKRCYCNLLILILMISECSVGILHYEKLSKIGLVKGVTR KYKIKSNPLTKDIVIKMIPNVSNMS QCTGSVMENYKTRLNGILTPIKGALEIYKNNTHDLVGDVRLAGVIMAGVAIGIATAAQITAGVALYEAMKNADNINKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDKISCKQTELSLDLALS KYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYETLLRTLGYATEDFDDLLESDSITGQIIYVDLSSYYIIVRVYFPILTEIQQAYIQELLPVSFNNDNSEWISIVPNFILV RNTLISNIEIGFCLITKRSVICNQDYATPMTNNMRECLTGSTEKCPRELVVSSHVPRFALSNGVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCPTAVLGNVIISLGKY LGSVNYNSEGIAIGPPVFTDKVDISSQISSMNQSLQQSKDYIKEAQRLLDTVNPSLISMLSMIILYVLSIASLCIGLITFISFIIVEKKRNTYSRLEDRRVRPTSSGDLYYIGT

[0016] As used herein, the term "N protein" refers to the Nipah virus nucleoprotein, which encapsulates the genome to protect it from degradation by nucleases. The encapsulated 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 shown in SEQ ID NO: 3.

[0017] SEQ ID NO:3>sp|Q9IK92|NCAP_NIPAV nucleoprotein OS=Nipah virus OX=121791 GN=N PE=1 SV=1

[0018] MSDIFEEAASFRSYQSKLGRDGRASAATATLTTKIRIFVPATNSPELRWELTLFALDVIRSPSAAESMKVGAAFTLISMYSERPGALIRSLLNDPDIEAVIIDVGSMVNGIPVMERRGDKAQEEMEGLMRILKTARDSSKGKTPFVDSRAYGLRITDM STLVSAVITIEAQIWILIAKAVTAPDTAEESETRRWAKYVQQKRVNPFFALTQQWLTEMRNLLSQSLSVRKFMVEILIEVKKGGSAKGRAVEIISDIGNYVEETGMAGFFATIRFGLETRYPALALNEFQSDLNTIKSLMLLYREIGPRAPYMVLLEES IQTKFAPGGYPLLWSFAMGVATTIDRSMGALNINRGYL EPMYFRLGQKSARHHAGGIDQNMANRLGLSSDQVAELAAAVQETSAGRQESNVQAREAKFAAGGVLIGGSDQDIDEGEEPIEQSGRQSVTFKREMSISSLANSVPSSSVSTSGGTRLTNSLLNLRSRLAAKAAKEAASSNATDDPAISNRTQGESEKKNNQDLKPAQNDLDFVRADV.

[0019] As used herein, the term "polynucleotide" refers to a nucleotide polymer of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may include modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, the nucleotide structure may be modified before or after polymer assembly. The term polynucleotide used herein refers to double-stranded and single-stranded molecules interchangeably. Unless otherwise specified or required, embodiments of any polynucleotide form described in the present invention include a double-stranded form and each single-stranded form of the two complementary single-stranded forms that make up the double-stranded form.

[0020] As used herein, the expression "derived from" refers to the process of separating, deriving or preparing a different second component (eg, a second polypeptide different from the first) from a first component (eg, a first polypeptide) or information from the first component.

[0021] As used herein, the term "coding" refers to the inherent properties of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA, used as a template for synthesizing other polymers and macromolecules with a determined nucleotide sequence (such as rRNA, tRNA and mRNA) or a determined amino acid sequence in a biological process, and the biological properties resulting therefrom. Therefore, the condition for a gene, cDNA or RNA to encode a protein is that the mRNA corresponding to the gene can produce a certain protein in a cell or other biological system after transcription and translation. The coding strand (whose nucleotide sequence is the same as the mRNA sequence, usually provided in the form of a sequence table) and the non-coding strand (used as a template for transcription of a gene or cDNA) can both be referred to as encoding a protein or other product corresponding to the gene or cDNA. Unless otherwise indicated, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are degenerate to each other and encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" may also include introns, because the nucleotide sequence encoding a protein may contain introns in some variants.

[0022] As used herein, the terms "vector", "cloning vector" and "expression vector" refer to vectors that can introduce DNA or RNA sequences (e.g., foreign genes) into host cells to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequences.

[0023] As used herein, the term "promoter / regulatory sequence" refers to a polynucleotide sequence (e.g., a DNA sequence) recognized by a cellular synthetic mechanism or an introduced synthetic mechanism, which is required to initiate specific transcription of the polynucleotide sequence so that a gene product operably linked to the promoter / regulatory sequence is expressed. In some cases, the sequence may be a core promoter sequence, and in other cases, the sequence may also include enhancer sequences and other regulatory elements required for gene product expression. For example, a promoter / regulatory sequence may be a sequence that expresses a gene product in a tissue-specific manner.

[0024] As used herein, the term "operably linked" or "transcriptional control" refers to a functional connection between a regulatory sequence and a heterologous polynucleotide sequence, resulting in the expression of the latter. For example, a first polynucleotide sequence is operably linked to a second polynucleotide sequence when the first polynucleotide sequence is functionally linked to 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 contiguous to each other, for example, in the case where it is desired to connect two protein coding regions, the operably linked DNA sequences can be in the same reading frame.

[0025] As used herein, the term "transformation" refers to the introduction of "foreign" (i.e., exogenous or extracellular) genes, DNA or RNA sequences into host cells, causing the host cells to express the introduced genes or sequences to produce a desired substance, usually a protein or enzyme encoded by the introduced genes or sequences. Host cells that accept and express the introduced DNA or RNA are referred to as "transformed" cells.

[0026] As used herein, the term "expression system" refers to a host cell and a compatible vector under appropriate conditions, for example, for expressing a protein encoded by foreign DNA carried by the vector and introduced into the host cell.

[0027] As used herein, the "percentage of identity" between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions × 100), while taking into account the number of gaps and the length of each gap that need to be introduced to achieve optimal alignment of the two sequences. The comparison of sequences and the determination of the percentage of identity between the two sequences can be completed using a mathematical algorithm as described below. The percentage of identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443–53.). The percentage of 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, when using EMBOSS Needle, the BLOSUM62 matrix can be used with a Gap Open Penalty of 10, a Gap Extension Penalty of 0.5, an False End Gap Penalty, an End Gap Open Penalty of 10, and an End Gap Extension Penalty of 0.5. In general, the "percent identity" is a function of the number of matched positions divided by the number of compared positions multiplied by 100. For example, two aligned sequences are 60% identical if 6 out of 10 sequence positions are identical. The percent identity is typically determined over the entire length of the query sequence being analyzed. Two molecules having the same primary amino acid sequence or polynucleotide sequence are identical, regardless of any chemical and / or biological modifications. According to the present invention, a first amino acid sequence that is at least 80% identical to a second amino acid sequence means that the first sequence is 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to the second amino acid sequence.

[0028] As used herein, the term "conjugate" or interchangeably "conjugated polypeptide" is intended to mean a composite or chimeric molecule formed by covalently linking one or more polypeptides. The term "covalently linked" or "conjugated" refers to a polypeptide and a non-peptide moiety that are covalently linked directly or indirectly through one or more intermediate moieties (such as a bridging moiety, a spacer, or a linking moiety). A special conjugate is a fusion protein.

[0029] As used herein, the term "fusion protein" refers to a protein produced by connecting two or more polypeptides derived from different proteins. In particular, fusion proteins can be produced by recombinant DNA technology and are commonly used in biological research or treatment. Fusion proteins can also be produced by chemical covalent fusion with or without a linker between the polypeptide portions of the fusion protein. In a fusion protein, two or more polypeptides are fused directly or through a linker.

[0030] As used herein, the term "directly" refers to the fusion of the first amino acid at the N-terminus of a first polypeptide with the last amino acid at the C-terminus of a second polypeptide. Such 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).

[0031] As used herein, the term "linker" has a general meaning in the art, and refers to an amino acid sequence that is long enough to ensure that the protein forms a suitable secondary and tertiary structure. In some embodiments, the linker is a peptide linker, which comprises at least one but is less than 30 amino acids, such as a peptide linker consisting of 2-30 amino acids, preferably consisting of 10-30 amino acids, more preferably consisting of 15-30 amino acids, more preferably consisting of 19-27 amino acids, and most preferably consisting of 20-26 amino acids. In certain embodiments, the connecting peptide 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 is a sequence that enables the compound to form a suitable conformation. The most suitable linker sequences (1) are able to form a flexible extended conformation, (2) do not show a tendency to form ordered secondary structures that could interact with the functional domains of the fusion protein, and (3) have minimal hydrophobicity or charge, properties that could promote interaction with the functional protein domains.

[0032] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules containing antigen binding sites that immunospecifically bind to antigens. In natural antibodies of rodents and primates, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains, λ (1) and κ (k). There are five main heavy chain categories (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains different sequence domains. In a typical IgG antibody, the light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of the light chain (VL) and the heavy chain (VH) determine the binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to Fc receptors (FcRs). The Fv fragment is the N-terminal portion of the immunoglobulin Fab fragment and consists of the variable portions of one light chain and one heavy chain. The specificity of an antibody lies in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is composed of residues primarily from the hypervariable regions or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable regions or framework regions (FRs) can participate in the formation of the antibody binding site or affect the overall domain structure and, therefore, the binding site. The complementarity determining regions or CDRs refer to the amino acid sequences that together define the binding affinity and specificity of the Fv region in the natural 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 includes six CDRs, which are composed of complementarity determining regions from one V region of a heavy chain and one of a light chain. Framework regions (FRs) refer to the amino acid sequences inserted between the CDRs. Thus, the variable regions of the light and heavy chains typically include four framework regions and three CDRs of the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The residues in the variable domains of antibodies are usually numbered according to the system devised by Kabat et al. This system was described in Kabat et al., 1987, "Protein Sequences of Immunological Interest", National Institutes of Health, U.S. Department of Health and Human Services (Kabat et al., 1992, here after "Kabat et al."). The Kabat residue names do not always correspond directly to the linear numbering of the amino acid residues in the SEQ ID sequence.The actual linear amino acid sequence may contain fewer or more amino acids than in the strict Kabat numbering, which is related to the shortening or insertion of structural components of the basic variable domain structure, whether framework or complementary determining regions (CDRs). The correct Kabat residue numbering for a given antibody can be determined by aligning the homologous residues in the antibody sequence with the "standard" Kabat numbering sequence. According to the Kabat numbering system, 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). For the agonist antibodies described below, the CDRs were determined using the CDR search algorithm of www.bioinf.org.uk. For specific methods, please refer to the "How to identify CDRs by viewing the sequence" section on the antibody page of this website.

[0033] As used herein, the term "immunoglobulin domain" refers to a globular region of an antibody chain (eg, a heavy chain antibody or a light chain), or a polypeptide consisting essentially of such a globular region.

[0034] 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 from position P230 to the carboxyl terminus of the IgG antibody. The residue numbering in the Fc region adopts the EU index numbering system of Kabat. The C-terminal lysine (residue K447) in the Fc region can be removed during, for example, the production or purification of the antibody. Therefore, the antibody composition of the present invention can include antibody groups having all K447 residues removed, antibody groups having no K447 residue removed, and antibody groups with and without a mixture of K447 residue numbers.

[0035] 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 region (i.e., heavy chain and / or light chain) or a portion thereof is altered, replaced or exchanged so that the antigen binding site (variable region) is connected to a constant region of a different or altered class, effector function and / or species, or to a completely different molecule that imparts new properties to the chimeric antibody, such as an enzyme, toxin, agonist molecule such as CD40 ligand, hormone, growth factor, drug, etc.; or (b) the variable region or a portion thereof is altered, replaced or exchanged with a variable region having a different or altered antigenic specificity. Chimeric antibodies also include primatized antibodies, particularly humanized antibodies. In addition, chimeric antibodies may contain residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. For more 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 US Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0036] As used herein, the term "humanized antibody" includes antibodies having 6 CDRs of a mouse antibody but having a humanized framework and constant region. More specifically, the term "humanized antibody" used herein may include antibodies constructed by transplanting CDR sequences derived from another mammalian species (such as a mouse) germline to human framework sequences.

[0037] 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-directed mutagenesis in vitro or somatic mutations in vivo). However, in one embodiment, the term "human monoclonal antibody" used herein does not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted to human framework sequences.

[0038] As used herein, the term "immune response" refers to the response of the immune system to an antigen in the host, including the production of antigen-specific antibodies and / or cytotoxic responses. The immune response to the initial antigen exposure (primary immune response) is usually detectable after a latent period of several days to two weeks; the immune response to re-stimulation of the same antigen (secondary immune response) is faster than the primary immune response. The immune response to the transgenic product may include humoral immune responses (such as antibody responses) and cellular immune responses (such as cytotoxic T cell responses), which may be triggered 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).

[0039] As used herein, the term "APCs" or "antigen presenting cells" refers to cells that are capable of activating T cells, including but not limited to certain macrophages, B cells, and dendritic cells.

[0040] As used herein, the term "dendritic cell" or "DCs" refers to any of a variety of morphologically similar cell types found in lymphoid or non-lymphoid tissues. These cells are characterized by their unique morphology and high levels of surface MHC-II class molecule expression (Steinman, et al., Ann. Rev. Immunol. 9:271 (1991); incorporated here in by reference for its description of such cells).

[0041] As used herein, the term "CD40" has its ordinary meaning in the art and refers to the human CD40 polypeptide receptor. In some embodiments, CD40 is a subtype of the human canonical sequence reported in UniProtKB-P25942 (also known as human TNR5).

[0042] As used herein, the term "CD40L" has a general meaning in the art and refers to a human CD40L polypeptide, for example, a human CD40L polypeptide as reported in UniProtKB-P25942, including its CD40 binding domain of SEQ ID NO: 4. CD40L can be expressed as a soluble polypeptide and is a natural ligand for the CD40 receptor.

[0043] SEQ ID NO:4>CD40L binding domain

[0044] MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL.

[0045] 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 is capable of (i) inducing proliferation of B cells, as measured in vitro by flow cytometry analysis or by replica dilution analysis of CFSE-labeled cells; and / or (ii) inducing secretion of cytokines, such as IL-6, IL-12 or IL-15, which can be measured in vitro by a dendritic cell activation assay.

[0046] As used herein, the term "Langerin" has a general meaning in the art and refers to a human C-type lectin domain family 4 member K polypeptide. In some embodiments, Langerin is a subtype of the human canonical sequence reported in UniProtKB-Q9UJ71 (also known as human CD207).

[0047] As used herein, the term "treatment" or "treatment" refers to preventive or prophylactic treatment as well as therapeutic or disease-modifying treatment, including treatment of patients at risk for or suspected of contracting a disease and patients who are ill or diagnosed with a disease or disease, and includes the inhibition of clinical relapse. Patients who are suffering from a disease or may eventually develop a disease may be treated to prevent, cure, delay the onset of the disease, reduce its severity, or improve the symptoms of one or more disorders or recurrent disorders, or prolong the patient's survival beyond what would be expected without such treatment. "Treatment regimen" refers to the pattern of treatment of a disease, such as the dosage pattern used during treatment. Treatment regimens may include induction regimens and maintenance regimens. The term "induction regimen" or "induction phase" refers to a treatment regimen (or part of a treatment regimen) used for the initial treatment of a disease. The overall goal of an induction regimen is to provide patients with high levels of drug during the initial phase of the treatment regimen. The induction regimen may (partially or fully) employ a "loading dose regimen," which may include larger doses of the drug than the physician uses during the maintenance regimen, more frequent dosing than the physician uses during the maintenance regimen, or both. The term "maintenance regimen" or "maintenance phase" refers to a regimen (or portion of a regimen) used to maintain a patient during treatment of a disease, e.g., to keep the patient in remission for an extended period of time (months or years). A maintenance regimen can be continuous (e.g., given at a fixed time, such as weekly, monthly, yearly, etc.) or intermittent (e.g., interrupted, intermittent, upon relapse, or after certain predetermined criteria (e.g., pain, disease manifestations, etc.) are met).

[0048] As used herein, the term "pharmaceutical composition" refers to the composition described herein or a pharmaceutically acceptable salt thereof, and other agents, such as carriers and / or excipients. The pharmaceutical compositions provided herein generally include a pharmaceutically acceptable carrier.

[0049] As used herein, the term "pharmaceutically acceptable carrier" includes all solvents, diluents or other liquid carriers, dispersants or suspending aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc. suitable for the specific dosage form desired. Remington's Pharmacy, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used to formulate pharmaceutical compositions and known preparation techniques thereof.

[0050] As used herein, the term "vaccination" or "vaccination" refers to, but is not limited to, the process of eliciting an immune response in a subject against a specific antigen.

[0051] As used herein, the term "vaccine composition" is intended to refer to a composition that can be administered to humans or animals to induce an immune system response; such an immune system response may result in the activation of certain cells, particularly APCs, T lymphocytes and B lymphocytes.

[0052] As used herein, the term "antigen" refers to a molecule that can be specifically bound by an antibody or T cell receptor (TCR) if processed and presented by an MHC molecule. Antigens can also be recognized by the immune system and / or can induce a humoral immune response and / or a cellular immune response, thereby activating B and / or T lymphocytes. Antigens can have one or more epitopes or antigenic sites (B- and T-epitopes).

[0053] 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 also generally refers to a substance that is generally used to accelerate, prolong or improve 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 the innate immune response by affecting the transient response of the innate immune response and enhances the long-term effects of the adaptive immune response by activating and maturing antigen presenting cells (APCs), particularly dendritic cells (DCs).

[0054] As used herein, the term "therapeutically effective amount" refers to an amount of the active ingredient of the present invention sufficient to induce an immune response at a reasonable benefit / risk ratio applicable to medical treatment.

[0055] Antibodies of the present invention:

[0056] The first object of the present invention relates to an antibody against an antigen on the surface of an antigen presenting cell, wherein the heavy chain and / or light chain of the antibody is coupled or fused to a polypeptide, and the polypeptide is the same as SEQ ID NO: 1 ("Niv (G) B The amino acid sequence of the peptides from amino acid residue 71 (Q) to amino acid residue 602 (T) in the extracellular domain ("extracellular domain") has at least 80% identity.

[0057] In some embodiments, the light chain of the antibody is coupled or fused to a polypeptide that is identical to SEQ ID NO: 1 ("Niv (G) B The amino acid sequence of the polypeptide having the amino acid residue at position 71 (Q) to the amino acid residue at position 602 (T) in the extracellular domain ("extracellular domain") has at least 80% identity.

[0058] In some embodiments, the heavy chain of the antibody is coupled or fused to a polypeptide that is identical to SEQ ID NO: 1 ("Niv (G) BThe amino acid sequence of the polypeptide having the amino acid residue at position 71 (Q) to the amino acid residue at position 602 (T) in the extracellular domain ("extracellular domain") has at least 80% identity.

[0059] The heavy and / or light chains of the antibody are bound to Niv(G) via their C-termini B In some embodiments, the heavy chain and / or light chain of the antibody is bound or fused to the extracellular domain of Niv(G) B N-terminal fusion of the extracellular domain.

[0060] In some embodiments, the heavy and / or light chains of the antibodies are chemically coupled to Niv(G) BThe antibody may be attached to or coupled to an outer domain. Several methods are known in the art for attaching or coupling an antibody to its coupling moiety. Examples of types of linkages used to couple a moiety to an antibody include, but are not limited to, hydrazone, thioether, ester, disulfide, and peptide-containing linkages, such as valine-citrate linkages. A linkage may be selected that is, for example, susceptible to cleavage under low pH conditions within a lysosomal compartment, or susceptible to cleavage by a protease, such as a protease preferentially expressed in tumor tissue, such as a cathepsin (e.g., cathepsin B, C, D). The coupling technology of polypeptides (especially antibodies) is well known in the art (see 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); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (Robinson et al. eds., Marcel Deiker, Inc., 2nd ed. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al. eds., 1985); "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al. eds., Academic Press, 1985); and Thorpe et al., 1982, Immunol. Rev. 62: 119-58; see also, eg, PCT publication WO 89 / 12624.). Typically, the peptide is covalently linked to a lysine or cysteine ​​residue on the antibody via an N-hydroxysuccinimide ester or maleimide functional group, respectively.It has been reported that the use of engineered cysteine ​​or the incorporation of unnatural amino acids in conjugation methods can improve the uniformity of the conjugates (Axup, JY, Bajjuri, KM, Ritland, M., Hutchins, BM, Kim, CH, Kazane, SA, Halder, R., Forsyth, JS, Santidrian, AF, Stafin, K., et al. (2012). Synthesis of site-specific antibody-drug conjugates using unnatural aminoacids. 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 an improved therapeutic index to target 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 conjugation method called "THIOMABs" (TDCs), which is said to have an improved therapeutic index compared to traditional conjugation methods. The conjugation of ADCs to non-natural amino acids that have been incorporated into antibodies is also being explored; however, the universality of this approach has not yet been determined (Axup et al., 2012). In particular, one skilled in the art can also envision that the Fc polypeptide modified by polypeptide engineering will carry a glutamine-containing tag (e.g., a Gin-containing peptide tag or a Q-tag) as an acyl donor, or contain endogenous glutamine that is reactive by polypeptide engineering (e.g., by amino acid deletion, insertion, substitution or mutation on the polypeptide). The transglutaminase can then be covalently cross-linked with an amine donor reagent (e.g., a small molecule containing or attached to a reactive amine) to form a stable and uniform population of engineered Fc-containing polypeptide conjugates, wherein the amine donor reagent is site-specifically conjugated to the Fc-containing polypeptide via an acyl donor glutamine tag or accessible / exposed / reactive endogenous glutamine (WO 2012059882).

[0061] In some embodiments, the heavy and / or light chains of the antibodies bind to Niv(G) through a dockerin domain or domains. B The extracellular domains are combined to allow non-covalent coupling to cohesive fusion proteins as described in US20160031988A1 and US20120039916A1.

[0062] In some embodiments, the heavy chain and / or light chain of the antibody is identical to Niv(G) B The extracellular domains are fused to form a fusion protein.

[0063] In some embodiments, the Niv(G) B The extracellular domain is fused directly or via a linker to the heavy chain and / or light chain. As used herein, the term "directly" refers to Niv(G) B The first amino acid at the N-terminal end of the extracellular domain is fused to the last amino acid at the C-terminal end of the heavy chain 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), (Babon et al., Nat. Med. 2016, PMID 27798614).

[0064] In some embodiments, Niv(G) B The N-terminus of the extracellular domain is fused directly or via a linker to the C-terminus of the heavy chain.

[0065] 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.

[0066] QTPTNTISVTPTNNSTPTNNSNPKPNP(flexV1, SEQ ID NO: 5);

[0067] SSVSPTTSVHPTPTSVPPTPTKSSP(f1, SEQ ID NO: 6);

[0068] PTSTPADSSTITPTATPTATPTIKG(f2, SEQ ID NO: 7);

[0069] TVTPTATATPSAIVTTITPTATTKP(f3, SEQ ID NO: 8);

[0070] TNGSITVAATAPTVTPTVNATPSAA (f4, SEQ ID NO: 9).

[0071] In some embodiments, an antibody of the invention comprises:

[0072] An antibody heavy chain coupled or fused to a polypeptide having the same sequence as SEQ ID NO: 1 (Niv (G) B The amino acid sequence of the extracellular domain of the polypeptide has at least 80% identity from amino acid residue 71 (Q) to amino acid residue 602 (T), and

[0073] An antibody light chain coupled or fused to a polypeptide, wherein the polypeptide is identical to SEQ ID NO: 2 (predicted epitope-rich peptide segment Niv (F) B ) has at least 80% identity from amino acid residue 45 (K) to amino acid residue 90 (I).

[0074] In some embodiments, the C-terminus of the antibody light chain is coupled or fused to the N-terminus of a polypeptide having the same sequence as SEQ ID NO: 2 ("predicted epitope-rich peptide Niv (F) B ”) has at least 80% identity with the amino acid sequence from amino acid residue 45 (K) to amino acid residue 90 (I) in SEQ ID NO: 1 (Niv (G) B The amino acid sequence of the extracellular domain of the present invention is at least 80% identical to that of the amino acid residue at position 71 (Q) to that at position 602 (T).

[0075] In some embodiments, an antibody of the invention comprises:

[0076] An antibody heavy chain coupled or fused to a polypeptide having at least 80% identity to the amino acid sequence from amino acid residue 71 (Q) to amino acid residue 602 (T) in SEQ ID NO: 1 (Niv(G)B extracellular domain), and

[0077] An antibody light chain coupled or fused to a polypeptide, wherein the polypeptide is identical to SEQ ID NO: 3 (predicted epitope-rich peptide segment Niv (F) B ) has at least 80% identity in the amino acid sequence from amino acid residue 318 (I) to amino acid residue 355 (L).

[0078] In some embodiments, the C-terminus of the antibody light chain is coupled or fused to the N-terminus of a polypeptide having the same sequence as SEQ ID NO: 3 ("predicted epitope-rich peptide Niv (N) B ”) has at least 80% identity to the amino acid sequence from amino acid residue 318 (I) to amino acid residue 355 (L), the C-terminus of the antibody heavy chain is coupled or fused to the N-terminus of the polypeptide, and the N-terminus of the polypeptide is identical to SEQ ID NO: 1 (Niv (G) B The amino acid sequence of the extracellular domain of the polypeptide has at least 80% identity from amino acid residue 71 (Q) to amino acid residue 602 (T).

[0079] In some embodiments, an antibody of the invention comprises:

[0080] An antibody heavy chain coupled or fused to a polypeptide having the same sequence as SEQ ID NO: 1 (Niv (G) B The amino acid sequence of the extracellular domain of the polypeptide has at least 80% identity from amino acid residue 71 (Q) to amino acid residue 602 (T), and

[0081] An antibody light chain coupled or fused to the following two polypeptides: (i) the polypeptide and SEQ ID NO: 2 (predicted epitope-rich peptide segment Niv (F) B ) has at least 80% identity with the amino acid sequence from amino acid residue 45 (K) to amino acid residue 90 (I) in SEQ ID NO: 3 (predicted epitope-rich peptide Niv (F) B ) has at least 80% identity in the amino acid sequence from amino acid residue 318 (I) to amino acid residue 355 (L).

[0082] In some embodiments, an antibody of the invention comprises:

[0083] An antibody heavy chain coupled or fused to a polypeptide having the same sequence as SEQ ID NO: 1 (Niv (G) B The amino acid sequence of the extracellular domain of the polypeptide has at least 80% identity from amino acid residue 71 (Q) to amino acid residue 602 (T), and

[0084] An antibody light chain coupled or fused to a fusion protein, wherein (i) is identical to SEQ ID NO: 3 (predicted epitope-rich peptide segment Niv (F) B ) is fused to (ii) a polypeptide having at least 80% identity with the amino acid sequence from amino acid residue 318 (I) to amino acid residue 355 (L) of SEQ ID NO: 2 (predicted epitope-rich peptide Niv (F) B ) has at least 80% identity in the amino acid sequence from amino acid residue 45 (K) to amino acid residue 90 (I).

[0085] In some embodiments, the light chain is coupled or fused to a fusion protein comprising a peptide sequence similar to SEQ ID NO: 2 ("predicted epitope-rich peptide Niv (F) B A polypeptide having at least 80% identity with the amino acid sequence from amino acid residue 45 (K) to amino acid residue 90 (I) in SEQ ID NO: 3 (predicted epitope-rich peptide Niv (F)) wherein the polypeptide is fused to the N-terminus of the polypeptide directly or through a linker peptide via its C-terminus. B ) has at least 80% identity in the amino acid sequence from amino acid residue 318 (I) to amino acid residue 355 (L).

[0086] In some embodiments, the light chain of the antibody is coupled or fused to the fusion protein via its C-terminus.

[0087] In some embodiments, the peptide sequence of SEQ ID NO: 3 (predicted epitope-rich peptide Niv (F) B ) in the amino acid sequence from amino acid residue 318 (I) to amino acid residue 355 (L) having at least 80% identity, fused to SEQ ID NO: 2 (predicted epitope-rich peptide Niv (F) via a linker B ) in a polypeptide having at least 80% identity in the amino acid sequence from amino acid residue 45 (K) to amino acid residue 90 (I).

[0088] In some embodiments, the linker consists of the amino acid sequence shown in SEQ ID NO:10.

[0089] SEQ ID NO: 10

[0090] AEAAAKEAAAKA

[0091] In some embodiments, an antibody of the invention comprises:

[0092] An antibody heavy chain coupled or fused to a polypeptide having the same sequence as SEQ ID NO: 1 (Niv (G) B The amino acid sequence of the extracellular domain of the polypeptide has at least 80% identity from amino acid residue 71 (Q) to amino acid residue 602 (T), and

[0093] An antibody light chain coupled or fused to a polypeptide having at least 80% identity to the amino acid sequence shown in SEQ ID NO:11.

[0094] SEQ ID NO: 11

[0095] KYKIKSNPLTKDIVIKMIPNVSNMSQCTGSVMENYKTRLNGILTPI AE AAAKEAAAKA IQTKFAPGGYPLLWSFAMGVATTIDRSMGALNINRGYL.

[0096] In some embodiments, the antibody is an IgG antibody, preferably an IgG1 or IgG4 antibody, even more preferably an IgG4 antibody.

[0097] In some embodiments, the antibody is a chimeric antibody, particularly a chimeric mouse / human antibody.

[0098] In some embodiments, the antibody is a humanized antibody.

[0099] Chimeric or humanized antibodies can be prepared based on the sequence of mouse monoclonal antibodies prepared as described above. The DNA encoding heavy chain and light chain immunoglobulins can be obtained from interested mouse hybridomas, and transformed into containing non-mouse (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, in order to prepare chimeric antibodies, methods known in the art can be used to connect mouse variable regions to human constant regions (see, e.g., US Patent No. 4,816,567 to Cabilly et al.). In order to prepare humanized antibodies, methods known in the art can be used to insert mouse CDR regions into human frameworks. See, e.g., U.S. Patent No. 5,225,539 of Winter, and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370 of Queen et al., for example.

[0100] In some embodiments, the antibody is a human antibody. In some embodiments, transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system can be used to identify human antibodies. These transgenic and transchromosomal mice include mice referred to herein as HuMAb mice and KM mice, respectively, collectively referred to herein as "human Ig mice". HuMAb (Medarex, Inc.) contains a human immunoglobulin gene minigene site encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, as well as targeted mutations that inactivate endogenous μ and κ light chain gene sites (see, for example, Lonberg, et al., 1994 Nature 368 (6474): 856-859). In another embodiment, mice carrying human immunoglobulin sequences on transgenic and transhomologous bodies can be used to produce human antibodies, such as mice carrying human heavy chain transgenics and human light chain transchromosomes. Such mice, referred to herein as "KM mice," are described in detail in PCT Publication WO 02 / 43478 by Ishida et al.

[0101] 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 a macrophage.

[0102] In some embodiments, the antibody is selected from the group consisting of one that specifically binds to DC immune receptor (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.

[0103] In some embodiments, the antibody is specific for CD40.

[0104] In some embodiments, the anti-CD40 antibody is derived from the 12E12 antibody and comprises:

[0105] A heavy chain comprising complementary 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),

[0106] and a light chain comprising complementary 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).

[0107] In some embodiments, the anti-CD40 antibody is derived from the 11B6 antibody and comprises:

[0108] A heavy chain comprising complementary 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), CDR3H has the amino acid sequence EDYVY (SEQ ID NO: 20), and

[0109] A light chain comprising complementary 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).

[0110] In some embodiments, the anti-CD40 antibody is derived from the 12B4 antibody and comprises:

[0111] A heavy chain comprising complementary 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), CDR3H has the amino acid sequence GYPAYSGYAMDY (SEQ ID NO: 26), and

[0112] A light chain comprising complementary 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).

[0113] In some embodiments, the anti-CD40 antibody is selected from the group consisting of mAb1, mAb2, mAb3, mAb4, mAb5, and mAb6 described in Table A.

[0114]

[0115] Table A: CD40 Antibodies

[0116] SEQ ID NO: 30 (amino acid sequence of humanized 11B6 heavy chain variable region (VH) (v2))

[0117] EVQLVQSGAEVKKPGASVKISCKASGYSFTGYYMHWVKQAHGQGLEWIGRINPYNGATSYNQNFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCAREDYVYWGQGTTVTVSSAS

[0118] SEQ ID NO: 31 (humanized 11B6 VL light chain variable region (VL) Vk (v2) amino acid sequence)

[0119] DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPWTFGGGTK

[0120] SEQ ID NO: 32 (amino acid sequence of humanized 11B6 heavy chain variable region VH (v3))

[0121] EVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYMHWVRQAPGQGLEWIGRINPYNGATSYNQNFKDRVTLTVDKSTSTAYMELSSLRSEDTAVYYCAREDYVYWGQGTTVTVSSAS

[0122] SEQ ID NO: 33 (VH amino acid sequence of mAb3 (12B4))

[0123] EVQLQQSGPELVKPGASVKMSCKASGYTFTDYVLHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGYPAYSGYAMDYWGQGTSVTVSSAS

[0124] SEQ ID NO: 34 (VL amino acid sequence of mAb3 (12B4))

[0125] DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCHHGNTLPWTFGGGTK

[0126] SEQ ID NO: 35 (VH amino acid sequence of mAb4 (24A3-HC))

[0127] DVQLQESGPDLVKPSQSLSLTCTVTGYSITSDYSWHWIRQFPGNKLEWMGYIYYSGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDSATYFCARFYYGYSFFDYWGQGTTLTVSSAS

[0128] SEQ ID NO: 36 (VL amino acid sequence of mAb4 (24A3 KC))

[0129] QIVLTQSPAFMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTK

[0130] SEQ ID NO: 37 (VH amino acid sequence of mAb5)

[0131] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSAS

[0132] SEQ ID NO: 38 (VL amino acid sequence of mAb5)

[0133] DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTK

[0134] SEQ ID NO: 39 (VH amino acid sequence of mAb6 (12E12 H3 humanized HC))

[0135] EVQLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQAPGKGLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARRGLPFHAMDYWGQGTLVTVSSAS

[0136] SEQ ID NO: 40 (VL amino acid sequence of mAb6 (humanized K2 12E12))

[0137] DIQMTQSPSSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQFNKLPPTFGGGTK

[0138] In some embodiments, the anti-CD40 antibody comprises: a heavy chain, wherein the variable domain has the amino acid sequence set forth in SEQ ID NO:30, and a light chain, wherein the variable domain has the sequence set forth in SEQ ID NO:31.

[0139] In some embodiments, the anti-CD40 antibody comprises: a heavy chain, wherein the variable domain has the amino acid sequence shown in SEQ ID NO:32, and a light chain, wherein the variable domain has the sequence shown in SEQ ID NO:31.

[0140] In some embodiments, the anti-CD40 antibody comprises: a heavy chain, wherein the variable domain has the amino acid sequence set forth in SEQ ID NO:33, and a light chain, wherein the variable domain has the sequence set forth in SEQ ID NO:34.

[0141] In some embodiments, the anti-CD40 antibody comprises: a heavy chain, wherein the variable domain has the amino acid sequence set forth in SEQ ID NO:35, and a light chain, wherein the variable domain has the sequence set forth in SEQ ID NO:36.

[0142] In some embodiments, the anti-CD40 antibody comprises: a heavy chain, wherein the variable domain has the amino acid sequence set forth in SEQ ID NO:37, and a light chain, wherein the variable domain has the sequence set forth in SEQ ID NO:38.

[0143] In some embodiments, the anti-CD40 antibody comprises: a heavy chain, wherein the variable domain has the amino acid sequence set forth in SEQ ID NO:39, and a light chain, wherein the variable domain has the sequence set forth in SEQ ID NO:40.

[0144] In some embodiments, the anti-CD40 antibody is a CD40 agonist antibody. CD40 agonist antibodies are described in WO2010 / 009346, WO2010 / 104747, and WO2010 / 104749. Other anti-CD40 agonist antibodies under development include the fully human IgG2 CD40 agonist antibody CP-870,893 developed by Pfizer. It binds to CD40 with a KD of 3.48×10 -10 M, but does not block the binding of CD40L (see, for example, US Pat. No. 7,338,660); SGN-40 is a humanized IgG1 antibody developed by Seattle Genetics from mouse antibody clone S2C6, which was prepared using a human bladder cancer cell line as an immunogen. Its KD for binding to CD40 is 1.0×10 -9 M, and exerts its effect by enhancing the interaction between CD40 and CD40L, thereby exhibiting a partial agonist effect (Francisco JA, et al., Cancer Res, 60:3225-31, 2000). More specifically, the CD40 agonist antibody is selected from the group consisting of mAb1, mAb2, mAb3, mAb4, mAb5 and mAb6 in Table A.

[0145] In some embodiments, the antibody is specific for Langerin. In some embodiments, the antibody is derived from antibody 15B10 with ATCC accession number PTA-9852. In some embodiments, the antibody is derived from antibody 2G3 with ATCC accession number PTA-9853. In some embodiments, the antibody is derived from antibody 91E7, 37C1 or 4C7 as described in WO2011032161.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] In some embodiments, the antibody is from Table B The method was selected from the group consisting of mAb7, mAb8 and mAb9 described above.

[0150]

[0151] SEQ ID NO: 41 (amino acid sequence of the variable heavy chain region (VH) of 15B10)

[0152] SVKMSCKASGYTFTDYVISWVKQRTGQGLEWIGDIYPGSGYSFYNE NFKGKATLTADKSSTTAYMQLSSLTSEDSAVYFCA

[0153] SEQ ID NO: 42 (variable light chain amino acid sequence (VL) 15B10)

[0154] ASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTNFTLKISRVEAEDLGLYFCS

[0155] SEQ ID NO: 43 (amino acid sequence of 2G3 variable heavy chain region (VH))

[0156] SSVKMSCKASGYTFTDYVISWVKQRTGQGLEWIGDIYPGSGYSFYN ENFKGKATLTADKSSSTTAYMQLSSLTSEDSAVYFCA

[0157] SEQ ID NO: 44 (amino acid sequence of variable light chain (VL) 2G3)

[0158] VTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRVSGVPARFSGSLIGDKAALTITGAQTEDEAIYFCA

[0159] SEQ ID NO: 45 (4C7 heavy chain amino acid sequence)

[0160] QVQLQQSGAELVRPGASVTLSKASGYTFIDHDMHWVQQTPVYGLEWIGAIDPETGDTGYNQKFKGKAILTADKSSRTAYMELRSLTSEDSAVYYCTIPFYYSNYSPFAYWGQG ALVTVSAAKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNP CPPLKECPPCADLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDAQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKT ISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGKAS

[0161] SEQ ID NO: 46 (4C7 light chain amino acid sequence)

[0162] QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQRKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPLTFGAGTKLELK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0163] In some embodiments, the anti-Langerin antibody comprises: a heavy chain, wherein the variable domain has the amino acid sequence shown in SEQ ID NO:41, and a light chain, wherein the variable domain has the sequence shown in SEQ ID NO:42.

[0164] In some embodiments, the anti-Langerin antibody comprises: a heavy chain, wherein the variable domain has the amino acid sequence shown in SEQ ID NO:43, and a light chain, wherein the variable domain has the sequence shown in SEQ ID NO:44.

[0165] In some embodiments, the anti-Langerin antibody comprises: a heavy chain having the amino acid sequence shown in SEQ ID NO:45 and a light chain having the sequence shown in SEQ ID NO:46.

[0166] The antibodies of the present invention can be produced by any technique known in the art per se, such as, but not limited to, any chemical, biological, genetic or enzyme techniques, used alone or in combination. Those skilled in the art will appreciate that the amino acid sequence of the desired sequence can easily produce the polypeptide by standard techniques for producing polypeptides. For example, the antibodies of the present invention can be synthesized by recombinant DNA technology, which is well known in the art. For example, after the DNA sequence encoding the desired (poly)peptide is incorporated into an expression vector and such vector is introduced into a suitable eukaryotic or prokaryotic host that will express the desired polypeptide, these fragments can be obtained as DNA expression products, from which well-known techniques can be subsequently used to separate.

[0167] In some embodiments, the amino acid sequences described herein comprise one or more sequences derived from restriction cloning sites present in the polynucleotide encoding the amino acid sequence. Typically, the sequence may consist of 2 amino acid residues, typically including AP, AS, AR, PR, SA, TR and TS sequences.

[0168] In some embodiments, the amino acid sequence described herein describes the sequence of a signal peptide. As used herein, the term "signal peptide" has a general meaning in the art and refers to a propeptide that exists as an N-terminal peptide in the form of a protein precursor. The function of the signal peptide is to promote the transport of the expressed polypeptide to which it is connected to the endoplasmic reticulum. In this process, the signal peptide is usually cleaved off. The signal peptide can be heterologous or homologous to the organism used to produce the polypeptide.

[0169] In some embodiments, an antibody of the invention comprises:

[0170] A heavy chain having at least 80% identity to the amino acid sequence shown in SEQ ID NO:47, and a light chain having at least 80% identity to the amino acid sequence shown in SEQ ID NO:48.

[0171] SEQ ID NO: 47

[0172] MGWSLILLFLVAVATRVHSEVQLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQAPGKGLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARRGLPFHAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS QTPTNTISVTPTNNSTPTNNSNPKPNP.

[0173] SEQ ID NO: 48

[0174] DIQMTQSPSSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQFNKLPPTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0175] In some embodiments, an antibody of the invention comprises:

[0176] 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.

[0177] SEQ ID NO: 49

[0178] DIQMTQSPSSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQFNKLPPTFGGGTKLEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECAS KYKI KSNPLTKDIVIKMIPNVSNMSQCTGSVMENYKTRLNGILTPIAEAAAKEAAAKAIQTKFAPGGYPLLWSFAMGVAT TIDRSMGALNINRGYL .

[0179] Polynucleotides, vectors and host cells of the present invention:

[0180] Another object of the present invention relates to polynucleotides encoding the heavy chain and / or light chain of the antibody of the present invention.

[0181] Typically, the polynucleotide is a DNA or RNA molecule, which may be contained in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, bacteriophage or viral vector.

[0182] Therefore, another object of the present invention relates to a vector comprising the polynucleotide of the present invention.

[0183] Such vectors may include regulatory elements, such as promoters, enhancers, terminators, etc., to cause or direct the expression of the antibody after administration to the subject. Examples of promoters and enhancers for animal cell expression vectors include early promoters and enhancers of SV40, LTR promoters and enhancers of Moloney mouse leukemia virus, promoters and enhancers of immunoglobulin H chains, etc. Any expression vector for animal cells can be used, as long as the gene encoding the human antibody C region can be inserted and expressed. Examples of suitable vectors include pAGE107, pAGE103, pHSG274, pKRC, pSG1βd2-4, etc. Other examples of plasmids include replication plasmids containing a replication origin, or integration plasmids, such as pUC, pcDNA, pBR, etc. Other examples of viral vectors include adenovirus vectors, retrovirus vectors, herpes virus vectors, and AAV vectors. This recombinant virus 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 virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-deficient recombinant viruses can be found in, for example, WO 95 / 14785, WO 96 / 22378, US5882877, US6013516, US4861719, US5278056, and WO 94 / 19478.

[0184] Another object of the present invention relates to a host cell which has been transfected, infected or transformed with a polynucleotide and / or a vector according to the present invention.

[0185] The polynucleotides of the present invention can be used to produce the antibodies of the present invention in a suitable expression system. Common expression systems include Escherichia 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, plant cells, etc.). Specific examples include Escherichia coli, Kluyveromyces or yeast. Mammalian host cells include Chinese hamster ovary (CHO cells), including dhfr-CHO cells (described in Urlaub and Chasin, 1980) used with DHFR selective markers, CHOK1-dhfr+ cell lines, NSO myeloma cells, COS cells and SP2 cells, such as GS-CHO cell lines and GS-XceedTM gene expression system (Lonza), or HEK cells.

[0186] The present invention also relates to a method for producing a recombinant host cell expressing the antibody of the present invention, the method comprising the following steps: (i) introducing the recombinant polynucleotide or vector as described above into a competent host cell in vitro or in vitro, (ii) culturing the obtained recombinant host cell in vitro or in vitro, and (iii) optionally screening cells expressing and / or secreting the antibody.

[0187] Such recombinant host cells can be used to produce the antibodies of the present invention.

[0188] Therefore, the host cells disclosed herein are particularly suitable for producing the antibodies of the present invention. In fact, when recombinant expression is introduced into mammalian host cells, the polypeptide is produced by culturing the host cells for a sufficient period of time so that the antibody is expressed in the host cells and optionally secreted into the culture medium in which the host cells are grown. The antibody can be recovered and purified from the culture medium after secretion using standard protein purification methods.

[0189] Drug and vaccine compositions:

[0190] The antibodies described herein can be administered as part of one or more pharmaceutical compositions. Unless any conventional carrier medium is incompatible with the antibodies of the invention, for example by producing any adverse biological effect or by producing a deleterious interaction with any other component of the pharmaceutical composition, its use should be within the scope of the present invention. Some examples of materials that can be used as pharmaceutically acceptable carriers include, but are not limited to, the following: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; gum arabic; malt; gelatin; talc; excipients, such as 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; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; at the discretion of the formulator, the composition may also contain ethanol and phosphate buffer solutions, as well as other non-toxic compatible lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coating agents, sweeteners, flavoring agents and fragrances, preservatives and antioxidants, etc.

[0191] The antibodies described herein are particularly suitable for the preparation of vaccine compositions. Therefore, another object of the present invention relates to a vaccine composition comprising the antibodies of the present invention.

[0192] In some embodiments, the vaccine composition of the present invention comprises an adjuvant. In some embodiments, the adjuvant is alum. In some embodiments, the adjuvant is incomplete Freund's adjuvant (IFA) or other oil-based adjuvants, and its content is 30-70%, preferably 40-60%, more preferably 45-55% weight ratio (w / w). In some embodiments, the adjuvant is polyinosinic acid-poly-L-lysine carboxymethyl cellulose (poly ICLC). In some embodiments, the vaccine composition of the present invention comprises at least one Toll-like receptor (TLR) agonist selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7 and TLR8 agonists.

[0193] Treatment:

[0194] 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.

[0195] Therefore, another object of the present invention relates to a method of vaccinating a subject in need thereof with a Nipah virus vaccine, comprising administering a therapeutically effective amount of the antibody of the present invention.

[0196] In some embodiments, the antibodies and pharmaceutical or vaccine compositions described herein are particularly suitable for treating Nipah virus infection.

[0197] In some embodiments, the subject can be a human or any other animal (e.g., birds and mammals) susceptible to Nipah virus infection (e.g., domestic animals, such as cats and dogs; livestock and farm animals, such as horses, cattle, pigs, chickens, etc.). Typically, the subject is a mammal, including non-primates (e.g., camels, donkeys, zebras, cattle, pigs, horses, goats, sheep, cats, dogs, rats, and mice) and primates, such as monkeys, chimpanzees, and humans. In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a farm animal or a 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 an adult human. In some embodiments, the subject is an elderly person. In some embodiments, the subject is a premature infant.

[0198] In some embodiments, the subject may be symptomatic or asymptomatic.

[0199] Typically, the active ingredients of the present invention (i.e., antibodies and drugs or vaccine compositions described herein) are administered to a subject in a therapeutically effective amount. It should 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 reasonable medical judgment. The specific therapeutically effective dosage level of any particular subject will depend on a variety of factors, including the disease being treated and the severity of the disease; the activity of the specific compound used; the specific ingredients used; the age, weight, general health, sex and diet of the subject; the administration time, route of administration and excretion rate of the specific compound used; the duration of treatment; drugs used in combination or simultaneously with the specific polypeptide used; and similar factors well known in the medical field. For example, a person skilled in the art can start administration at a dosage level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the product can vary in a wide range of 0.01 to 1000 mg per adult per day. In particular, the composition contains 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 active ingredient for adjusting the dosage according to the specific symptoms of the patient. The drug usually contains about 0.01 mg to about 500 mg of active ingredient, especially 1 mg to about 100 mg of active ingredient. The effective dose of the drug is usually supplied at a dosage level of 0.0002 mg / kg to about 20 mg / kg body weight per day, especially about 0.001 mg / kg to 7 mg / kg body weight per day.

[0200] The antibodies and pharmaceutical or vaccine compositions described herein can be administered to a subject by any route of administration, in particular by oral, nasal, rectal, topical, buccal (e.g., sublingual), parenteral (e.g., subcutaneous, intramuscular, intradermal or intravenous) and transdermal administration, although the most appropriate route of administration in any given case will depend on the nature and severity of the disease being treated and the nature of the specific active agent being used.

[0201] The present invention will be further illustrated by the following drawings and examples. However, these examples and drawings should not be interpreted as limiting the scope of the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0202] Figure 1, Epitope mapping of NiV G antigen associated with CD40 monoclonal antibody. In the genome of NiV, ORFs encoding G, F, and N proteins are highlighted in blue, orange, and red, respectively. Full-length aa sequences were screened for prediction of HLA-I (NetMHC4.0) and HLA-II (NetMHCII 2.3) as well as linear B-cell epitopes (BepiRed 2.0). For HLA-I and HLA-II, color intensity represents the density of predicted epitopes, covering at least 30% of the global G protein HLA (Top) exosomes associated with Gen-1 and Gen-2 vaccines.

[0203] Figure 2 , Epitope mapping of NiV F and N antigens associated with CD40 monoclonal antibodies. Representation of the F and N selection domains associated with the Gen-2 vaccine.

[0204] Figure 3 Schematic diagram of the first-generation (Gen-1) and second-generation (Gen-2) vaccines.

[0205] Figure 4 SDS-PAGE profiles of Gen-1, Gen-2 and IgG4 control vaccines under non-reducing (NR) and reducing (R) conditions.

[0206] Figure 5 , G antigen fused to the Gen-1 vaccine was confirmed by Western Blot using sera from Niv(G)-immunized mice and internally produced G protein as a positive control. The left lane shows the molecular weight (kDa) marker. Arrows indicate the expected sizes of heavy chains (HC) and light chains (LC) in the vaccine construction, respectively.

[0207] Figure 6 Size exclusion chromatography (SEC) of Gen-1, Gen-2 vaccines with standard protein molecular weight markers. Recombinant monoclonal antibodies are indicated by arrows.

[0208] Figure 7 Binding assay of CD40.NiV vaccine to mouse cells expressing human CD40 receptor. Mouse splenocytes from hCD40My / Hu-Tg mice were incubated with 10nM CD40.Niv Gen-1 monoclonal antibody and subsequently labeled with T, B and DC cell surface markers, respectively. Untreated and non-targeted IgG4.Niv treated splenocytes were used as negative controls.

[0209] Figure 8 , CD40.NiV vaccine and AGM PBMCs binding test. Figure 7The same method was used, using PBMCs from unvaccinated AGM. Two homemade anti-CD40 monoclonal antibodies (clones VH3 and 11B6) that do not bind to protein G were used as positive controls. The results showed that the vaccine bound to B cells expressing CD40 and, to a lesser extent, also to monocyte / macrophage populations.

[0210] Fig. 9 , Gen-1 and Gen-2 CD40.NiV vaccines in hCD40 Hy / Mu Humoral immune responses were induced in transgenic mice. G-specific IgG titers were measured by ELISA on days 0, 21, and 28 after mice were immunized twice with 10 ug CD40.NiV (Gen-1), Gen-2, or molar equivalents of NiV (G) protein alone, and were measured with (solid circles) and without Poly ICLC (open circles) at the time of vaccination. The median AUC [Min-Max] is represented and compared with control animals (Poly ICLC alone).

[0211] Fig.10 Neutralizing activity of sera from vaccinated mice detected by Luminex-based pseudoneutralization assay on days 0, 21, and 28 after immunization. Statistical analysis and Fig. 9 same.

[0212] Fig.11 In vitro neutralization assays were performed on cells infected with NiV-B using serially diluted sera collected on day 28 after immunization. IC50 values ​​were calculated and reported as titers. Fig. 9 same.

[0213] Fig.12 , Niv(G)-specific B cells in dLN were stained using biotinylated Niv(G) protein. (Left) Representative scatter plot. Niv(G)-specific B cells were considered double positive for two anti-biotin monoclonal antibodies. (Right) Percentage (%) of GC B cells (FAS+GL-7+) and percentage of Niv(G)-specific B cells in this population. Poly-ILCCs were co-immunized with 30ug CD40.NiV(Gen-2) and compared with the Poly-ILC-negative group. Mann-Whitney unpaired t-test was used, ** indicates p<0.01; *** indicates p<0.0001.

[0214] Fig.13, Gen-1, and Gen-2 CD40.NiV vaccines induced cellular immune responses in hCD40Hy / Mu transgenic mice. One week after boosting, IFN-g T cell responses to overlapping peptide pools of G, F, and N were assessed by ELISpot. (Left) Specific IFN-g responses to G antigen in mice immunized with Gen-1 and Gen-2, respectively. Number of spots reported per million splenocytes (Right) Total specific IFN-g responses to the three antigens of Gen-2 in mice immunized with 10 and 30 ug of vaccine, respectively. (Bottom right) Percentage of G, F, and N specific responses when 10 and 30 ug of vaccine were administered, respectively. Solid circles indicate the presence of Poly ICLCs, and open circles indicate the absence of Poly ICLCs.

[0215] Fig.14 , immune responses of AGM vaccinated with CD40.Niv Gen-2. Schematic representation of the study design. Diamonds represent sampling points. Nine AGM were vaccinated with CD40.NiV(Gen-2) and Poly ICLC at a 3-week interval. Niv(G)-specific IgG (left) and IgA (right) titers were measured in the Poly ICLC group of animals (n=3, left) and the vaccinated group of animals (n=9, right) at 0, 10, 21, 35, and 56 days after pretreatment. Individual Ig titers (log(1 / EC50)) are expressed, and whiskers indicate the median [Min-Max] for each group and time point, respectively. Primary and booster immunizations are indicated by arrows, respectively. Dashed lines indicate the threshold for Ig titer detection.

[0216] Fig.15 ,and Fig.10 Likewise, the neutralizing activity of AGM sera was measured by an indirect inhibition assay.

[0217] Fig.16 , in vitro neutralization effect of serum from AGM inoculation on NiV-B.

[0218] Fig.17 ,and Fig.13 Similarly, the total specific IFN-γ response to G, F and N antigens was measured in the circulating PBMCs of AGM collected on days 21 and 35 after immunization. The results are expressed as the number of spots per million PBMC. The dotted line represents the positive threshold defined by the average response (+3SD) of the unvaccinated AGM group. (Right) Percentage (%) of specific T cell responses for each vaccine antigen (mean ± standard deviation).

[0219] Fig.18In vitro neutralizing activity of AGM serum against NiV-B after immunization and challenge (80 days after immunization): Poly-ICLC group (n=3, black circles); CD40.NiV (Gen-2) combined with Poly-ICLC group (n=9, white circles); The thin black line indicates the downward trend of neutralizing antibodies after the peak of the reaction (35 days).

[0220] Fig.19 , AGM protection test. Survival curve. 9 AGM were immunized twice with CD40.NiV (Gen-2) and challenged with 102 Pfu-NiV-B (intratracheal route). Unimmunized animals served as controls. Gehan-Breslow-Wilcoxon test was used, **** indicates p < 0.0001.

[0221] Fig. 20 : Clinical scores and body temperatures of unimmunized (black circles) and immunized AGMs (white circles). Dashed lines indicate the scoring threshold based on ethical considerations (left); Dashed lines: median body temperature on day 0 (right).

[0222] Figure 21: Virus transmission after challenge. (A) (left) Quantification of viral NiV RNA in peripheral blood mononuclear cells (PBLs) by RT-PCR (N gene). Values ​​were normalized to the value of the GADPH housekeeping gene. Genomic RNA in nasal (middle) and pharyngeal (right) swabs was measured by RT-qPCR. The results are expressed as the mean (± standard error) of the non-immunized group (black circles, n=7) and the CD40.NiV (Gen-2) immunized group (white circles, n=9). (B) Same as A, but tested on fluid samples collected at autopsy. Example

[0223] method:

[0224] Molecular cloning and production:

[0225] First generation: The extracellular domain of NiV-B G protein ( Figure 1 , Bangladesh strain (NiVB) (GenBank: AY988601.1) (Harcourt BH et al., Emerging Infect. Dis. 2005) The extracellular domain is highlighted) was cloned into the UCOE vector, located at the C-terminus of the heavy chain of the anti-CD40 monoclonal antibody (clone 12E12 / VH3) and connected with a flexible linker (see Figure 3 ).

[0226] Second generation: AA sequences of F and N proteins (Bangladesh strain) were screened using online software (NetMHC) to determine the density of predicted MHC-I and MHC-II epitopes and the diversity of HLA ( Figure 2 The sequence was further resolved by analyzing the presence of predicted linear B-cell epitopes and screening for non-hydrophobic stabilizing protein domains. One peptide from each of the F and N proteins was linked to the linker domain and bound to the C-terminus of the anti-CD40 light chain (clone 12E12 / VH3) (see Figure 3 on the right).

[0227] CHO cell line production:

[0228] CHO cells were transfected with plasmids expressing the H and L chains (see Figure 3 ). The culture supernatant was collected, protein A was captured and purified on FPLC. The purified vaccine batches were tested for their endotoxin levels (<0.5 ng / mg protein).

[0229] Biochemical Quality Control:

[0230] The quality of vaccine batches was analyzed by the following methods: (1) SDS-PAGE analysis (under reducing and non-reducing conditions) followed by Coomassie Brilliant Blue staining ( Figure 4 ); and (ii) SEC analysis using standard proteins ( Figure 6 The Gen1 vaccine was evaluated by Western blot using serum from C57BL / 6 mice immunized with in-house produced G protein (+CpG) ( Figure 5 ). The heavy chain fused to the G protein was detected in mouse sera.

[0231] Combination test:

[0232] Gen-1 antibody and 12E12 / VH3 clone without any antigen were labeled with fluorescent dye. Clone 11B6 was also tested in parallel as a positive control. Mouse spleen cells from hCD40-Tg mice ( Figure 7 ) and PBMCs from AGM ( Figure 8 ) were incubated with fluorescent vaccines in vitro. Cell phenotypes were analyzed by FACS.

[0233] Immune response of hCD40-Tg mice:

[0234] hCD40Hy / Mu Tg mice (CO-1704hTnfrsf5-OST5(CD40) knock-in HOM) were immunized twice by subcutaneous injection with or without adjuvant, with the immunizing antigens CD40.NiV(G) (Gen-1 or Gen-2) or NiV(G) protein alone (see Table 1). Serum was collected at 0, 3, and 4 weeks. Splenocytes were collected at 4 weeks. The content of G-specific IgG in serum was measured by ELISA. Antibody titers were determined by serial dilution method ( Fig. 9 The neutralizing activity of serum antibodies was determined by an in-house Luminex assay ( Fig.10 ) or in vitro NiV-B neutralization assay ( Fig.11 ) were measured. The neutralization effect of serum on NiV infection was determined by serial dilution method. The proportion of germinal center B cells and G-specific B cells in this cell population was evaluated by FACS ( Fig.12 ).

[0235] Splenocytes were in vitro treated with the selected domains of NiV G, F and N proteins included in the vaccine ( Figure 1 or 2) an overlapping peptide pool (OVLP, 1 μg / mL; see Fig.13 IFN-g production was assessed by ELISpot.

[0236] Immune Responses in AGM:

[0237] Nine AGM were vaccinated with CD40.NiV (Gen-2) vaccine twice, 3 weeks apart. Three unimmunized animals were used as controls. Blood was collected regularly at various time points after immunization, and serum G-specific IgG and IgA were measured by ELISA (Figure 14). The neutralizing activity of serum was determined in the same way as in the mouse experiment ( Fig.15 , 16 and 18). PBMCs collected on days 21 and 35 after immunization were subjected to IFNg-ELISPOT assay ( Fig.17 ).

[0238] Challenge test:

[0239] Intratracheal injection 10 2 pfu of Niv-B, vaccinated animals (n=9) and unvaccinated AGM (n=8) were challenged. Survival was observed within three weeks ( Fig.19 ).

[0240] Clinical and blood biochemical follow-up:

[0241] After the virus challenge, animals were clinically examined daily and scored for the following parameters: body temperature, body weight, dehydration, respiration, responsiveness, fecal examination, and neurological symptoms. For ethical reasons, animals were euthanized when the total score reached >15 ( Fig. 20 ).

[0242] virology

[0243] Extraction of RNA from infected AGM PBL:

[0244] Blood samples collected from infected AGMs in EDTA tubes were diluted in Pharmalyse buffer, vortexed, and incubated in the dark for 15 min. The pellets were then centrifuged at 200 × g for 5 min and washed with 2 mL of PBS containing 1% FBS and 2 mM EDTA. Finally, the pellets were resuspended in 600 μL RLT lysis buffer and purified according to the manufacturer's instructions (Macherey-Nagel). Samples were stored at -80 °C until use.

[0245] RT-qPCR:

[0246] Viral RNA was extracted from serum, swab, urine, BAL, vitreous, and pleural effusion samples using the Qiamp viral RNA kit (Qiagen), and from PBMCs and organ samples using the Nucleospin kit (Macherey-Nagel). When necessary, viral load was assessed by one-step RT-qPCR using NiV-N-specific primers and GAPDH primers (Figure 21).

[0247] result:

[0248] Screening of NiV predictive immunogenic peptides and design of CD40.NiV vaccine.

[0249] Two CD40.NiV vaccine candidates have been successfully produced. While Gen-1 and Gen-2 contain the gold standard Niv G (glycoprotein) surface antigen, the Gen-2 vaccine also incorporates peptides from the F and N proteins that were screened by computer simulation and predicted to be rich in T-cell and B-cell epitopes ( Figures 1 to 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 extracellular domain (ECD) to identify vaccine epitopes using NetMHC 4.0 and NetMHCII 2.3 software, which predict T cell epitopes that bind to a large number 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 3522 T cell epitopes and 15 B cell epitopes, respectively. Due to their conservation between NiV strains, we further identified vaccine epitopes for the fusion protein (F) and nucleocapsid protein (N). Regions with strong binding epitopes and the highest HLA coverage, as well as linear B cell epitopes, are highlighted. The peptides of NiV F (aa 45-90) and NiV N (aa 318-355) retained after screening contained 356 and 266 predicted T cell epitopes, respectively, and 3 linear B cell epitopes. Globally, these amino acid sequences were screened for homology with other Henipaviruses, and the homology of F and N peptides between different Nipah strains was 100%, and the homology of NiV G ECD exceeded 98%. ( Figures 1 to 8 ) Next, we designed a vector expressing NiV-B G ECD fused to the C-terminus of the heavy chain (HC) of the anti-human CD40 humanized 12E12 IgG4 monoclonal antibody, and NiV F and NiV N peptides fused to the C-terminus of the light chain (LC) (named CD40.NiV)( Figure 3 The vaccine was produced in CHO cells and its quality was controlled. Here, we demonstrated that the CD40.NiV vaccine binds to human CD40 and AGM CD40 receptors in vitro using spleen cells of mice transgenic for the human CD40 receptor (CD40Hy / Mu transgenic mice; hCD40Tg) and PBMCs of AGM, respectively ( Figure 7-8 ).

[0250] CD40.NiV induced specific T cell and B cell responses in hCD40Tg mice.

[0251] We first evaluated hCD40 My / Hu Immunogenicity in Tg mice. hCD40Tg mice received two subcutaneous (SC) injections of CD40.NiV (Gen-1 or Gen-2) vaccine (10 μg) or an equivalent amount of NiV G protein on days 0 and 21, along with poly-ICLC (50 μg). Antibody-mediated immune responses were first assessed by Luminex assay. Our results showed that G-specific IgG titers were high for both vaccines ( Fig. 9). Three weeks after the primary immunization, mice immunized with NiV G protein targeting CD40 showed significantly higher anti-NiV G IgG levels (P<0.01), highlighting the benefit of the DC-targeted system. This antibody level was significantly enhanced in both the following two situations: (i) when the G protein was targeted to APCs, and (ii) when the mice were immunized with adjuvant. Next, we used a multiplex immunoassay approach to compare the affinity of the enhanced NiV G protein-specific IgG. Surprisingly, the affinity index was significantly improved when the NiV G protein was targeted to the CD40 receptor (P<0.05), indicating that this targeting effect has an advantage in inducing B cell affinity maturation (data not shown). Notably, we established an in-house Luminex-based detection method to evaluate the inhibitory effect of circulating IgG in serum on the binding of G protein to the Ephrin B2 receptor. Our results showed that immune serum has a strong ability to mimic and neutralize this G protein / receptor interaction ( Fig.10 Surprisingly, neutralization assays performed at BSL-4 confirmed the neutralizing capacity of vaccine-induced IgG ( Fig.11 ).

[0252] G-specific B cells were detected in the germinal center B cell population of immunized mice by FACS ( Fig.12 We further confirmed the B cell responses induced in CD40.NiV(+poly-ICLC)-vaccinated mice by detecting germinal center (GC) B cells in draining lymph nodes (dLN) (P<0.001 compared with the Poly-ICLC control group) ( Fig.12 ). In addition, FACS staining showed a significant population of NiV G-specific GC B cells in the dLN of vaccinated animals (P < 0.01). To demonstrate specific T cell responses against various CD40.NiV antigens, we performed IFN-g ELISpot assays on splenocytes using overlapping peptide pools ( Fig.13 ). We detected dose-dependent IFN-g-producing cells specific for NiV g ECD as well as NiV F and N peptides. Finally, we demonstrated by IFN-g ELISpot on mouse splenocytes that the Gen-2 vaccine induced specific cellular responses to the impaired G, F, and N peptides within the vaccine, respectively ( Fig.13 ). Overall, these results demonstrate the immunogenicity of the CD40.NiV vaccine candidate.

[0253] CD40.NiV vaccine induced early and strong humoral and T cell responses in AGMs.

[0254] We then evaluated the cellular and humoral responses of the Gen-2 vaccine in the African green monkey (AGM) model, the most relevant model for testing infection with the Bangladeshi NiV strain. Twelve animals have been imported and maintained at the Bioprim Animal Center (Toulouse). Two groups of animals have been immunized following a homologous immunization scheme of prime-boost, as follows: three non-human primates (AGM) were immunized with Poly ICLC (Poly-ICLC) alone, while the other nine AGM were immunized with 200 μg of the second generation vaccine + Poly-ICLC. Blood samples have been collected serially as follows: (i) serum for G-specific Ig titers and neutralization responses, and (ii) PBMCs for ELISpot ( Fig.14 We demonstrated that our vaccine candidate induced a strong and durable humoral response with circulating IgG neutralizing the virus in vitro (assessed in BSL-4)( Figures 14 to 16 All vaccinated animals showed specific and significant IgG and IgA titers 10 days after vaccination ( Fig.14 IgG titers increased over time and then remained stable until day 56, while serum IgA levels decreased. Vaccination triggered the neutralization capacity of NiV G-specific IgG on day 10 after primary immunization, which reached a significant level two weeks after booster immunization (mean neutralization titer 3.2 (±0.2), P < 0.001) and was maintained until day 56 ( Fig.15 , 16 and 18). We estimated that neutralization titers decreased significantly by an average of 0.016 log units per day and therefore predicted that neutralization titers would remain above the detectable threshold for 100 days after booster immunization (mean log titer 2.2 (±0.1)). Neutralization capacity of specific antibodies was confirmed using an alternative inhibition assay based on Luminex technology after primary and booster immunization (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) as well as HeV by in vitro infection of VeroE6 cells with recombinant (F / G) VSV particles (data not shown). Two weeks after booster immunization, all animals showed similar cross-reactive humoral responses to NiV (F / G) proteins and also showed positive cross-reactive responses to Hendra virus (data not shown). PBMCs were collected on days 21 (before booster vaccination) and 35 after primary immunization and IFN-g ELISpot assays were performed using peptide combinations targeting each vaccine antigen ( Fig.17). Two weeks after the booster, the overall response was significant (P<0.05), with 5 / 8 animals tested positive. Our results highlight the polyclonal IFN-g T cell response to all vaccine antigens ( Fig.17 ).

[0255] Overall, CD40.NiV(Gen-2) induced cellular responses associated with strong, rapid, and durable humoral responses in the AGM model and produced high titers of neutralizing antibodies.

[0256] Clinical outcomes and survival of challenged animals.

[0257] We then tested vaccine-induced protection against Niv infection in African green monkeys (AGM). Control and vaccinated AGM were moved to BSL-4 and inoculated with Niv (10 2 pfu intratracheal, Bangladesh strain) were used for the challenge experiment. The challenge dose was selected based on previous experiments that included five unvaccinated animals infected under the same conditions. Surprisingly, we observed that the vaccinated animals were completely protected (at day 22 post-inoculation, n = 8), while all unvaccinated mice died between D7 and D11 (n = 8) ( Fig.19 From a clinical perspective, vaccinated animals showed only mild and transient clinical signs, with the mean clinical score remaining below 6, primarily attributable to the lack of reactivity ( Fig. 20 ). The control group had higher clinical scores, manifested by apathy, tachypnea, dyspnea, and gastrointestinal symptoms, as well as fever (body temperature above 38.9°C in all animals). During the challenge phase, animals showed various hematological and serum biochemical changes (data not shown). During the critical disease period in the control group, enzyme activity levels (aspartate aminotransferase, AST; creatine kinase, CK) reflecting liver disease were higher than normal values, but not in the vaccinated animals. We also observed disturbances in the white blood cell and red blood cell counts in the control group, manifested by lymphopenia and thrombocytopenia, while we did not observe obvious abnormalities in the vaccine recipients after vaccination and during the challenge phase. Necropsy studies revealed the pathological and pathophysiological processes of NiV-B infection in the control animals (data not shown). Lung tissue examination showed interstitial pneumonia, edema, and vasculitis with inflammatory cell infiltration in all control groups, but these lesions were rarely seen in the vaccinated animals (observed in only 2 of 9 animals, namely AGM#S1134 and #O1376, data not shown).

[0258] Unvaccinated individuals showed follicular depletion in the spleen, whereas eight CD40.NiV-vaccinated AGMs showed follicular hyperplasia, suggesting a robust adaptive immune response induced after challenge. Notably, we observed no relevant lesions in the frontal cortex of any animal. From a clinical perspective, CD40.NiV (Gen-2) appears to provide complete protection against disease progression and mortality.

[0259] Plasma and tissue viral loads of AGMs after challenge

[0260] We regularly tested PBLs of vaccinated and control animals for NiV viremia by qRT-PCR, and analyzed viral loads in organs, fluids, and swabs at the end of the study (euthanasia or 28 dpc). All control groups (n = 3 animals receiving poly-ICLC plus n = 4 unvaccinated AGM) showed high levels of NiV-B in PBLs (range 4.5-5.5 log10 copies / mL), and the virus was detectable as early as 7 to 8 days after challenge, while we did not detect the virus in vaccinated subjects until 22 days after challenge ( Fig.21A ).

[0261] Except for one animal (AGM#S1134) in which NiV-B transcripts were detected in the lung by RT-PCR, NiV-B replication was not detected in nasal and nasopharyngeal swabs, fluids (including bronchoalveolar lavage (BAL), serum, urine, and pleural secretions), and organs (lung, spleen, and neural tissue) of the immunized AGMs, whereas high levels of NiV-B replication were detected in the control group ( Fig. 21B ). This strong antiviral effect was confirmed by the lack of NiV N protein expression in lung and spleen tissues assessed by tissue immunofluorescence (data not shown). Viral syncytia were detected in the lungs of unvaccinated animals. Notably, NiV-B infection of the brain could not be confirmed by tissue immunofluorescence. Overall, vaccination with CD40.NiV(Gen-2) induced sterile immunity in AGMs and limited viral dissemination and viral shedding.

[0262] Immunological and cytokine characteristics of challenged animals

[0263] We further characterized the immune response after challenge by analyzing the immune phenotype of cell populations from 0 to 22 dpc after challenge. Dead animals showed profound and significant defects in lymphoid cell populations (CD20+B cells and CD3+T cells) (data not shown). In T cells, the percentage of CD8+T cells was significantly reduced during NiV infection (data not shown). These changes were transitional and not significant in the vaccinated group. Innate immunity was also affected by NiV-B infection, with circulating monocytes in dying animals almost completely disappearing, while the number of inflammatory monocytes, intermediate monocytes, and classical monocytes in vaccine recipients remained stable after the critical period of infection (7 to 9 dpc) (data not shown). Overall, these data indicate that challenge with NiV results in dramatic disturbances in innate and adaptive cellular immunity, which are attenuated and / or not significant in animals immunized with CD40.NiV.

[0264] Changes in gene expression in blood cells after AGM inoculation.

[0265] To parse the early changes in gene expression associated with vaccination, we performed RNA sequencing analysis on peripheral whole blood from animals receiving adjuvanted CD40.NiV (Gen-2) at D0 (before primary immunization) and one day after primary immunization (PP, D1), one day before boost immunization (D21), and one day after boost immunization. Given the small number of animals in the adjuvant control group, we analyzed the differentially expressed genes (DEGs) of vaccinated animals at different time points relative to baseline. Principal component analysis (PCA) showed changes in gene abundance PP and PB relative to baseline (pre-injection D0) (data not shown). A total of 773 DEGs were significantly regulated at D22 (one day after boost immunization). Interestingly, among these DEGs, 437 were found at both D1 and D22 (one day PP and PB). An additional 236 DEGs were common on day 1 of PP and day 1 of PB (data not shown). From D0 to D22, the most upregulated genes included genes with major roles in antiviral innate immunity and proinflammatory responses (e.g., ISG15, MX1, IFI44, CXCL10, and IL-27), as well as genes with major roles in adaptive immunity (e.g., SIGLEC-1 for antigen uptake, CCL8 for T cell chemotaxis, and CXCL11) (data not shown). The majority of these transcripts were found to be upregulated in both PP and PB (data not shown). A range of genes with the highest downregulated levels were also associated with features of primary or secondary immune responses to the vaccine (e.g., CD1c, CD79A, CCR6, and CXCR4). Globally, the most upregulated and downregulated genes in vaccinated animals were primarily associated with immune system processes and pathways for defense against viruses and other organisms (data not shown). Circulating immunoglobulin-mediated humoral immune responses specifically promoted PB (data not shown), highlighting the significant antibody-mediated immune response after the second injection of CD40.NiV (Gen-2).

[0266] Overall, the gene module associated with D22 antiviral defense was the most significantly upregulated (data not shown). Detailed dynamics of the genes involved in this module showed that all PP and PB were rapidly and significantly activated (data not shown). Ten transcripts, including interferon-induced antiviral proteins (RSAD2 and RIG1), were significantly upregulated in PB relative to PP. Other gene network modules were also highlighted after PP and PB (e.g., response to viruses, cytokine signaling, neutrophil degranulation, and regulation of leukocyte activation). Although we cannot distinguish the effects of vaccine and adjuvant on vaccine recipients, our transcriptome analysis highlights vaccine-induced gene signatures.

[0267] in conclusion:

[0268] Here, we demonstrate the efficacy of an innovative DC-targeted vaccine candidate in preventing NiV-B infection in challenge experiments in the AGM model. More than three decades after the discovery of the immunological properties of DCs, we show for the first time that targeting viral antigens to professional APCs can be effective as a preventive measure against a lethal dose of the virus. In contrast to most other NiV vaccine platforms, the DC-targeting strategy allowed us to design a subunit construct that contains immunogenic and cross-reactive epitopes from the F and NNiV proteins in addition to the NiV G ECD (Bossart et al., 2012; Foster et al., 2022; Geisbert et al., 2021; 287 Loomis et al., 2020; Mohammed et al., 2020; Woolsey et al., 2023; Yoneda et al., 2013).

[0269] The CD40.NiV vaccine induced both IgG and IgA antibodies in AGMs as early as 10 days after vaccination. We also showed that neutralization responses could be sustained, with mean log titers estimated to be stable at approximately 2.2 (±0.1) 100 days after the peak antibody response.

[0270] Results showed that the vaccine elicited responses that cross-neutralized multiple NiV strains and also cross-neutralized HeV strains. Despite low levels detected, we found that the CD40.NiV vaccine elicited T cell responses against NiVG ECD in two preclinical models and downregulated screened F and N peptides. Transcriptomic analysis revealed a common set of DEGs, including antiviral innate DEGs (ISG15, MX1, IFI44) and adaptive immunity DEGs, one day after primary and boost immunization (D1 and D22). As described for other vaccine strategies (Hagan et al., 2022), differentially expressed genes in vaccinated animals were primarily associated with pathways involved in the immune process and pathways involved in innate immune antiviral defense at early time points (D1, PP). Interestingly, regulation of gene pathways associated with humoral responses was significant after boost immunization on day 22.

[0271] Overall, these results highlight vaccine features that are associated with protective effects.

[0272] References:

[0273] Throughout this application, various references describe the prior art to which the present invention pertains. The disclosures of these references are incorporated herein by reference.

Claims

1. An antibody against an antigen on the surface of an antigen presenting cell, wherein: The heavy chain and / or light chain of the antibody is coupled or fused to a polypeptide having the same sequence as SEQ ID NO: 1 ("Niv (G) B The amino acid sequence of the polypeptide having the amino acid residue at position 71 (Q) to the amino acid residue at position 602 (T) in the extracellular domain ("extracellular domain") has at least 80% identity.

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

3. The method according to claim 1, wherein: The heavy chain of the antibody is coupled or fused to a polypeptide having at least 80% identity to the amino acid sequence from amino acid residue 71 (Q) to amino acid residue 602 (T) in SEQ ID NO: 1 ("Niv(G)B extracellular domain").

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

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

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

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

8. The method according to 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 according to any one of claims 1 to 8, wherein The antibody comprises: An antibody heavy chain coupled or fused to a polypeptide having the same sequence as SEQ ID NO: 1 (Niv (G) B The amino acid sequence of the extracellular domain of the polypeptide has at least 80% identity from amino acid residue 71 (Q) to amino acid residue 602 (T), and An antibody light chain coupled or fused to a polypeptide, wherein the polypeptide is identical to SEQ ID NO: 2 (predicted epitope-rich peptide segment Niv (F) B ) has at least 80% identity from amino acid residue 45 (K) to amino acid residue 90 (I).

10. The antibody according to any one of claims 1 to 8, wherein The antibody comprises: An antibody heavy chain coupled or fused to a polypeptide having the same sequence as SEQ ID NO: 1 (Niv (G) B The amino acid sequence of the extracellular domain of the polypeptide has at least 80% identity from amino acid residue 71 (Q) to amino acid residue 602 (T), and An antibody light chain coupled or fused to a polypeptide, wherein the polypeptide is identical to SEQ ID NO: 3 (predicted epitope-rich peptide segment Niv (F) B ) has at least 80% identity in the amino acid sequence from amino acid residue 318 (I) to amino acid residue 355 (L).

11. The antibody according to any one of claims 1 to 8, wherein The antibody comprises: An antibody heavy chain coupled or fused to a polypeptide having the same sequence as SEQ ID NO: 1 (Niv (G) B The amino acid sequence of the extracellular domain of the polypeptide has at least 80% identity from amino acid residue 71 (Q) to amino acid residue 602 (T), and An antibody light chain coupled or fused to the following two polypeptides: (i) the polypeptide and SEQ ID NO: 2 (predicted epitope-rich peptide segment Niv (F) B ) has at least 80% identity with the amino acid sequence from amino acid residue 45 (K) to amino acid residue 90 (I) in SEQ ID NO: 3 (predicted epitope-rich peptide Niv (F) B ) has at least 80% identity in the amino acid sequence from amino acid residue 318 (I) to amino acid residue 355 (L).

12. The antibody according to any one of claims 1 to 8, wherein The antibody comprises: An antibody heavy chain coupled or fused to a polypeptide having the same sequence as SEQ ID NO: 1 (Niv (G) B The amino acid sequence of the extracellular domain of the polypeptide has at least 80% identity from amino acid residue 71 (Q) to amino acid residue 602 (T), and An antibody light chain coupled or fused to a fusion protein, wherein (i) is identical to SEQ ID NO: 3 (predicted epitope-rich peptide segment Niv (F) B ) in the amino acid sequence from amino acid residue 318 (I) to amino acid residue 355 (L) having at least 80% identity with (ii) and SEQ ID NO: 2 (predicted epitope-rich peptide Niv (F) B ) are fused to polypeptides having at least 80% identity in the amino acid sequence from amino acid residue 45 (K) to amino acid residue 90 (I).

13. The antibody according to claim 12, wherein and SEQ ID NO: 3 (predicted epitope-rich peptide Niv (F) B ) in the amino acid sequence from amino acid residue 318 (I) to amino acid residue 355 (L) having at least 80% identity, connected by a linker to SEQ ID NO: 2 (predicted epitope-rich peptide Niv (F) B ) are fused to polypeptides having at least 80% identity in the amino acid sequence from amino acid residue 45 (K) to amino acid residue 90 (I).

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

10.

15. The antibody according to claim 14, wherein The antibody comprises: An antibody heavy chain coupled or fused to a polypeptide having the same sequence as SEQ ID NO: 1 (Niv (G) B The amino acid sequence of the extracellular domain of the polypeptide has at least 80% identity from amino acid residue 71 (Q) to amino acid residue 602 (T), and An antibody light chain coupled or fused to a fusion protein having at least 80% identity to the amino acid sequence shown in SEQ ID NO:

11.

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

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

18. The antibody according to claim 17, wherein Anti-CD40 antibodies are derived from 12E12 antibodies and include: A heavy chain comprising complementary 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), and a light chain comprising complementarity determining regions CDR1L, CDR2L, and CDR3L, CDR1L having the amino acid sequence SASQGISNYLN (SEQ ID NO: 15), CDR2L having the amino acid sequence YTSILHS (SEQ ID NO: 16), and CDR3L having the amino acid sequence QQFNKLPPT (SEQ ID NO: 17); Anti-CD40 antibodies are derived from 11B6 antibodies and include: A heavy chain comprising complementary 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), CDR3H has the amino acid sequence EDYVY (SEQ ID NO: 20), and a light chain comprising complementary 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); Anti-CD40 antibodies are derived from 12B4 antibodies and include: A heavy chain comprising complementary 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), CDR3H has the amino acid sequence GYPAYSGYAMDY (SEQ ID NO: 26), and A light chain comprising complementary 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).

19. The antibody according to claim 17, wherein Anti-CD40 antibodies were selected from Table A mAb1, mAb2, mAb3, mAb4, mAb5 and mAb6 described in.

20. The antibody of claim 1, comprising: A heavy chain having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 47, and A light chain having at least 80% identity to the amino acid sequence shown in SEQ ID NO:

48.

21. The antibody of claim 1, comprising: A heavy chain having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 47, and A light chain having at least 80% identity to the amino acid sequence shown in SEQ ID NO:

49.

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

23. A vector comprising the polynucleotide of claim 22.

24. A host cell which has been 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 1-21.

26. A method for 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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