Vaccine molecule

Abstract

To provide DNA vaccines that demonstrate high efficacy.SOLUTION: Provided is a DNA vaccine comprising first and second nucleic acid constructs. Here, the first and second nucleic acid constructs encode first and second fusion proteins, respectively. The first and second fusion proteins each comprise a targeting unit, a heterodimerization unit, and an antigenic unit in operable association. The antigenic unit for each of the first and second fusion proteins are variant target antigenic proteins. When the first and second nucleic acid constructs are introduced into a cell, the first and second fusion proteins are expressed and associate to form a first heterodimeric protein via association of the heterodimerization units.SELECTED DRAWING: None

Description

Technical Field

[0001] Provided herein are technologies related to vaccines, compositions, methods, and uses, particularly but not limited to, vaccines of heterodimeric vaccine molecules formed from monomers comprising a variant targeting unit and a variant antigenic unit, linked by a heterodimerization unit.

Background Art

[0002] Influenza A viruses circulate among human populations and cause significant morbidity and mortality. Most inactivated and live attenuated vaccines against seasonal and novel influenza are strain-specific and require constant updating of the strains used in the annual multivalent vaccine formulations. Also, in zoonotic infections where there is no immunity in the human population, pandemic strains may sometimes emerge, such as the swine influenza in 2009 [1][2]. Human infections with avian H5N1 and H7N9 viruses further highlight the potential risks posed by the animal reservoirs of influenza A viruses. Therefore, there is an urgent need for influenza vaccines that induce broad neutralizing antibodies against all serotypes of influenza.

[0003] Known methods for increasing the immunogenicity of protein antigens in subunit vaccines involve incorporating the antigen chemically [3-5] or genetically [6-9] into antibodies or antibody fragments that target antigen-presenting cells. This principle has been extended to DNA vaccination by constructing DNA plasmids encoding APC-specific fusion proteins. Thus, cells transfected in vivo by DNA vaccination secrete a fusion protein that facilitates delivery of the antigen to APCs, resulting in an improved immune response [10-14]. Electroporation of the injection site of DNA vaccines ensures efficient uptake and translation into vaccine proteins

[12] . To further enhance immunogenicity, Bogen and co-workers developed a dimeric target DNA vaccine [12,13]. The dimeric form, containing two targeting units and two antigenic units, increased binding activity. This divalency, combining heterologous sequences found in this homodimeric vaccine, increased the antibody response compared to monomeric vaccines in short-term assays

[15] .

SUMMARY OF THE INVENTION

[0004] Provided herein are technologies related to vaccines, compositions, methods, and uses thereof, particularly but not limited to, heterodimeric vaccine molecules formed from monomers comprising variant targeting units and variant antigenic units, linked by heterodimerization units.

[0005] Accordingly, in some embodiments, the invention is a DNA vaccine comprising a first nucleic acid construct and a second nucleic acid construct, wherein the first nucleic acid construct and the second nucleic acid construct encode a first fusion protein and a second fusion protein, and the first fusion protein and the second fusion protein comprise an operably linked targeting unit, heterodimerization unit, and antigenic unit. The antigenic units in each of the above-mentioned first fusion protein and the above-mentioned second fusion protein are variant antigen target proteins, When the above-mentioned first nucleic acid construct and the above-mentioned second nucleic acid construct are introduced into cells, the above-mentioned first fusion protein and the above-mentioned second fusion protein are expressed, and through the association of the above-mentioned heterodimerization unit, a first heterodimer protein is formed, providing a DNA vaccine.

[0006] In some embodiments, the heterodimerization unit in one of the above-mentioned first nucleic acid construct and the above-mentioned second nucleic acid construct is an ACID heterodimerization unit, and the heterodimerization unit in the other of the above-mentioned first nucleic acid construct and the above-mentioned second nucleic acid construct is a BASE heterodimerization unit that interacts to form an ACID / BASE heterodimerization domain, which is the first heterodimer protein. In some embodiments, the heterodimerization unit in one of the above-mentioned first nucleic acid construct and the above-mentioned second nucleic acid construct is a barnase heterodimerization unit, and the heterodimerization unit in the other of the above-mentioned first nucleic acid construct and the above-mentioned second nucleic acid construct is a barstar heterodimerization unit that interacts to form a barstar / barnase heterodimerization domain, which is the first heterodimer protein.

[0007] In some embodiments, the targeting units of the first fusion protein and the second fusion protein are the same. In some embodiments, the targeting units of the first fusion protein and the second fusion protein are different. In some embodiments, the targeting unit is an antigen-binding protein. In some embodiments, the antigen-binding protein is a scFv. In some embodiments, the targeting unit is an antigen-presenting cell (APC) targeting unit. In some embodiments, the APC targeting unit binds to a target selected from the group consisting of MHC-II molecules, CD40, CD11c, CD14, HLA-DP, Toll-like receptors, and chemokine receptors.

[0008] In some embodiments, the variant antigen target protein has a sequence identity of greater than about 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or has a conserved region of greater than about 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% sequence identity in the variant antigen target protein, with a length exceeding 8, 10, 12, 15, 20, 30, 40, 50 or 60 amino acids and a maximum length of 100 - 200 amino acids. In some embodiments, the different variant antigen target proteins are from different strains or serotypes of an organism. In some embodiments, the organism is a pathogenic organism. In some embodiments, the organism is selected from the group consisting of viruses, bacteria, fungi and protozoa. In some embodiments, the different variant antigen target proteins are variants of hemagglutinin (HA). In some embodiments, the vaccine comprises variants of HA from at least 3, 4, 5 or 6 and a maximum of 12 or 18 strains or serotypes of influenza virus. In some embodiments, the influenza virus is selected from the group consisting of influenza virus group 1 and influenza virus group 2. In some embodiments, the influenza virus group 1 is selected from the group consisting of H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17 and H18, and the influenza virus group 2 is selected from the group consisting of H3, H4, H7, H10, H14 and H15. In some embodiments, the different variant antigen target proteins are variants of cancer antigens (e.g., neo - epitopes).

[0009] In some embodiments, a DNA vaccine further comprising at least a third nucleic acid construct and a fourth nucleic acid construct, wherein the third nucleic acid construct and the fourth nucleic acid construct encode a third fusion protein and a fourth fusion protein, and the third fusion protein and the fourth fusion protein comprise an operably linked targeting unit, heterodimerization unit, and antigenic unit, and the antigenic unit in each of the third fusion protein and the fourth fusion protein is a variant antigen target protein. In some embodiments, expression of the third fusion protein and the fourth fusion protein intracellularly using the first fusion protein and the second fusion protein produces a mixture of heterodimeric proteins. In some embodiments, when the first nucleic acid construct and the second nucleic acid construct are expressed intracellularly, production of the heterodimeric protein is characterized by the substantial absence of production of a homodimeric protein containing the same antigen target protein. In some embodiments, when the first nucleic acid construct and the second nucleic acid construct and at least the third nucleic acid construct and the fourth nucleic acid construct are expressed intracellularly, production of the heterodimeric protein is characterized by the substantial absence of production of a homodimeric protein containing the same antigen target protein.

[0010] In some embodiments, the sequence encoding the fusion protein is operably linked to a promoter, preferably an exogenous promoter.

[0011] In some embodiments, the present invention is a vaccine composition comprising a heterodimeric protein molecule, wherein the heterodimeric protein molecule comprises a first fusion protein monomer and a second fusion protein monomer, and the first fusion protein monomer and the second fusion protein monomer comprise an operably linked targeting unit, heterodimerization unit, and antigenic unit, The antigenic units in each of the above first fusion protein monomers and the above second fusion protein monomers differ by encoding different variant antigen target proteins, The above heterodimeric protein molecule provides a vaccine composition comprising two of the above monomers linked by the association of the above dimerization domains.

[0012] In some embodiments, the heterodimerization unit in one of the above first fusion protein monomer and the above second fusion protein monomer is an ACID heterodimerization unit, and the heterodimerization unit in the other of the above first fusion protein monomer and the above second fusion protein monomer is a BASE heterodimerization unit that interacts to form an ACID / BASE heterodimerization domain which is the above first heterodimeric protein. In some embodiments, the heterodimerization unit in one of the above first fusion protein monomer and the above second fusion protein monomer is a barnstar heterodimerization unit, and the heterodimerization unit in the other of the above first fusion protein monomer and the above second fusion protein monomer is a barnase heterodimerization unit that interacts to form a barnstar / barnase heterodimerization domain which is the above first heterodimeric protein.

[0013] In some embodiments, the targeting units of the first fusion protein and the second fusion protein are the same. In some embodiments, the targeting units of the first fusion protein and the second fusion protein are different. In some embodiments, the targeting unit is an antigen-binding protein. In some embodiments, the antigen-binding protein is a scFv. In some embodiments, the targeting unit is an antigen-presenting cell (APC) targeting unit. In some embodiments, the APC targeting unit binds to a target selected from the group consisting of MHC-II molecules, CD40, CD11c, CD14, HLA-DP, Toll-like receptors, and chemokine receptors.

[0014] In some embodiments, the different variant antigen target proteins have a sequence identity of greater than about 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, or have a conserved region of greater than 8, 10, 12, 15, 20, 30, 40, 50 or 60 amino acids in length and up to 100 - 200 amino acids in length among the variant antigen target proteins having a sequence identity of greater than about 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%. In some embodiments, the different variant antigen target proteins are from different strains or serotypes of an organism. In some embodiments, the organism is a pathogenic organism. In some embodiments, the organism is selected from the group consisting of viruses, bacteria, fungi and protozoa. In some embodiments, the different variant antigen target proteins are variants of hemagglutinin (HA). In some embodiments, the vaccine comprises variants of HA from at least 3, 4, 5 or 6 and up to 12 or 18 strains or serotypes of influenza virus. In some embodiments, the influenza virus is selected from the group consisting of influenza virus group 1 and influenza virus group 2. In some embodiments, the influenza virus group 1 is selected from the group consisting of H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17 and H18, and the influenza virus group 2 is selected from the group consisting of H3, H4, H7, H10, H14 and H15. In some embodiments, the different variant antigen target proteins are variants of cancer antigens.

[0015] In some embodiments, a vaccine composition further comprising at least a third fusion protein monomer and a fourth fusion protein monomer, wherein the third fusion protein monomer and the fourth fusion protein monomer comprise a targeting unit, a heterodimerization unit, and an antigenic unit that are operably linked, and the antigenic units in each of the third fusion protein monomer and the fourth fusion protein monomer are different by encoding different types of the variant antigen target protein. In some embodiments, the vaccine comprises a mixture of heterodimeric proteins characterized by having all possible combinations of the first monomer, the second monomer, and at least the third monomer and the fourth monomer. In some embodiments, the vaccine composition is characterized by substantially no homodimeric proteins containing the same antigen target protein. In some embodiments, the vaccine composition is characterized by substantially no homodimeric proteins containing the same antigen target protein.

[0016] In some embodiments, the present invention provides a pharmaceutical formulation comprising the DNA vaccine or vaccine composition described above and a pharmaceutically acceptable carrier.

[0017] In some embodiments, the present invention provides a vaccine formulation comprising the vaccine composition described above and an adjuvant.

[0018] In some embodiments, the present invention provides a method of conferring immunity to, or inducing an immune response against, a variant antigen target protein in a subject, comprising administering to the subject a DNA vaccine, vaccine composition, pharmaceutical preparation, or vaccine preparation according to any one of claims 1 to 45. In some embodiments, the method further comprises a second administration of the above-mentioned DNA vaccine, vaccine composition, pharmaceutical preparation, or vaccine preparation to the subject. In some embodiments, the method further comprises administering to the subject a second DNA vaccine, vaccine composition, pharmaceutical preparation, or vaccine preparation comprising one or more non-target variant antigen target protein subunits.

[0019] In some embodiments, the present invention provides the use of the above-mentioned DNA vaccine, vaccine composition, pharmaceutical preparation, or vaccine preparation for conferring immunity or inducing an immune response in a subject in need thereof.

[0020] In some embodiments, the present invention provides the use of the above-mentioned DNA vaccine, vaccine composition, pharmaceutical preparation, or vaccine preparation as a vaccine for preventing or treating infection by a pathogen.

[0021] In some embodiments, the present invention provides the use of the above-mentioned DNA vaccine, vaccine composition, pharmaceutical preparation, or vaccine preparation as a vaccine for preventing or treating cancer.

[0022] Further embodiments will be apparent to those skilled in the art based on the teachings contained herein.

Brief Description of the Drawings

[0023] These and other features, aspects, and advantages of the present invention will be better understood by reference to the following drawings:

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[0024] It should be understood that the drawings are not necessarily drawn to scale, and that the objects in the drawings are not necessarily drawn to scale relative to each other. The drawings are a depiction intended to clarify and understand various embodiments of the devices, systems, and methods disclosed herein. Identical reference numerals are used, wherever possible, to represent the same or similar configurations throughout the drawings. Further, it should be understood that the drawings are in no way intended to limit the scope of the present teachings. Detailed Description of the Invention

[0025] Provided herein are vaccines, compositions, methods, and uses related thereto, particularly, but not limited to, vaccines of heterodimeric vaccine molecules formed from monomers comprising variant targeting units and variant antigenic units, linked by a heterodimerization unit.

[0026] The section headings used herein are for organizational purposes only and should in no way be construed as limiting the subject matter being described.

[0027] In this detailed description of various embodiments, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. However, one of ordinary skill in the art will understand that these various embodiments may be practiced with or without these specific details. In other instances, structures and devices are shown in block diagram form. Further, one of ordinary skill in the art will readily understand that the specific arrangements in the methods shown and executed are exemplary, and that the arrangements may be varied and still be within the spirit and scope of the various embodiments disclosed herein.

[0028] All documents and similar materials cited in this application, including but not limited to patents, patent applications, treatises, books, papers, and Internet web pages, are hereby expressly incorporated by reference in their entirety for any purpose. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the various embodiments described herein belong. In the event that the definitions of terms in the incorporated references are clearly different from the definitions provided in this teaching, the definitions provided in this teaching shall prevail.

[0029] 〔Definitions〕 To facilitate understanding of this technology, some terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

[0030] Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly indicates otherwise. As used herein, the phrase "in one embodiment" does not necessarily refer to the same embodiment, but it may. Further, as used herein, the phrase "in another embodiment" does not necessarily refer to a different embodiment, but it may. Thus, as described below, the various embodiments of the present invention can be readily combined without departing from the scope or spirit of the present invention.

[0031] Also, as used herein, the term "or" is an inclusive "or" and is equivalent to the term "and / or" unless the context clearly dictates otherwise. The term "based on" is not limiting and admits that the subject matter may be based on additional factors not recited, unless the context clearly dictates otherwise. Also, throughout this specification, the meanings of "a", "an", and "the" include the plural, and the meaning of "in" includes "in" and "on".

[0032] As used in this application, the terms "subject" and "patient" refer to any animal, including mammals such as dogs, cats, birds, livestock, and preferably humans.

[0033] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent and an inert or active carrier that renders the composition particularly suitable for therapeutic use.

[0034] As used herein, the terms "pharmaceutically acceptable" or "pharmacologically acceptable" refer to compositions that, when administered to a subject, do not substantially produce adverse reactions, such as toxic, allergic, or immunological reactions.

[0035] As used herein, the term "treating" includes reducing or alleviating at least one harmful effect or symptom of a disease or disorder by introducing a therapeutic composition of the present technology into the body or on the body of a subject in any manner. "Treatment" refers to both therapeutic treatment and prophylactic or preventive measures, where the aim is to prevent or delay (e.g., minimize or alleviate) a targeted pathological condition or disorder. Those in need of treatment include those already having a disorder, as well as those having a tendency to have a disorder or those in whom the disorder should be prevented.

[0036] As used herein, the term "antibody" is used in its broadest sense to refer to intact antibodies, monoclonal antibodies (including human, humanized, or chimeric antibodies), polyclonal antibodies, and antibody fragments capable of binding an antigen (e.g., Fab’, F’(ab)2, Fv, single-chain antibodies), and includes the above-described complementarity determining regions (CDRs) so long as they exhibit the desired biological activity.

[0037] As used herein, the term "antibody fragment" includes a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0038] A molecule that "specifically binds" to another molecule, or that is "specific for" another molecule, is a molecule that binds to that particular molecule without substantially binding to any other molecule.

[0039] As used herein, the term "in vitro" refers to an artificial environment and processes or reactions that occur within an artificial environment. In vitro environments include, but are not limited to, test tubes and cell culture media. The term "in vivo" refers to a natural environment (e.g., an animal or a cell) and processes or reactions that occur within a natural environment.

[0040] As used herein, the term "administration" refers to the act of giving a drug, prodrug, antibody, vaccine, or other agent to a physiological system (e.g., a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs), or to a therapeutic treatment. Exemplary routes of administration to the human body can be through the eye (ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lung (inhalant), oral mucosa (sublingual tablet), ear, injection (e.g., intravenous, subcutaneous, intratumoral, intraperitoneal, etc.), and the like. "Combination" refers to the administration of multiple chemical agents or therapeutic treatments (e.g., radiation therapy) to a physiological system (e.g., a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs). Administration "in combination with" one or more additional therapeutic agents as used herein includes simultaneous (parallel) administration and sequential administration in any order. The "combination" of therapeutic treatments may be simultaneous, may be in any temporal order, or may be a physical combination.

[0041] As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to the exposed cells or mammals at the employed dosage and concentration. Often, a physiologically acceptable carrier is a pH buffered aqueous solution. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants.

[0042] As used herein, the terms "protein", "polypeptide", and "peptide" refer to a molecule containing amino acids linked via peptide bonds. Generally, "peptide" is used to refer to a sequence of 20 or fewer amino acids, and "polypeptide" is used to refer to a sequence of more than 20 amino acids.

[0043] As used herein, the terms "synthetic polypeptide", "synthetic peptide", and "synthetic protein" refer to peptides, polypeptides, and proteins produced by recombinant processing (i.e., expression of exogenous nucleic acids encoding peptides, polypeptides, or proteins in organisms, host cells, or cell-free systems) or by chemical synthesis.

[0044] As used herein, the term "protein of interest" refers to a protein encoded by a nucleic acid of interest.

[0045] As used herein, the term "native" (or wild-type), when used with respect to a protein, refers to a protein encoded by the genome of a cell, tissue, or organism other than the genome engineered to produce a synthetic protein.

[0046] As used herein, a "domain" (typically a sequence of three or more, generally five or more, or seven or more amino acids) refers to a portion of a molecule, such as a protein or encoding nucleic acid, that is structurally and / or functionally distinct from other portions of the molecule and is identifiable. For example, a domain can include a polypeptide chain that can form an independently folded structure within a protein composed of one or more structural motifs, and / or a portion of a polypeptide chain recognized by a functional activity such as proteolytic activity. As described above, a domain refers to a folded protein structure that retains its tertiary structure independently of the rest of the protein. Generally, domains are involved in distinct functional properties of a protein and can often be added, removed, or transferred to other proteins without loss of function of the remainder of the protein and / or the domain.

[0047] A protein can have one or more different domains. For example, a domain can be identified, defined, or distinguished by the homology of the sequence in that domain to related family members (such as homology to motifs defining a protease domain or a gla domain). In another embodiment, a domain can be distinguished by its function (such as proteolytic activity) or its ability to interact with biomolecules (such as DNA binding, ligand binding, and dimerization). A domain can exhibit a biological function or activity independently, and as a result, a domain can exhibit an activity such as proteolytic activity or ligand binding independently or when fused to another molecule. A domain can be a linear sequence of amino acids or a non-linear sequence of amino acids. Many polypeptides contain multiple domains. Some domains are known and can be identified by those skilled in the art. It should be understood that recognizing a particular domain by name is well within the knowledge of those skilled in the art. If necessary, appropriate software can be used to identify domains.

[0048] As used herein, the term "host cell" refers to any eukaryotic cell (such as mammalian cells, avian cells, amphibian cells, plant cells, fish cells, insect cells, yeast cells) and bacterial cells, etc., regardless of whether they are located in vitro or in vivo (such as in a transgenic organism). The term "host cell" refers to any cell that can replicate and / or transcribe and / or translate a heterologous gene. Thus, the "host cell" refers to any eukaryotic or prokaryotic cell, regardless of whether it is located in vitro or in vivo. For example, a host cell can be located in a transgenic animal.

[0049] As used herein, the term "cell culture" refers to any in vitro culture of cells. This term includes continuous cell lines (e.g., having an immortalized phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro, including oocytes and embryos.

[0050] The term "isolated," when used in reference to a nucleic acid or polypeptide or protein, refers to a nucleic acid or polypeptide or protein sequence that has been identified and separated from at least one contaminating nucleic acid or polypeptide or protein with which it is ordinarily associated in its natural source. An isolated nucleic acid or polypeptide or protein is a molecule that exists in a form or configuration different from that in which it is found in nature. In contrast, an unisolated nucleic acid or polypeptide or protein is found in the state in which it exists in nature.

[0051] The term "antigen" refers to a molecule (e.g., a protein, glycoprotein, lipoprotein, lipid, nucleic acid, or other substance) that reacts with an antibody specific for a portion of the molecule.

[0052] The term "antigenic determinant" refers to the portion of an antigen that contacts a particular antibody (e.g., an epitope). When a protein or fragment of a protein is used to immunize a host animal, multiple regions of the protein may induce the production of antibodies that specifically bind to a given region or three-dimensional structure on the protein, and these regions or structures are referred to as antigenic determinants. Antigenic determinants may compete with the intact antigen (e.g., the "immunogen" used to induce an immune response) when binding to an antibody. In some embodiments, an "antigenic determinant" or "epitope" is a "neoepitope" or novel epitope.

[0053] The terms "protein" and "polypeptide" refer to compounds containing amino acids linked via peptide bonds and are used interchangeably. A "protein" or "polypeptide" encoded by a gene is not limited to the amino acid sequence encoded by the gene and includes post-translational modifications of the protein.

[0054] When the term "amino acid sequence" described herein refers to the amino acid sequence of a protein molecule, the terms "amino acid sequence" and similar terms such as "polypeptide" or "protein" do not mean to limit the amino acid sequence to the complete native amino acid sequence associated with the described protein molecule. Further, the "amino acid sequence" can be deduced from the nucleic acid sequence encoding the protein.

[0055] The term "portion", when used with respect to a protein (as in "a portion of a given protein"), refers to a fragment of that protein. The size of the fragment can range from 4 amino acid residues to one less than the entire amino acid sequence (e.g., the size can include from 4, 5, 6, 7, 8, 9, 10, or 11 amino acids to one less than the entire amino acid sequence).

[0056] As used herein, a "vaccine" comprises one or more immunogenic antigens that are intentionally administered to induce acquired immunity in a recipient (e.g., a subject).

DETAILED DESCRIPTION OF THE INVENTION

[0057] It should be understood that the description herein refers to specific exemplary embodiments, but these embodiments are presented by way of example and not by way of limitation.

[0058] In some embodiments, the present invention is A DNA vaccine comprising a first nucleic acid construct and a second nucleic acid construct, wherein the first nucleic acid construct and the second nucleic acid construct encode a first fusion protein and a second fusion protein, and the first fusion protein and the second fusion protein comprise an operably linked targeting unit, heterodimerization unit, and antigenic unit, to provide a DNA vaccine. In some embodiments, the antigenic unit in each of the first fusion protein and the second fusion protein is a variant antigen target protein. In some embodiments, when the first nucleic acid construct and the second nucleic acid construct are introduced into a cell, the first fusion protein and the second fusion protein are expressed, and a first heterodimeric protein is formed through the association of the heterodimerization unit. This is schematically shown, for example, in FIGS. 1 and 8. As seen in these figures, introduction of the vaccine nucleic acid construct into a host cell results in the production of monomeric units comprising a targeting unit (exemplified by scFvs such as anti-MHCII and anti-NIP), a heterodimerization unit (exemplified by ACID / BASE domains and barstar / barnase domains), and different antigenic units (exemplified by Fv315 and HA from PR8 and Cal07). Upon expression, the monomer having an ACID domain pairs with the monomer having a BASE domain, and similarly, the monomer having a barstar domain pairs with the monomer having a barnase domain to form a heterodimeric protein molecule. Exemplary sequences of the fusion proteins of the present invention are provided herein. For example, SEQ ID NOs: 1 and 2 provide the nucleic acid sequences of constructs encoding a fusion protein having a MIP1α targeting unit and an M315 antigenic unit separated by a hinge region and an ACID-ACID region. SEQ ID NO: 3 provides the corresponding amino acid sequence of the fusion protein. SEQ ID NOs: 4 and 5 provide the nucleic acid sequences of constructs encoding a fusion protein having a MIP1α targeting unit and an M315 antigenic unit separated by a hinge region and a BASE-BASE domain. SEQ ID NO: 6 provides the corresponding amino acid sequence of the fusion protein.The exemplified constructs and fusion proteins are modular, for example, the exemplified targeting unit may be replaced with different targeting units as described in more detail herein, the exemplified antigenic unit may be replaced with different antigenic units as described in more detail herein, and the heterodimerization domain may be replaced with different heterodimerization domains as described in more detail herein. Additional antigenic units are exemplified by those provided as SEQ ID NOs: 7 - 13.

[0059] It will be recognized that the constructs can also be used to produce polypeptide vaccine compositions by expressing a suitable expression vector containing the construct in a host cell. Accordingly, the present invention also provides a vaccine composition comprising a heterodimeric protein molecule, the heterodimeric protein molecule comprising a first fusion protein monomer and a second fusion protein monomer, the first fusion protein monomer and the second fusion protein monomer comprising an operably linked targeting unit, a heterodimerization unit, and an antigenic unit, wherein the antigenic units in each of the first fusion protein monomer and the second fusion protein monomer differ by encoding different variant antigen target proteins, and the heterodimeric protein molecule comprises two such monomers linked by association of the dimerization domain.

[0060] In preferred embodiments, the different antigenic units are variants of a target antigen protein, such as an antigen protein derived from a pathogen (e.g., a virus, bacterium, fungus or protozoan) or a target cancer antigen. In some embodiments, the cancer antigen is a neoepitope resulting from a somatic mutation or passenger mutation within a tumor that gives rise to a novel epitope or neoepitope. Neoepitopes are recognized as "mutated self" by the adaptive immune system and serve as a means for the immune system to distinguish cancer from normal cells. Thus, neoepitopes can be strong candidates for personalized cancer immunotherapy vaccines. In some embodiments, the variant of the target antigen protein is a variant of the target antigen protein derived from different strains of the target pathogen (e.g., different strains of a virus, bacterium, fungus, or protozoan, etc.). In some embodiments, the variant of the target antigen protein is a variant of the target antigen protein derived from different species or genera of the target pathogen (e.g., different species or genera of a virus, bacterium, fungus, or protozoan, etc.). In some embodiments, the variant can be defined by the degree of sequence identity of the sequences shared by the variant of the target antigen protein. For example, variant antigen target proteins derived from different strains of the same pathogenic organism are very diverse in sequence, so a high degree of sequence identity is not required. In some embodiments, the variant can have a sequence identity of greater than about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%. Accordingly, the DNA vaccines and vaccine compositions of the present invention can comprise sequences encoding 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 variant antigen target proteins, or 2 - 10, 2 - 20, 2 - 30, 2 - 50, 3 - 10, 3 - 20, 3 - 30, 3 - 50, 4 - 10, 4 - 20, 4 - 30, 4 - 50, 5 - 10, 5 - 20, 5 - 30, 5 - 50, 6 - 10, 6 - 20, 6 - 30, or 6 - 50 variant antigen target proteins, or 2, 3, 4, 5, 6, 7, 8, 9 or 10 variant antigen target proteins.

[0061] The present invention is not limited to a specific mechanism of action. In fact, understanding the mechanism of action is not essential for practicing the present invention. However, FIG. 14 provides a model for the induction of an immune response using the vaccine of the present invention. Referring to FIG. 14, after electroporation following DNA vaccination, host cells can take up the DNA and transcribe the vaccine protein. See FIG. 14A. The targeting unit on the vaccine effectively binds to and activates APCs, is taken up intracellularly, and is processed to present antigens to CD4+ T cells against MHCII. Antigen-specific BCRs recognize epitopes on the antigenic unit, whereby the vaccine protein is taken up by B cells and processed. By presenting antigens against MHCII on the B cell surface, B cells can receive assistance from CD4+ T cells, activate B cells to differentiate into plasma cells, and produce antibodies. Furthermore, the targeted vaccine dimer can form a synapse between B cells and APCs. The bivalent targeted vaccine allows one BCR to bind to two arms of the vaccine dimer. As shown in FIG. 14D, when a vaccine dimer bound to multiple APCs and BCRs bind in series, a synapse is formed and the BCRs are immobilized with respect to each other in the B cell membrane. Thereby, the BCRs remain close to each other in the membrane. It is thought that this amplifies the BCR signal, enhances the activation of B cells, and ultimately increases the secretion of antigen-specific antibodies. As shown in FIG. 14C, the monovalent targeted vaccine can promote synapse formation but cannot form a strong binding form with the same BCR. The BCRs can move through the membrane relative to each other (indicated by the arrows), causing proximity of the less constrained BCRs. The non-targeted vaccine does not stabilize the B cell-APC synapse (FIG. 14B), and moreover, cannot constrain the BCRs in the B cell membrane.

[0062] The constructs, the vaccine of the constructs, the vaccine, and the use of the vaccine are described in more detail below.

[0063] 〔Antigenic unit〕 The vaccine molecule of the present invention preferably comprises different antigenic units that are variants of a target antigen protein (e.g., an antigen protein derived from a pathogen such as a virus, bacterium, fungus, or protozoan) or a target cancer antigen. As described above, a variant can be defined by the degree of sequence identity of a variant of the target antigen protein or a sequence shared by conserved regions of the target antigen protein. In some embodiments, the variant can have a sequence identity of greater than about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%. In still other embodiments, a conserved region within a variant target protein that is greater than 8, 10, 12, 15, 20, 30, 40, 50, or 60 amino acids in length and up to 100 - 200 amino acids in length can have a sequence identity of greater than about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. According to the present invention, an antigenic unit comprises an antigen, which is a substance that elicits the production of one or more antibodies in an organism such as a subject. Each antibody binds to a specific antigen. In some contexts, the term antigenic unit refers to any molecule or molecular fragment that can bind to the major histocompatibility complex (MHC) and be presented to a T cell receptor.

[0064] In some contexts, an immunogen is a specific type of antigen. An immunogen is a substance that induces an adaptive immune response when injected alone (or, for example, as part of an antigenic unit of a dimeric vaccine molecule). Thus, an immunogen induces an immune response, while an antigen, once produced, binds to the products of an immune response (e.g., antibodies). As one aspect of the art, the term "antigen" is used in its broadest sense, whether or not it can be characterized as an immunogen and / or an antigen, and refers to a molecule, substance, chemical, or macromolecule such as a protein, polypeptide, and / or peptide that induces an immune response in a subject, for example, as a prophylactic means or as a treatment.

[0065] At the molecular level, sometimes antigens can be characterized by their ability to "bind" to the antigen-binding sites of antibodies. Antibodies recognize specific molecular structures present on the surface of antigens. Antigens are usually proteins (such as polypeptides, peptides, proteins) or polysaccharides that exist as parts of bacteria, viruses, and other microorganisms (e.g., outer shells, capsules, cell walls, flagella, pili, and toxins). Lipids and nucleic acids can be made antigenic by combining them with proteins and polysaccharides.

[0066] Cells present immunogenic antigens to the immune system via histocompatibility molecules. Depending on the type of antigen and histocompatibility molecule presented, several types of immune cells can be activated. By endocytosis or phagocytosis, exogenous antigens are taken up by antigen-presenting cells (APCs) and processed into fragments. Next, the APCs present the fragments to helper T cells (CD4+) using class II histocompatibility molecules on their surface. Some T cells are specific for the peptide:MHC complex. They become activated and begin to secrete cytokines. Cytokines are substances that can activate cytotoxic T lymphocytes (CTLs), B cells that secrete antibodies, macrophages, and other cells.

[0067] According to embodiments of the technology, the dimeric vaccine molecule can be subjected to general medical treatment by inducing an immune response against any polypeptide of any origin. Any antigenic sequence can be incorporated if the antigenic sequence is of sufficient length to allow proper folding of the polypeptide. This sequence can be derived, for example, from a pathogenic protein of cancer. In some embodiments, the target antigen protein is a protein (or nucleic acid encoding a protein) that is therapeutically used to induce an immune response that rescues or treats the progression of a disease (e.g., viral infection, autoimmune disease, or cancer).

[0068] In some embodiments, the molecule of interest is an antigen derived from a pathogen (e.g., an antigen derived from a pathogen or a nucleic acid or polypeptide encoding the antigen). Exemplary pathogenic organisms include, but are not limited to, bacteria, viruses, fungi, and protozoa. In some preferred embodiments, the antigen derived from a pathogen is influenza hemagglutinin (HA). In some particularly preferred embodiments, the HA is derived from a group 1 influenza virus. In some embodiments, the group 1 influenza virus is selected from the group consisting of two or more (i.e., two or more, three or more, four or more, five or more, or all twelve) of H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18. In still other embodiments, the HA is derived from a group 2 influenza virus selected from the group consisting of two or more (i.e., two or more, three or more, four or more, five or more, or all six) of H3, H4, H7, H10, H14, and H15.

[0069] Antigens from other pathogenic organisms may also be used in the fusions of the present invention. Exemplary pathogens from which antigens can be obtained include, but are not limited to, in some embodiments, as microorganisms, Bacillus (including Bacillus anthracis); Vibrio (e.g., V. cholera); Escherichia (e.g., enterotoxigenic E. coli); Shigella (e.g., S. dysenteriae); Salmonella (e.g., S. typhi); Mycobacterium (e.g., M. tuberculosis, M. leprae); Clostridium (e.g., C. botulinum, C. tetani, C. difficile, C. perfringens); Cornyebacterium (e.g., C. diphtheria); Streptococcus, S. pyogenes, S. pneumonia); Staphylococcus (e.g., S. aureus); Haemophilus (e.g., H. influenza); Neisseria (e.g., N. meningitidis, N. gonorrhoeae); Yersinia (e.g., Y. lamblia, Y. pestis); Pseudomonas (e.g., P. aeruginosa, P. putida); Chlamydia (e.g., C. trachomatis); Bordetella (e.g., B. pertussis); Treponema (e.g., T. palladium); B. anthracis, Y. pestis, Brucella spp., F. tularensis, B. mallei, B. pseudomallei, B. mallei, B. pseudomallei, C. botulinum, Salmonella spp., SEB V. cholera toxin B, E. coli O157:H7, Listeria spp., Trichosporon beigelii, Rhodotorula species, Hansenula anomala, Enterobacter sp., Klebsiella sp., Listeria sp., Mycoplasma ssp., Francisella spp., Bartonella spp., Borrelia spp., Campylobacter spp., Chlamydia spp., Simkania spp., Ehrlichia spp., Enterococcus spp., Coccidioides spp., Bordetella spp., Coxiella spp., Ureaplasma spp., Trichomatis spp., Helicobacter spp., Legionella spp., Mycobacterium spp., Corynebacterium spp., Rhodococcus spp., Rickettsia spp., Arcanobacterium spp., Listeria spp., Treponema spp., Brucella spp., Campylobacter spp., Pasteurella spp., Pseudomonas ssp., Burkholderii spp.etc., orthomyxoviruses (e.g., influenza viruses), paramyxoviruses (e.g., respiratory syncytial virus, mumps virus, measles virus), adenoviruses, rhinoviruses (human and porcine), coronaviruses, reoviruses, togaviruses (e.g., rubella virus), parvoviruses, poxviruses (e.g., variola virus, vaccinia virus), enteroviruses (e.g., poliovirus, coxsackievirus), hepatitis viruses (including types A, B, C, and E), herpesviruses (e.g., herpes simplex virus, HV-I and HV-II, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus), rotaviruses, norwalk virus, hantaviruses, arenaviruses, rhabdoviruses (e.g., rabies virus), retroviruses (HIV-1, HIV-2, HTLV-I, and HLTV-II), papovaviruses (e.g., papillomavirus), polyomaviruses, picornaviruses, dengue viruses, filoviruses (e.g., Marburg virus and Ebola virus), hantaviruses, Lassa fever virus, HHV-8, human papillomavirus, bovine leukemia virus, influenza virus, Guanarito virus, Lassa fever virus, measles virus, rubella virus, mumps virus, varicella (varicella virus), monkeypox, Epstein-Barr virus, parvovirus B19, Hunter virus, Sin Nombre virus, Venezuelan equine encephalitis, Sabia virus, West Nile virus, yellow fever virus, etc., causative agents of transmissible spongiform encephalopathies, Creutzfeldt-Jakob pathogen, variant Creutzfeldt-Jakob pathogen, Candida strains (C. glabrata, C. albicans, C. krusei, C. lusitaniae, and C.(including maltosa), and Aspergillus, Cryptococcus, Histoplasma, Coccidioides, Blastomyces and Penicillium, Cryptcooccus, Cryptosporidium, Giardia lamblia, Microsporidia, Plasmodium vivax, Plasmodium falciparum, Pneumocystis carinii, Toxoplasma gondii, Trichophyton mentagrophytes, Enterocytozoon bieneusi, Cyclospora cayetanensis, Encephalitozoon hellem, Encephalitozoon cuniculi, Ancylostama, Strongylus, Trichostrongylus, Haemonchus, Ostertagia, Ascaris, Toxascaris, Uncinaria, Trichuris, Dirofilaria, Toxocara, Necator, Enterobius, Strongyloides and Wuchereria; Acanthamoeba and other amoeba, Cryptosporidium, Fasciola, Hartmanella, Acanthamoeba, Giardia lamblia, Isospora belli, Leishmania, Naegleria, Plasmodium spp., Pneumocystis carinii, Schistosoma spp., Toxoplasma gondii, and Trypanosoma spp., other viruses, bacteria, archaea, protozoa, fungi, etc.

[0070] The fusion molecules described herein further include mammalian-derived antigens, including but not limited to proteins, subunits, motifs, and / or epitopes belonging to the following list of target antigens (where the following list includes transmembrane receptors and includes both soluble factors such as cytokines and membrane-bound factors): 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2, BMP-2a, BMP-3 osteogenin, BMP-4, BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, welchii toxin, CKb8-1, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, Cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Putrefaction promoting factor, des(1-3)-IGF-I (Brain IGF-1), Dhh, Digoxin, DNAM-1, DNase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, Endothelin receptor, Enkephalinase, eNOS, Eot, Eotaxin 1, EpCAM, Ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, Factor IIa, Factor VII, Factor VIIIc, Factor IX, Fibroblast activation protein (FAP), Fas, FcR1, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, Follicle-stimulating hormone, Fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (Myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, Glucagon, Glut 4, Glycoprotein IIb / IIIa (GP IIb / IIIa), GM-CSF, gp130, gp72, GRO, Growth hormone releasing factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMVgB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparinase, Her2, Her2 / neu (ErbB-2), Her3(ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin chain, insulin B chain, insulin-like growth factor 1, integrin alpha2, integrin alpha3, integrin alpha4, integrin alpha4 / beta7, integrin alpha4 / beta7, integrin alpha5(alphaV), integrin alpha5 / beta1, integrin alpha5 / beta3, integrin alpha6, integrin beta1, integrin beta2, interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP(TGF-1), latent TGF-1, latent TGF-1bp1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotoxin Beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloprotease, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Müllerian inhibitor, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, neurotrophin-4, or neurotrophin-6, Neurturin, nerve growth factor (NGF)), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, prolactin, protein C, PS, PSA, PSCA, prostate specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSVFgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, serine, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, Pan-specific TGF-beta, TGF-beta R1 (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R3 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, T RICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF R1 CD120a, p55 - 60), TNFRSF1B (TNF RII CD120b, p75 - 80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo - 1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4 - 1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo - 3, LARD, TR - 3, TRAMP, WSL - 1), TNFSF10 (TRAIL Apo - 2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo - 3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF - a connectin, DIF, TNFSF2), TNFSF1B (TNF - b LTa, TNFSF1)), TNFSF3 (LTbTNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, tumor-associated antigen-expressing Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (fit-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, and receptors for hormones and growth factors.

[0071] One of ordinary skill in the art will understand that the above list of target antigens refers not only to specific proteins and biomolecules, but also to biochemical pathways that include them. For example, reference to CTLA-4 as a target antigen means that the ligands and receptors that make up the T cell co-stimulatory pathway, including CTLA-4, B7-1, B7-2, CD28, and other undiscovered ligands or receptors that bind to these proteins, are also targets. Thus, as used herein, a target refers not only to a specific biomolecule, but also to a set of proteins that interact with the above target, and members of the biochemical pathway to which the above target belongs. One of ordinary skill in the art will further understand that any of the above target antigens, ligands or receptors that bind to them, or other members of their corresponding biochemical pathways, can be operably linked to the Fc variants of the present invention to generate an Fc fusion. Thus, for example, an Fc fusion targeting EGFR can be constructed by operably linking an Fc variant to EGF, TGF-β, or other known or undiscovered ligands that bind to EGFR. Accordingly, the Fc variants of the present invention can be operably linked to EGFR to generate an Fc fusion that binds to EGF, TGF-β, or other known or undiscovered ligands that bind to EGFR. Thus, in essence, although not limited to the above targets, any polypeptide (ligand, receptor, or some other protein or protein domain) that includes these and the proteins that make up their corresponding biochemical pathways can be operably linked to the Fc variants of the present invention to develop an Fc fusion.

[0072] The choice of suitable antigens depends on the desired application. In some embodiments, the constructs described herein target pathogen antigens. For anti-cancer therapy, it is desirable to have a target whose expression is limited to cancer cells. Among the targets that have proven particularly suitable for antibody therapy are those with signaling functions. Other therapeutic antibodies exert their effect by blocking receptor signaling by inhibiting the binding of the receptor to its cognate ligand. Another mechanism of action of therapeutic antibodies is to cause down-regulation of the receptor. Other antibodies do not act via signaling through their target antigen. In some cases, antibodies against infectious agents are used.

[0073] In one embodiment, the fusion protein of the present invention is used for the treatment of autoimmune, inflammatory, or transplantation indications. Target antigens, clinical products, and candidates associated with such diseases include anti-α4β7 integrin antibodies such as LDP-02, anti-β2 integrin antibodies such as LDP-01, anti-complement (C5) antibodies such as 5G1.1, anti-CD2 antibodies such as BTI-322, anti-CD3 antibodies such as MEDI-507, OKT3, SMART anti-CD3 antibodies, anti-CD4 antibodies such as IDEC-151, MDX-CD4, OKT4A, anti-CD11a antibodies, anti-CD14 antibodies such as IC14, anti-CD18 antibodies, anti-CD23 antibodies such as IDEC152, anti-CD25 antibodies such as Zenapax, anti-CD40L antibodies such as 5c8, anti-CD64 antibodies such as Antova, IDEC-131, MDX-33, anti-CD80 antibodies such as IDEC-114, anti-CD147 antibodies such as ABX-CBL, anti-E-selectin antibodies such as CDP850, anti-gpIIb / IIIa antibodies such as ReoPro / Abcixima, anti-ICAM-3 antibodies such as ICM3, anti-ICE antibodies such as VX-740, anti-FcR1 antibodies such as MDX-33, anti-IgE antibodies such as rhuMab-E25, anti-IL-4 antibodies such as SB-240683, anti-IL-5 antibodies such as SB-240563, SCH55700, anti-IL-8 antibodies such as ABX-IL8, anti-interferon γ antibodies, anti-TNF (TNF, TNFa, TNFa, TNF-α) antibodies such as CDP571, CDP870, D2E7, infliximab, MAK-195F, anti-VLA-4 antibodies such as Antegren, but are not limited thereto.

[0074] 〔Targeting Unit〕 One way to increase the immunogenicity of a protein antigen is to incorporate the antigen into an antibody or antibody fragment that targets immune system cells, such as antigen-presenting cells (APCs). APCs process antigens and present them to T cells to produce antibodies against those antigens. Thus, delivering an antigen to APCs provides an efficient route for inducing an immune response against the antigen.

[0075] Antigen-presenting cells (APCs) are cells that present antigen complexes with major histocompatibility complex (MHC) on their surface. APCs take up antigens, perform antigen processing, and return all or part of the antigen (e.g., epitope) back to the surface of the APC in MHC class II molecules that present the antigen. The CD4 receptor carried by naive helper T cells binds to MHC class II. The epitope (within the MHC class II molecule) imprints the T cell receptor (TCR) of the naive helper T cell and remembers that epitope.

[0076] T cells cannot recognize isolated antigens and thus cannot react with isolated antigens. T cells can react only with antigens processed and presented by cells via MHC molecules. Most cells in the body can present antigens to CD8+ T cells via MHC class I molecules, so they are APCs. In some contexts, the term APC refers to specialized cells that can prime T cells (e.g., activate naive T cells, which are T cells not exposed to antigens and are called "naive" T cells). These cells generally express both MHC class II and MHC class I molecules and can stimulate CD4+ ("helper") cells as well as CD8+ ("cytotoxic") T cells, respectively.

[0077] APCs take up antigens very efficiently, either by phagocytosis or receptor-mediated endocytosis, and then present fragments of the antigens bound to class II MHC molecules on their membranes. T cells recognize and interact with the antigen-class II MHC molecule complex on the membrane of the APC. Subsequently, additional co-stimulatory signals are generated by the APC, triggering T cell activation. APCs include dendritic cells, which present antigens over the broadest range. Activated DCs are particularly potent Th cell activators because they express co-stimulatory molecules such as B7 as part of their composition. In addition, APCs include macrophages, B cells, and some activated epithelial cells. Cells that can act as APCs when stimulated by certain cytokines (e.g., IFN-γ) include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells (brain), pancreatic β cells, and vascular endothelial cells.

[0078] For example, in some embodiments, the targeting unit comprises a single-chain variable region fragment targeting unit specific for MHC class II molecules. A single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy and light chains of an immunoglobulin connected by a short linker peptide (e.g., about 10 to about 25 amino acids). The linker is usually rich in glycine for flexibility and rich in serine or threonine for solubility. The linker can connect the N-terminus of the heavy chain to the C-terminus of the light chain or vice versa. scFvs can be made directly from subcloned heavy and light chains derived from hybridomas (e.g., from mammalian cell culture or bacterial cell culture such as E. coli culture).

[0079] An scFv with targeting function can be derived from B cell hybridomas expressing monoclonal antibodies (mAbs) that bind to surface molecules on APCs, or can be derived from any source (e.g., phage display library). By using scFvs derived from B cell hybridomas as targeting moieties, a wide range can be targeted due to the extensive collection of B cell hybridomas producing mAbs that bind to different surface molecules on APCs. Furthermore, by employing agonist or antagonist mAbs, the nature of the signal given to the target cells can be selected. Increasing knowledge about the Ab-Ag interaction would allow the binding affinity of such mAbs for the Ag to be improved by amino acid substitutions at the binding site. This can be done by conventional site-directed mutagenesis. For the purpose of a vaccine, there is an attractive way to make the target of the dimeric vaccine molecule a surface molecule that is only limitedly expressed on a subset of dendritic cells (DCs) that can initiate a strong specific immune response against the patient's own Id. Examples of such target surface molecules on APCs include MHCII molecules, CD40, CD14, CD11c, HLA-DP, Toll-like receptors, and chemokine receptors. Thus, in some embodiments, the scFv is anti-MHC-II or anti-HLA (e.g., HLA-DP), anti-CD14, anti-CD11c, anti-CD40, or anti-Toll-like receptor (anti-Toll-like receptor 2). In some embodiments, the targeting unit is a ligand (e.g., soluble CD40 ligand or chemokine (e.g., RANTES, MIP-1α, Flt3-L, GM-SCF or Xcl1)), bacterial antigen (e.g., flagellin).

[0080] The targeting scFv is inserted into the V cassette of the expression vector pLNOH2 (Norderhaug, Olafsen et al. 1997) and can thus be easily exchanged with other scFvs.

[0081] Alternatively, the antigen can target antigen-presenting cells (APCs) by binding the antigen to an antibody or other antibody fragment against surface molecules (e.g., receptors) on various types of APCs. Other targeting units include anti-immunoglobulins (e.g., anti-IgG, anti-IgA, anti-IgM, IgD, etc.), and Fab or Fab’ fragments.

[0082] 〔Heterodimerization Unit〕 The dimeric vaccine molecules of the present technology include a heterodimerization domain. In particular, each polypeptide of the dimeric vaccine molecule includes a heterodimerization unit that specifically interacts with a heterodimerization unit on another polypeptide to form a heterodimerization domain. Thus, as used herein, the term “heterodimerization domain” refers to a domain in a dimeric molecule formed by the interaction of two heterodimerization units, such as ACID and BASE heterodimerization units, or barstar and barnase heterodimerization units, as described in more detail below. Specific examples of heterodimerization domains include, but are not limited to, ACID / BASE heterodimerization domains and barstar / barnase heterodimerization domains.

[0083] In some embodiments, the heterodimerization domain is a leucine zipper used in an ACID / BASE heterodimerization system, etc. (see Busch, et al. 2002 “Stabilization of soluble, low-affinity HLA-DM / HLA-DR1 complexes by leucine zippers” J Immunol Methods 263: 111). In this system, one heterodimerization unit includes an acidic leucine zipper domain, such as an ACID module (e.g., AcidP1 or Fos), and the other heterodimerization unit includes a basic leucine zipper domain, such as a BASE module (e.g., BaseP1 or Jun), which specifically interact with each other to form the heterodimerization domain of the heterodimeric vaccine molecule.

[0084] In some embodiments, the heterodimerization domain comprises a bacterial barnase module and a bacterial barstar module. For example, Spang HCL, Braathen R, Bogen B (2012) Heterodimeric Barnase-Barstar Vaccine Molecules: Influence of One versus Two Targeting Units Specific for antigen Presenting Cells. PLoS ONE 7(9): e45393, incorporated herein by reference in its entirety. The proteins barnase and barstar from Bacillus amyloliquefaciens bind to each other with a very high affinity (K D :~10~14M) comparable to the affinity between biotin and streptavidin. The barnase-barstar module can be adapted for the fusion of the scFv N-termini of barnase and barstar, as well as the scFv

[29] or the second barnase C-terminus of barnase.

[0085] However, the technology is not limited to heterodimeric vaccine molecules comprising ACID / BASE or barstar / barnase domains. For example, any dimerization domain can be used that comprises two dimerization domains specific for each other. In some embodiments, a “knobs and holes” system is used (Xie, et al. 2005 “A new format of bispecific antibody: highly efficient dimerization, expression “and tumor cell lysis.” J Immunol Methods 296: 95, see reference). This system generates complementary knobs and holes that provide a dimerization domain for a dimeric vaccine molecule based on the CH3 domain of the human IgG1 Fc fragment. One polypeptide contains a knob module and the other polypeptide contains a hole module.

[0086] It will be understood that the heterodimerization domains of at least the first construct and the second construct encoding the vaccine monomer of the present invention are different and compatible with each other. For example, if the DNA vaccine of the present invention includes a first diffusion construct and a second nucleic acid construct (or a third construct and a fourth construct, etc.), one of the constructs has an ACID (or baluster) unit and the other construct of the dimer pair has a BASE (or barnase) unit. Similarly, the heterodimeric vaccine polypeptide molecule of the present invention includes a pair of monomers, where one of the monomers has an ACID (or baluster) unit and the other monomer of the dimer pair has a BASE (or barnase) unit.

[0087] [DNA Vaccine] The techniques provided herein provide a DNA vaccine comprising a nucleic acid encoding a dimeric vaccine molecule. In some embodiments, a single nucleic acid comprises two polypeptides that dimerize to form a dimer. In some embodiments, two separate nucleic acids comprise two polypeptides that dimerize to form a dimer (e.g., one nucleic acid encodes one polypeptide and the other nucleic acid encodes the other polypeptide). In various embodiments, the nucleic acid can be any nucleic acid that can be introduced into a cell and can express a polypeptide in vivo.

[0088] In these embodiments, the DNA vaccine comprises, alone or in combination with other desired sequences, a DNA molecule encoding the above-described fusion construct (i.e., a targeting unit operably linked to an antigenic unit via a dimerization domain and / or other linker). Unlike recombinant protein vaccines in which the antigen is taken up by antigen-presenting cells and expressed mainly in association with MHC class II, the DNA in the nucleic acid vaccine is directly taken up by antigen-presenting cells and expressed, and antigen presentation occurs via both naturally processed MHC class I and II epitopes.

[0089] In some embodiments, the DNA vaccine comprises a nucleic acid encoding the fusion construct described herein within a vector suitable for nucleic acid expression. In some embodiments, the nucleic acid is expressed in an expression cassette. In certain embodiments, the expression cassette is a eukaryotic expression cassette. The term "eukaryotic expression cassette" refers to an expression cassette that enables the expression of an open reading frame in a eukaryotic cell. A eukaryotic expression cassette includes regulatory sequences capable of controlling the expression of an open reading frame in a eukaryotic cell, preferably a promoter and a polyadenylation signal. The promoter and polyadenylation signal included in the recombinant DNA molecule are selected to be functional within the cells of the subject being immunized. Exemplary promoters suitable for the production of DNA vaccines, particularly for humans, include promoters derived from CMV such as the strong cytomegalovirus (CMV) immediate early promoter, promoters derived from simian virus 40 (SV40), promoters derived from mouse mammary tumor virus (MMTV), promoters derived from human immunodeficiency virus (HIV) such as the HIF long terminal repeat (LTR) promoter, promoters derived from Moloney virus, promoters derived from Epstein-Barr virus (EBV), and promoters derived from Rous sarcoma virus (RSV), as well as promoters derived from human genes such as human actin, human myosin, human hemoglobin, human muscle creatine, and human metallothionein, but are not limited thereto. In certain embodiments, the eukaryotic expression cassette includes a CMV promoter. In the context of the present invention, the term "CMV promoter" refers to the strong cytomegalovirus immediate early promoter.

[0090] Exemplary polyadenylation signals suitable for the production of DNA vaccines, particularly for humans, include, but are not limited to, the bovine growth hormone (BGH) polyadenylation site, the SV40 polyadenylation signal, and the LTR polyadenylation signal.

[0091] Other elements may also be included in the recombinant DNA molecule. Such additional elements include enhancers. Enhancers can be, for example, human actin, human myosin, human hemoglobin, human muscle creatine enhancers, and viral enhancers such as those derived from CMV, RSV, and EBV.

[0092] Regulatory sequences and codons are generally species-dependent, and thus, to maximize protein production, the regulatory sequences and codons are preferably selected to be efficient in the species to be immunized. One of ordinary skill in the art can produce a recombinant DNA molecule that is functional in a given target species.

[0093] The present invention is not limited by a particular dosage form of the vaccine composition. When the vaccine is a DNA vaccine, the vaccine can preferably be provided in physiological saline or other physiologically acceptable solutions or buffers. In some embodiments, the DNA is administered intramuscularly (i.m.) by needle injection into one or more tissues. In some embodiments, the DNA vaccination is i.m. into each quadriceps femoris muscle. In some embodiments, electroporation is performed immediately after injection, as disclosed in Liu et al. (2008, J Virol 82: 5643-5649), by delivering pulses from electrodes inserted i.m. adjacent to the injection site using an Elgen electroporator device (Elgen, Inovio Biomedical Co.) (needle EP), and the electrical pulses are given as 5×60 ms at intervals of 50 V / 400 mA, 200 ms. In other embodiments, electroporation is performed intradermally at the left and right flanks, followed by electroporation using a DermaVax (Cyto Pulse Sciences, Inc) system (the pulses are 450 V / cm×2.5 μs twice and 110 V / cm×8.1 ms eight times). Electroporation is performed to increase the translation of the DNA vaccine into vaccine proteins. In some embodiments, an efficient route is intramuscular injection into the quadriceps femoris or tibialis anterior muscles, followed by intradermal injection. These routes typically elicit a strong antigen-specific Th1-biased humoral and cellular immune response. In other embodiments, a gene gun is utilized. In these embodiments, the DNA vaccine described above is coated onto particles, preferably gold particles.

[0094] Generally, the delivery method determines the dose required to elicit an efficient immune response. For injection in saline, variable amounts of DNA from 10 μg to 1 mg are required, while for delivery by gene gun, 100 to 1000 times more are needed. Generally, 0.2 μg to 20 μg are required, although amounts as low as about 16 ng have also been reported. These amounts vary by species. For example, mice require about 10 times less DNA than primates. Saline injection requires more DNA because the DNA is carried extracellularly to the target tissue (usually muscle) and must overcome physical barriers (such as the basal lamina and large amounts of connective tissue, details of which are not worth mentioning) before being taken up by the cells. On the other hand, gene gun directly injects DNA into the cells, so there is less "waste". Electroporation also reduces the amount of DNA required (for example, 0.04 μg to 12.50 μg per plasmid).

[0095] [[Vaccine]] Use is found in pharmaceuticals comprising a dimeric vaccine molecule as provided by the techniques provided herein, a composition that is a vaccine or a vaccine component, and / or a dimeric vaccine molecule according to the present technique (for example, a dimeric polypeptide molecule), a DNA / RNA sequence, or an expression vector. Optionally, the pharmaceutical further comprises a pharmaceutically acceptable carrier. Suitable carriers and dosage forms in such pharmaceuticals are known to those skilled in the art. Suitable carriers are, for example, phosphate buffered saline, water, emulsions (for example, oil / water emulsions), wetting agents, sterile solutions, etc. The pharmaceutical can be administered orally or parenterally. Parenteral administration methods include topical administration, intraarterial administration, intramuscular administration, subcutaneous administration, intramedullary administration, intrathecal administration, intraventricular administration, intravenous administration, intraperitoneal administration, or intranasal administration. Suitable dosages are determined by the attending physician and depend on different factors (such as the patient's age, sex and weight, type of administration, etc.).

[0096] In one aspect, the vaccine or vaccine component is used to immunize mice to produce hybridomas. In some embodiments, the vaccine is for an infectious disease, and in other embodiments, the vaccine is a cancer therapeutic vaccine. Infectious diseases against which the vaccine can be constructed include viral diseases (including rotavirus, norovirus, rabies, influenza virus, herpes virus, etc.), bacterial diseases (e.g., gonorrhea, streptococcal pneumonia, tuberculosis, tularemia, etc.), fungal diseases (e.g., histoplasmosis, blastomycosis, and candidiasis), and protozoal diseases (e.g., cryptosporidiosis, leishmaniasis, filariasis, etc.), but are not limited thereto. Examples of cancers that can respond to therapeutic vaccination include, for example, cervical cancer, melanoma, myeloma, and breast cancer. In some embodiments, the vaccine is for human use, and in some embodiments, it is for the vaccination of animals (e.g., livestock, companion animals, and any other type of animal (fish, wildlife, etc.)). The above vaccine can also be applied in vitro to cells derived from a subject (e.g., a patient) to cause APC binding and presentation; then, the cells can be returned to the host (subject, patient) of origin.

[0097] In some embodiments, the nucleic acid expressing the polypeptide of the dimeric vaccine molecule is present in host cells in vitro for the production of the dimeric vaccine molecule. Recombinant methods for producing polypeptides in cell culture are well known in the art. For example, in some embodiments, the polypeptide of the dimeric vaccine molecule is expressed in bacterial culture such as the culture of E. coli, and the polypeptide of the dimeric vaccine molecule is purified and isolated from the culture to provide the vaccine. In some embodiments, the host cell is a eukaryotic cell (e.g., NSO cells, 293E cells, and cells transfected with Cos-7 cells) maintained in cell culture, and in some embodiments, it may or may not be a transformed cell.

[0098] In one embodiment, the dimeric vaccine is administered parenterally. In another embodiment, the dimeric vaccine molecule is administered to a mucosal surface such as the nasal cavity or other mucosae. In another specific embodiment, the dimeric vaccine molecule is administered orally so as to enable presentation to the oral mucosa or gastrointestinal mucosa. In some forms of oral administration, the dimeric vaccine molecule is encapsulated in enteric capsules or gel capsules. In yet other embodiments, the dimeric vaccine molecule is combined in a chewable form. When delivered to an animal, the dimeric vaccine molecule can be incorporated into feed or food. In some embodiments, the dimeric vaccine molecule can be topically applied to the skin.

[0099] In some embodiments, the present invention provides a vaccine composition comprising the dimeric vaccine provided herein. The present invention is not limited by a particular dosage form of the composition comprising the dimeric vaccine molecule. Indeed, the vaccine composition of the present invention may comprise one or more different agents in addition to the dimeric vaccine molecule. These agents or cofactors include, but are not limited to, adjuvants, surfactants, additives, buffers, solubilizing agents, chelating agents, oils, salts, therapeutic agents, drugs, bioactive agents, antibacterial agents, and antimicrobial agents (e.g., antibiotics, antiviral agents, etc.). In some embodiments, the vaccine composition comprising the dimeric vaccine molecule comprises an agent or cofactor (e.g., an adjuvant) that increases the ability of the antigenic unit to induce an immune response. In some preferred embodiments, the presence of one or more cofactors or agents reduces the amount of antigenic units required to induce an immune response (e.g., a protective immune response (e.g., protective immunization)). In some embodiments, the presence of one or more cofactors or agents skews the immune response to a cellular (e.g., T cell-mediated) or humoral (e.g., antibody-mediated) immune response. The present invention is not limited by the type of cofactor or agent used in the therapeutic agent of the present invention.

[0100] Adjuvants are generally described in Vaccine Design - the Subunit and Adjuvant Approach, edited by Powell and Newman, Plenum Press, New York, 1995, which is hereby incorporated by reference in its entirety for all purposes. The present invention is not limited by the type of adjuvant utilized (e.g., for use in a composition (e.g., a pharmaceutical composition)). For example, in some embodiments, suitable adjuvants include aluminum salts such as aluminum hydroxide gel (e.g., alum), or aluminum phosphate. In some embodiments, the adjuvant may be a salt of calcium, iron, or zinc, or may be acylated tyrosine or acylated sugar, a cationic or anionic derivatized polysaccharide, or an insoluble suspension of polyphosphazene.

[0101] Generally, an immune response is elicited against an antigen through the interaction of the antigen with cells of the immune system. The immune response is broadly classified into two categories: the humoral immune response and the cell-mediated immune response (e.g., traditionally characterized by defense mechanisms by antibodies and cellular effectors, respectively). These categories of responses are referred to as the Th1 type response (cell-mediated) and the Th2 type immune response (humoral response).

[0102] Stimulation of the immune response can result from direct or indirect reactions (e.g., exposure to antigenic units) of cells or components of the immune system to a medical intervention. The immune response can be measured in many ways, including activation, proliferation, or differentiation of immune system cells (e.g., B cells, T cells, dendritic cells, APCs, macrophages, NK cells, NKT cells, etc.); upregulated or downregulated expression of markers and cytokines; stimulation of IgA, IgM, or IgG titers; splenomegaly (including increased splenic cell hyperplasia); hyperplasia and mixed cell infiltration in various organs. Other responses, cells, and components of the immune system that can be evaluated for immune stimulation are known in the art.

[0103] Understanding of the mechanism is not essential for practicing the present invention, and the present invention is not limited to any particular mode of action. In some embodiments, the compositions and methods of the present invention induce the expression and secretion of cytokines (e.g., by macrophages, dendritic cells, and CD4+ T cells). Modulation of the expression of specific cytokines can occur locally or systemically. It is known that the cytokine profile can determine T cell regulation and effector functions in the immune response. In some embodiments, Th1-type cytokines can be induced, and thus the immune-stimulating compositions of the present invention can promote Th1-type antigen-specific immune responses, including cytotoxic T cells (e.g., thereby avoiding unwanted Th2-type immune responses (e.g., formation of Th2-type cytokines (e.g., IL-13) involved in disease exacerbation (e.g., induction of mucus formation by IL-13))).

[0104] Cytokines play a role in directing T cell responses. Helper (CD4+) T cells regulate the mammalian immune response through the production of soluble factors that act on other immune system cells, including B cells and other T cells. Most mature CD4+ T helper cells express one of two cytokine profiles, namely Th1 or Th2. Th1-type CD4+ T cells secrete IL-2, IL-3, IFN-γ, GM-CSF, and high levels of TNF-α. Th2 cells express IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, GM-CSF, and low levels of TNF-α. Th1-type cytokines promote both cell-mediated and humoral immunity, which is characterized by immunoglobulin class switching to IgG2a in mice and IgG1 in humans. Th1 responses can also be associated with delayed-type hypersensitivity and autoimmune diseases. Th2-type cytokines mainly induce humoral immunity and induce class switching to IgG1 and IgE. The isotypes of antibodies associated with Th1 responses generally have the ability to neutralize and opsonize, while the isotypes of antibodies associated with Th2 responses are related to allergic reactions.

[0105] Several factors have been shown to influence the skewing of the immune response towards either a Th1-type or Th2-type reaction. The regulators that best exhibit such properties are cytokines. IL-12 and IFN-γ are positive Th1 regulators and negative Th2 regulators. IL-12 promotes IFNγ production, and IFN-γ provides positive feedback to IL-12. IL-4 and IL-10 appear to be important in establishing the Th2 cytokine profile and downregulating the production of Th1 cytokines.

[0106] Accordingly, in a preferred embodiment, the present invention provides a method of stimulating a Th1-type immune response in a subject, the method comprising administering to the subject a composition comprising an antigenic unit (e.g., a dimeric vaccine molecule provided by the present technology described). However, in other embodiments, the present invention provides a method of stimulating a Th2-type immune response in a subject (e.g., when balancing a T cell-mediated response is desired), the method comprising administering to the subject a composition comprising an antigenic unit (e.g., a dimeric vaccine molecule provided by the present technology described). In a further preferred embodiment, an adjuvant may be used (e.g., co-administered with the composition of the present invention) to skew the immune response towards either a Th1-type or Th2-type immune response. For example, adjuvants that induce a Th2 response or a weak Th1 response include, but are not limited to, alum, saponin, and SB-As4. Adjuvants that induce a Th1 response include, but are not limited to, MPL, MDP, ISCOMS, IL-12, IFNγ, and SB-AS2.

[0107] In the compositions and methods of the present invention, several other types of Th1-type immunogens can be used (e.g., as adjuvants). These include, but are not limited to, the following. In some embodiments, monophosphoryl lipid A (e.g., in particular, 3-de-O-acylated monophosphoryl lipid A (3D-MPL)) is used. 3D-MPL is a well-known adjuvant manufactured by Ribi Immunochem, Montana. It is often supplied as a mixture of 3-de-O-acylated monophosphoryl lipid A having either 4, 5, or 6 acylated chains. In some embodiments, diphosphoryl lipid A and its 3-O-deacylated variants are used. Each of these immunogens can be purified and prepared by the methods described in GB2122204B, which is hereby incorporated by reference in its entirety. Other lipopolysaccharides that are purified and synthesized are described (e.g., U.S. Pat. No. 6,005,099 and EP 0 729 473; Hilgers et al., 1986, Int. Arch. Allergy. Immunol., 79(4):392-6; Hilgers et al., 1987, Immunology, 60(1):141-6; and EP 0 549 074, see (each of these documents is hereby incorporated by reference in its entirety into this specification)). In some embodiments, 3D-MPL is used in the form of a particulate dosage form (e.g., having a small particle size of less than 0.2 μm as described in EP 0 689 454, which is hereby incorporated by reference in its entirety into this specification)).

[0108] In some embodiments, saponin is used as an immunogen (e.g., a Th1 type adjuvant) in the compositions of the invention. Saponin is a well-known adjuvant (see, e.g., Lacaille-Dubois and Wagner (1996) Phytomedicine vol 2 pp 363-386). Exemplary saponins include Quil A (derived from the bark of the South American tree Quillaja Saponaria Molina) and its fractions (see, e.g., U.S. Pat. No. 5,057,540; Kensil, Crit Rev Ther Drug Carrier Syst, 1996, 12 (1-2):1-55; and EP 0 362 279, each of which is incorporated herein by reference in its entirety). Hemolytic saponins QS7, QS17, and QS21 (HPLC purified fractions of Quil A; see, e.g., Kensil et al. (1991). J. Immunology 146,431-437, U.S. Pat. No. 5,057,540; WO 96 / 33739; WO 96 / 11711 and EP 0 362 279, each of which is incorporated herein by reference in its entirety) are also thought to be beneficial in the present invention. Combinations of QS21 with polysorbate or cyclodextrin are also thought to be useful (see, e.g., WO 99 / 10008, which is incorporated herein by reference in its entirety).

[0109] In some embodiments, an immunogenic oligonucleotide comprising unmethylated CpG dinucleotides (“CpG”) is used as an adjuvant. CpG is an abbreviation of the cytosine-guanosine dinucleotide motif present in DNA. CpG is known in the art as an adjuvant when administered by both systemic and mucosal routes (e.g., WO 96 / 02555, EP 468520, Davis et al., J. Immunol, 1998, 160(2): 870-876; McCluskie and Davis, J. Immunol., 1998, 161(9):4463-6; and U.S. Pat. App. No. 20050238660, incorporated herein by reference in their entireties). For example, in some embodiments, the immunostimulatory sequence is purine-purine-C-G-pyrimidine-pyrimidine, where the CG motif is not methylated.

[0110] Understanding of the mechanism is not essential to practice the invention, and the invention is not limited to any particular mechanism of action. In some embodiments, the presence of one or more CpG oligonucleotides activates various immune subsets including natural killer cells (producing IFN-γ) and macrophages. In some embodiments, the CpG oligonucleotide is formulated in the compositions of the invention to induce an immune response. In some embodiments, a free solution of CpG is co-administered with an antigen (e.g., present in an aqueous solution (e.g., WO 96 / 02555, incorporated herein by reference). In some embodiments, the CpG oligonucleotide is covalently linked to an antigen (e.g., WO 98 / 16247, incorporated herein by reference), or formulated with a carrier such as aluminum hydroxide (e.g., Brazolot-Millan et al., Proc. Natl. Acad. Sci., USA, 1998, 95(26), 15553-8, incorporated herein by reference).

[0111] In some embodiments, complete Freund's adjuvant and incomplete Freund's adjuvant, cytokines (e.g., interleukins (e.g., IL-2, IFN-γ, IL-4, etc.), macrophage colony-stimulating factor, tumor necrosis factor, etc.), detoxified mutants of bacterial ADP-ribosylating toxins such as cholera toxin (CT), pertussis toxin (PT) or Escherichia coli heat-labile toxin (LT), particularly LT-K63 (lysine is substituted at position 63 relative to the wild-type amino acid), LT-R72 (arginine is substituted at position 72 relative to the wild-type amino acid), CT-S109 (serine is substituted at position 109 relative to the wild-type amino acid), and PT-K9 / G129 (lysine is substituted at position 9 and glycine is substituted at position 129 relative to the wild-type amino acid) (e.g., see WO93 / 13202 and WO92 / 19265, each of which is incorporated herein by reference), and other immunogenic substances (e.g., enhancing the efficiency of the compositions of the present invention) are used in combination with the compositions containing the dimeric vaccine molecules of the present invention.

[0112] Further examples of adjuvants used in the present invention include poly(di(carboxylatophenoxy))phosphazene (PCPP polymer; Virus Research Institute, USA); derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, Mont), muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania elongation factor (purified Leishmania protein; Corixa Corporation, Seattle, Wash).

[0113] An adjuvant may be added to a composition comprising a dimeric vaccine molecule, or the adjuvant may be formulated with a carrier, such as a liposome or a metal salt (e.g., an aluminum salt (e.g., aluminum hydroxide)), or combined with or co-administered with the composition prior to combination with the composition.

[0114] In some embodiments, a composition comprising a dimeric vaccine molecule comprises a single adjuvant. In other embodiments, the composition comprises two or more adjuvants (e.g., WO 94 / 00153; WO 95 / 17210; WO 96 / 33739; WO 98 / 56414; WO 99 / 12565; WO 99 / 11241; and WO 94 / 00153, incorporated herein by reference in their entirety).

[0115] In some embodiments, a composition comprising a dimeric vaccine molecule comprises one or more mucoadhesives (e.g., see U.S. Pat. App. No. 20050281843, incorporated herein by reference in its entirety). The present invention is not limited by the type of mucoadhesive used. Indeed, various mucoadhesives including poly(acrylic acid) (e.g., carbopol and polycarbophil), polyvinyl alcohol, polyvinylpyrrolidone, polysaccharides (e.g., alginates and chitosan), hydroxypropylmethylcellulose, lectins, fimbrial proteins, and cross-linked derivatives of carboxymethylcellulose are contemplated to be useful in the present invention, but are not limited thereto. It is not essential to understand the mechanism by which the present invention is practiced, and the present invention is not limited to any particular mechanism of action. In some embodiments, when a mucoadhesive is used, the use of the mucoadhesive (e.g., in a composition comprising a dimeric vaccine molecule) increases the duration and / or amount of exposure of the subject to antigenic units as compared to the duration and / or amount of exposure of the dimeric vaccine molecule in the absence of the mucoadhesive, thereby enhancing the induction of an immune response in the subject (e.g., to whom the composition of the present invention is administered).

[0116] In some embodiments, the composition of the present invention may include a sterile water preparation. Acceptable vehicles and solvents include, but are not limited to, water, Ringer's solution, phosphate buffered saline, and isotonic sodium chloride solution. Further, sterile non-volatile oil can be commonly used as a solvent or suspension solvent. For this purpose, any non-irritating non-volatile mineral oil or non-mineral oil containing synthetic mono- or diglycerides can be used. Further, in injectable preparations, fatty acids such as oleic acid can be used. Carrier formulations suitable for administration by mucosal, subcutaneous, intramuscular, intraperitoneal, intravenous, or other routes can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.

[0117] The composition containing the dimeric vaccine molecule of the present invention can be used for therapeutic purposes (e.g., to enhance the immune response) or prophylactic purposes (e.g., for immunization (e.g., to prevent the signs or symptoms of a disease)). The composition containing the dimeric vaccine molecule of the present invention can be administered to a subject via many different delivery routes and methods.

[0118] For example, the composition of the present invention can be administered to a subject (e.g., mucosal mucosa, vaginal mucosa, etc.) by a number of methods including, but not limited to, suspending in a solution and applying to a surface; suspending in a solution and spraying onto a surface using a spray applicator; mixing with a mucoadhesive and applying to a surface (e.g., mucosal surface) (e.g., spraying or rubbing); placing or immersing on a nasal and / or vaginal applicator and applying; applying by a controlled release mechanism; applying as a liposome; or applying onto a polymer.

[0119] In some embodiments, the compositions of the invention are administered mucosally (e.g., using standard techniques; e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th edition, 1995 (for mucosal delivery techniques including, for example, intranasal, pulmonary, vaginal, and rectal techniques), and European Publication No. 517,565 and Illum et al., J. Controlled Rel., 1994, 29:133-141 (for intranasal administration techniques), incorporated herein by reference in their entirety). Alternatively, the compositions of the invention can be administered dermally or transdermally using standard techniques (e.g., Remington: The Science arid Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th edition, 1995, incorporated herein by reference). The invention is not limited by the route of administration.

[0120] It is not essential to understand the mechanism for practicing the present invention, and the present invention is not limited to any specific mechanism of action. In some embodiments, mucosal vaccination is an administration route in which mucosal administration of an antigen has been shown to induce a protective immune response (e.g., mucosal immunity) at the mucosal surface, which is the entry route of many pathogens. Further, mucosal vaccination such as intranasal vaccination can induce mucosal immunity not only in the nasal mucosa but also in distant mucosal sites such as the genital mucosa (see, for example, Mestecky, Journal of Clinical Immunology, 7:265-276, 1987). In addition to inducing a mucosal immune response, mucosal vaccination also induces systemic immunity. In some embodiments, parenteral administration (e.g., intramuscular administration of a vaccine) provides an efficient and convenient method for accelerating systemic immunity (e.g., when multiple boosts are used to maintain an active systemic immunity induced by parenteral or mucosal vaccination).

[0121] In some embodiments, a composition comprising a dimeric vaccine molecule of the invention is used to protect or treat a subject who is susceptible to or suffering from a disease by means of administering the composition of the invention via a mucosal route (e.g., oral / digestive or nasal route). Alternative mucosal routes include intravaginal and intrarectal routes. In some embodiments of the invention, the nasal administration route is used, which is referred to herein as "intranasal administration" or "intranasal vaccination". Methods of intranasal vaccination are well known in the art and include administration of the vaccine in the form of droplets or sprays to the nasopharynx of the subject to be immunized. In some embodiments, a nebulized or aerosolized composition is provided. Enteric formulations such as gastric-resistant capsules for oral administration, suppositories for rectal or vaginal administration also form part of the invention. The composition of the invention can also be administered via the oral route. Under these conditions, the composition comprising the dimeric vaccine molecule can comprise pharmaceutically acceptable excipients and / or can comprise an alkaline buffer or an enteric capsule. Formulations for nasal delivery can include formulations having dextran or cyclodextran and saponin as adjuvants.

[0122] The composition of the invention can also be administered via the vaginal route. In such cases, the composition comprising the dimeric vaccine molecule can comprise pharmaceutically acceptable excipients and / or emulsifiers, polymers (e.g., CARBOPOL), and other known stabilizers for vaginal creams and suppositories. In some embodiments, the composition of the invention is administered via the rectal route. In such cases, the composition may comprise excipients and / or waxes and polymers known in the art for forming rectal suppositories.

[0123] In some embodiments, the same administration route (e.g., mucosal administration) is selected for both the primary vaccination and the booster vaccination. In some embodiments, multiple administration routes are used (e.g., simultaneously or alternatively, sequentially) to stimulate an immune response.

[0124] For example, in some embodiments, a composition comprising a dimeric vaccine is administered to the mucosal surface of a subject in either a primary vaccination or a booster vaccination dosing schedule. Alternatively, in some embodiments, the composition is administered systemically in either a primary vaccination or a booster vaccination dosing schedule. In some embodiments, a composition comprising a dimeric vaccine molecule is administered to a subject via mucosal administration in a primary vaccination dosing schedule and via systemic administration in an additional dosing schedule. In some embodiments, a composition comprising a dimeric vaccine molecule is administered to a subject via systemic administration in a primary vaccination dosing schedule and via mucosal administration in an additional dosing schedule. Exemplary systemic administration routes include, but are not limited to, oral, intramuscular, intradermal, transdermal, subcutaneous, intraperitoneal, or intravenous administration. Compositions comprising a dimeric vaccine can be used for both prophylactic and therapeutic purposes.

[0125] In some embodiments, the compositions of the invention are administered by pulmonary delivery. For example, the compositions of the invention can be delivered to the lungs of a subject (e.g., a human) via inhalation (e.g., thereby reaching the bloodstream across the lung epithelium) (see, e.g., Adjei, et al. Pharmaceutical Research 1990; 7:565-569; Adjei, et al. Int. J. Pharmaceutics 1990; 63:135-144; Braquet, et al. J. Cardiovascular Pharmacology 1989 143-146; Hubbard, et al. (1989) Annals of Internal Medicine, Vol. III, pp. 206-212; Smith, et al. J. Clin. Invest. 1989;84:1145-1146; Oswein, et al. “Aerosolization of Proteins”, 1990; Proceedings of Symposium on Respiratory Drug Delivery II Keystone, Colorado; Debs, et al. J. Immunol. 1988; 140:3482-3488; and U.S. Pat. No. 5,284,656 to Platz, et al, each of which is incorporated herein by reference in its entirety). Methods and compositions for pulmonary delivery of agents with systemic effects are described in U.S. Pat. No. 5,451,569 to Wong, et al. (incorporated herein by reference). See also U.S. Pat. No. 6,651,655 to Licalsi et al. (incorporated herein by reference in its entirety).

[0126] For use in the practice of the present invention, a wide range of mechanical devices designed for pulmonary and / or nasal mucosal delivery of pharmaceuticals are further contemplated, including, but not limited to, nebulizers, metered dose inhalers, and powder inhalers (all of which are well known to those of skill in the art). Some specific exemplary commercially available devices suitable for the practice of the present invention include the Ultravent nebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acorn II nebulizer (Marquest Medical Products, Englewood, Colo.); the Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park, N.C.); and the Spinhaler powder inhaler (Fisons Corp., Bedford, Mass.). All such devices require the use of formulations suitable for the dispensing of therapeutic agents. Typically, each formulation is specific to the type of device used and may include the use of appropriate propellant materials in addition to conventional diluents, adjuvants, surfactants, carriers, and / or other agents effective in therapy. Also contemplated is the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers.

[0127] Accordingly, in some embodiments, a composition comprising a dimeric vaccine molecule of the invention may be used to protect and / or treat a subject susceptible to or afflicted with a disease by means of administering the composition by a mucosal, intramuscular, intraperitoneal, intradermal, transdermal, pulmonary, intravenous, subcutaneous or other route of administration described herein. As methods of systemic administration of vaccine preparations, conventional syringes and needles, or devices designed for ballistic delivery of solid vaccines (e.g., see WO 99 / 27961, incorporated herein by reference), or needleless pressurized liquid jet devices (e.g., see U.S. Pat. No. 4,596,556; U.S. Pat. No. 5,993,412, each incorporated herein by reference), or transdermal patches (e.g., see WO 97 / 48440; WO 98 / 28037, each incorporated herein by reference) may be included. The invention may also be used to enhance the immunogenicity of antigens applied to the skin (transdermal or transcutaneous delivery, e.g., see WO 98 / 20734; WO 98 / 28037, each incorporated herein by reference). Accordingly, in some embodiments, the invention provides a delivery device for systemic administration pre-filled with a vaccine composition of the invention.

[0128] The present invention is not limited by the type of subject to which it is administered (e.g., for stimulating an immune response (e.g., for eliciting a protective immunity (e.g., mucosal and / or systemic immunity))). Indeed, a wide variety of different subjects are intended to benefit from administration of the compositions of the present invention. In a preferred embodiment, the subject is a human. In some embodiments, the human subject is of any age (e.g., adult, child, infant, etc.) that has been or is likely to be exposed to a microorganism (e.g., E. coli). In some embodiments, the human subject is a subject that is more likely to be directly exposed to a pathogenic microorganism or is more likely to exhibit signs and symptoms of a disease after exposure to a pathogen (e.g., an immunosuppressed subject). In some embodiments, the compositions of the present invention are administered (e.g., vaccinated) to the general public (e.g., for preventing the occurrence or spread of a disease). For example, in some embodiments, the compositions and methods of the present invention are used to vaccinate groups of people (e.g., populations of regions, cities, states, and / or countries) for their own health (e.g., for preventing or treating a disease). In some embodiments, the subject is a non-human mammal (e.g., pig, cow, goat, horse, sheep, or other livestock; or mouse, rat, rabbit, or other animal). In some embodiments, the compositions and methods of the present invention are used in a research environment (e.g., using research animals).

[0129] The compositions of the present invention can be formulated for administration by any route, such as mucosal, oral, transdermal, intranasal, parenteral, or other routes described herein. The compositions can be in any one or more different forms, including, but not limited to, tablets, capsules, powders, granules, troches, foams, creams, or liquid preparations.

[0130] The topical formulations of the present invention may be provided, for example, as ointments, creams or lotions, foams, and aerosols, and may contain suitable conventional additives such as preservatives, solvents (e.g., for assisting penetration), and emollients in ointments and creams.

[0131] The topical formulation may also contain an agent that enhances the penetration of the active ingredient through the skin. Exemplary agents include a combination of two components, N-(hydroxyethyl)pyrrolidone and a cell-envelope disordering compound, a combination of a sugar ester and a sulfoxide or phosphine oxide, and sucrose monooleate, decyl methyl sulfoxide, and alcohol.

[0132] Other exemplary substances that enhance skin penetration include polyoxyethylene sorbitan monooleate (polysorbate 80); sorbitan monooleate (span 80); p-isooctyl polyoxyethylene phenol polymer (Triton WR-1330); polyoxyethylene sorbitan trioleate (Tween 85); dioctyl sodium sulfosuccinate; and sodium sarcosinate (Sarcosyl NL-97); and surfactants or wetting agents including, but not limited to, other pharmaceutically acceptable surfactants.

[0133] In certain embodiments of the present invention, the composition may further comprise one or more alcohols, zinc-containing compounds, skin softeners, wetting agents, thickeners and / or gelling agents, neutralizing agents, and surfactants. The water used in the formulation is preferably ion-exchanged water having a neutral pH. Further additives in the topical formulation include, but are not limited to, silicone fluids, dyes, fragrances, pH adjusters, and vitamins.

[0134] The topical formulation may also include a suitable conventional carrier such as a cream or ointment base and ethanol or oleyl alcohol for lotions. Such carriers can be present from about 1% to about 98% of the formulation. The ointment base can include one or more of petrolatum, mineral oil, ceresin, lanolin alcohol, panthenol, glycerin, bisabolol, cocoa butter, and the like.

[0135] In some embodiments, the pharmaceutical composition of the present invention can be formulated and used as a form. Pharmaceutical forms include, but are not limited to, formulations such as emulsions, microemulsions, creams, jellies, and liposomes. These formulations are basically of the same nature, but differ in components and the softness of the final product.

[0136] The composition of the present invention may further contain other auxiliary components conventionally found in pharmaceutical compositions. Thus, for example, the composition may contain additional compatible pharmaceutical active substances such as, for example, antipruritics, astringents, local anesthetics, or anti-inflammatory agents, or may contain additional materials effective in physically formulating the various dosage forms of the composition of the present invention such as dyes, fragrances, preservatives, antioxidants, opacifiers, thickeners and stabilizers. However, when such materials are added, they preferably do not unduly interfere with the biological activity of the components of the composition of the present invention. The formulation can be sterilized and, if desired, mixed with adjuvants (such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts affecting osmotic pressure, buffers, colorants, fragrances and / or aromatic substances, etc.) that do not interact detrimentally with the antigenic units or other components of the formulation. In some embodiments, the immunostimulatory composition of the present invention is administered in the form of a pharmaceutically acceptable salt. When using salts, the salts should be pharmaceutically acceptable, but pharmaceutically unacceptable salts may be conveniently used to prepare their pharmaceutically acceptable salts. Such salts include, but are not limited to, salts prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Also, such salts can be prepared as alkali metal salts or alkaline earth salts such as sodium salts, potassium salts or calcium salts of carboxylic acid groups.

[0137] Suitable buffers include, but are not limited to, acetic acid and salts (1 - 2 w / v%); citric acid and salts (1 - 3 w / v%); boric acid and salts (0.5 - 2.5 w / v%); and phosphoric acid and salts (0.8 - 2 w / v%). Suitable preservatives may include benzalkonium chloride (0.003 - 0.03 w / v%); chlorobutanol (0.3 - 0.9 w / v%); parabens (0.01 - 0.25 w / v%) and thimerosal (0.004 - 0.02 w / v%).

[0138] In some embodiments, the vaccine composition is co - administered with one or more antibiotics. For example, one or more antibiotics can be administered simultaneously with, before, and / or after the administration of the composition. The present invention is not limited by the type of co - administered antibiotic. Indeed, various antibiotics including, but not limited to, β - lactam antibiotics, penicillins (natural penicillins, aminopenicillins, penicillinase - resistant penicillins, carboxypenicillins, ureidopenicillins, etc.), cephalosporins (first - generation, second - generation, and third - generation cephalosporins), and other β - lactams (imipenem, monobactams, etc.), β - lactamase inhibitors, vancomycin, aminoglycosides and spectinomycin, tetracyclines, chloramphenicol, erythromycin, lincomycin, clindamycin, rifampin, metronidazole, polymyxin, doxycycline, quinolones (e.g., ciprofloxacin), sulfonamides, trimethoprim, and quinoline can be co - administered.

[0139] Numerous antibacterial agents are currently available for use in the treatment of bacterial, fungal, and viral infections. For comprehensive academic papers on such drugs in general classes and their mechanisms of action, those skilled in the art can refer to Goodman & Gilman’s “The Pharmacological Basis of Therapeutics” Eds. Hardman et al., 9th Edition, Pub. McGraw Hill, chapters 43 through 50, 1996 (which is hereby incorporated by reference in its entirety). Generally, these agents include agents that inhibit cell wall synthesis (e.g., penicillin, cephalosporin, cycloserine, vancomycin, bacitracin); and imidazole antifungal agents (e.g., miconazole, ketoconazole, and clotrimazole); agents that directly affect the disruption of the cell membranes of microorganisms (e.g., detergents such as polymyxin and colistimethate, and the antifungal agents nystatin and amphotericin B); agents that act on ribosomal subunits to inhibit protein synthesis (e.g., chloramphenicol, tetracycline, erythromycin, and clindamycin); agents that modify protein synthesis and result in cell death (e.g., aminoglycosides); agents that affect nucleic acid metabolism (e.g., rifamycin and quinolone); antimetabolites (e.g., trimethoprim and sulfonamide); and nucleic acid analogs such as zidovudine, ganciclovir, vidarabine, and acyclovir that act to inhibit viral enzymes essential for DNA synthesis. Various combinations of antibacterial agents can be employed.

[0140] The present invention also includes a method comprising co - administration of a vaccine composition comprising a dimeric vaccine molecule with one or more additional active agents and / or immunostimulants (e.g., a composition comprising different antigenic units, antibiotics, antioxidants, etc.). Indeed, it is a further aspect of the present invention to provide a method for improving prior art immunostimulation methods (e.g., immunization methods) and / or pharmaceutical compositions by co - administering the compositions of the present invention. In the co - administration procedure, the agents can be administered simultaneously or sequentially. In one embodiment, the compositions described herein are administered prior to other active agents. The pharmaceutical formulations and modes of administration can be any of those described herein. Further, two or more co - administered agents can each be administered using different modes (e.g., routes) or different formulations. The additional co - administered agents (e.g., antibiotics, adjuvants, etc.) can be any of the agents well - known in the art, including but not limited to those currently in clinical use.

[0141] In some embodiments, a composition comprising a dimeric vaccine molecule is administered to a subject via two or more routes. For example, a subject for whom it would be beneficial to have a protective immune response (e.g., immunity) against a pathogenic microorganism may receive mucosal administration (e.g., nasal administration or other mucosal routes described herein), and further, it may be beneficial to receive one or more other routes of administration (e.g., parenteral or pulmonary administration (e.g., via a nebulizer, inhaler, or other methods described herein)). In some preferred embodiments, administration via the mucosal route is sufficient to induce both mucosal and systemic immunity to the antigenic unit or the organism from which the antigenic unit is derived. In other embodiments, administration via multiple routes serves to provide both mucosal and systemic immunity. Thus, understanding the mechanism is not essential for practicing the invention, and the invention is not limited to any particular mechanism of action. In some embodiments, a subject to whom the composition of the invention is administered via multiple routes of administration (e.g., immunization (e.g., mucosal administration of the composition as well as airway or parenteral administration)) may be thought to have a stronger immune response to the antigenic unit than a subject to whom the composition is administered via only one route.

[0142] Other delivery systems may include time-release, delayed-release, or sustained-release delivery systems. Such systems can avoid repeated dosing of the composition and increase convenience for the subject and the physician. Many types of release delivery systems are available and are known to those of skill in the art. They include polymeric systems such as poly(lactide-glycolide), copolioxalate, polycaprolactone, polyesteramide, polyorthoester, polyhydroxybutyrate, and polyanhydride. Microcapsules of the aforementioned polymers containing a drug are described, for example, in U.S. Pat. No. 5,075,109 (incorporated herein by reference). Delivery systems also include lipids such as cholesterol, cholesterol esters and fatty acids, or sterols such as mono-glycerides, di-glycerides and tri-glycerides; hydrogel release systems; sylastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; non-polymeric systems such as partial fusion implants, etc. Specific examples include (a) an erosion system in which the agent of the present invention is contained in a matrix as described in U.S. Pat. Nos. 4,452,775, 4,675,189, and 5,736,152 (each of these documents is incorporated herein by reference), and (b) a diffusion system in which the active ingredient permeates from a polymer at a controlled rate as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686 (these documents are incorporated herein by reference), but are not limited thereto. Furthermore, pump system hardware delivery systems can be used, some of which are compatible with implantation.

[0143] In some embodiments, the vaccine composition of the invention is formulated in a concentrated dosage that can be diluted prior to administration to a subject. For example, a dilution of the concentrated composition may be administered to the subject such that any one or more of the specific dosages provided herein are dosed to the subject. In some embodiments, the dilution of the concentrated composition can be prepared (e.g., in a single dose) such that a composition containing 0.5-50% nanoemulsion and antigenic units in the concentrated composition is administered. The concentrated composition is believed to be useful in settings where a large number of subjects can be administered the composition of the invention (e.g., immunization in a clinic, hospital, school, etc.). The composition (e.g., the concentrated composition) containing the dimeric vaccine molecule of the invention is stable for at least one week at room temperature in some embodiments, at least two weeks in some embodiments, at least three weeks in some embodiments, at least four weeks in some embodiments, at least five weeks in some embodiments, and at least six weeks in some embodiments.

[0144] In some embodiments, following the first administration (e.g., the first vaccination) of the composition of the invention, one or more booster immunizations may be administered to the subject (e.g., about 2 weeks later, about 3 weeks later, about 4 weeks later, about 5 weeks later, about 6 weeks later, about 7 weeks later, about 8 weeks later, about 10 weeks later, about 3 months later, about 4 months later, about 6 months later, about 9 months later, about 1 year later, about 2 years later, about 3 years later, about 5 years later, about 10 years later) subsequent to the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, and / or 11th and subsequent administrations. Understanding the mechanism is not essential for practicing the invention, and the invention is not limited to any particular mechanism of action. In some embodiments, reintroducing the antigenic unit by booster immunization enables a strong systemic immunity in the subject. The booster immunization may use the same formulation given for the primary immune response, or a different formulation containing the antigenic unit or the minimal immunogenic epitope. The dosing regimen is also determined at least in part by the needs of the subject and depends on the physician's judgment.

[0145] The unit of dosage may vary proportionally based on several factors including, but not limited to, the weight, age, and health status of the subject. Further, the unit of dosage may vary in subsequent administrations (e.g., booster immunizations).

[0146] The compositions and methods of the present invention may be used in a variety of environments including research settings. For example, the compositions and methods of the present invention may also be used in the study of the immune system (e.g., adaptive immune responses (e.g., defensive immune responses (e.g., mucosal or systemic immunity))). The use of the compositions and methods provided by the present invention encompasses human and non-human subjects as well as samples from these subjects, and also encompasses research applications using these subjects. The compositions and methods of the present invention are also useful for studying and optimizing albumin variants, antigenic units, and other components, as well as screening for novel components. Accordingly, the present invention is not limited to any particular subject matter and / or application environment.

[0147] The present invention further provides a kit comprising the vaccine composition included herein. In some embodiments, the kit includes all of the components necessary, sufficient, or useful for administering the vaccine. For example, in some embodiments, the kit includes a device for administering the vaccine (e.g., a needle or other injection device), a temperature control member (e.g., a refrigerator or other cooling member), a sanitary member (e.g., an alcohol swab for sanitizing the injection site), and instructions for administering the vaccine.

[0148] The present invention can also be configured as follows.

[0149] 〔1〕 A DNA vaccine comprising a first nucleic acid construct and a second nucleic acid construct, wherein the first nucleic acid construct and the second nucleic acid construct encode a first fusion protein and a second fusion protein, The first fusion protein and the second fusion protein include a targeting unit, a heterodimerization unit, and an antigenic unit that are operably linked. The antigenic unit in each of the first fusion protein and the second fusion protein is a variant antigen target protein. When the first nucleic acid construct and the second nucleic acid construct are introduced into a cell, the first fusion protein and the second fusion protein are expressed, and a first heterodimer protein is formed through the association of the heterodimerization unit, a DNA vaccine.

[0150] [2] The heterodimerization unit in one of the first nucleic acid construct and the second nucleic acid construct is an ACID heterodimerization unit, and the heterodimerization unit in the other of the first nucleic acid construct and the second nucleic acid construct is a BASE heterodimerization unit that interacts to form an ACID / BASE heterodimerization domain that is the first heterodimer protein, or The heterodimerization unit in one of the first nucleic acid construct and the second nucleic acid construct is a barstar heterodimerization unit, and the heterodimerization unit in the other of the first nucleic acid construct and the second nucleic acid construct is a barnase heterodimerization unit that interacts to form a barstar / barnase heterodimerization domain that is the first heterodimer protein, or When the first nucleic acid construct and the second nucleic acid construct are expressed intracellularly, the production of the heterodimer protein is characterized by the substantial absence of the production of a homodimer protein containing the same antigen target protein, or The sequence encoding the fusion protein is operably linked to a promoter, the DNA vaccine of [1].

[0151] [3] The targeting units of the above-mentioned first fusion protein and the above-mentioned second fusion protein are the same, or, The targeting units of the above-mentioned first fusion protein and the above-mentioned second fusion protein are different, the DNA vaccine of [1].

[0152] 〔4〕 The targeting unit is an antigen-binding protein, preferably, the antigen-binding protein is an scFv, or, The targeting unit is an antigen-presenting cell (APC) targeting unit, preferably, the APC targeting unit binds to a target selected from the group consisting of MHC-II molecules, CD40, CD11c, CD14, HLA-DP, Toll-like receptors and chemokine receptors, the DNA vaccine of [1].

[0153] 〔5〕 The variant antigen target protein has a sequence identity of about 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 95%, or among the variant antigen target proteins having a sequence identity of about 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 95%, it has a conserved domain with a length exceeding 8, 10, 12, 15, 20, 30, 40, 50 or 60 amino acids and a maximum length of 100 - 200 amino acids, the DNA vaccine of [1].

[0154] 〔6〕 The different variant antigen target proteins are derived from different strains or serotypes of an organism, preferably, the organism is a pathogenic organism, or, The organism is selected from the group consisting of viruses, bacteria, fungi and protozoa, the DNA vaccine of [1].

[0155] 〔7〕 The different variant antigen target proteins are variants of hemagglutinin (HA). Preferably, the vaccine comprises variants of HA from at least 3, 4, 5 or 6 and up to 12 or 18 strains or serotypes of influenza virus. Preferably, the influenza virus is selected from the group consisting of influenza viruses of group 1 and influenza viruses of group 2. Preferably, the influenza virus of group 1 is selected from the group consisting of H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17 and H18, and the influenza virus of group 2 is selected from the group consisting of H3, H4, H7, H10, H14 and H15. The DNA vaccine of [1].

[0156] [8] The different variant antigen target proteins are variants of cancer antigens or neoepitopes. The DNA vaccine of [1].

[0157] [9] A DNA vaccine further comprising at least a third nucleic acid construct and a fourth nucleic acid construct, The third nucleic acid construct and the fourth nucleic acid construct encode a third fusion protein and a fourth fusion protein, The third fusion protein and the fourth fusion protein comprise a targeting unit, a heterodimerization unit and an antigenic unit that are operably linked, The antigenic unit in each of the third fusion protein and the fourth fusion protein is a variant antigen target protein, Preferably, the expression of the third fusion protein and the fourth fusion protein in cells using the first fusion protein and the second fusion protein produces a mixture of heterodimeric proteins. Preferably, when the first nucleic acid construct, the second nucleic acid construct, and at least the third nucleic acid construct and the fourth nucleic acid construct are expressed in cells, the production of the heterodimeric protein is characterized in that there is substantially no production of homodimeric proteins containing the same antigen target protein. The DNA vaccine of [1].

[0158] 〔10〕 A vaccine composition comprising a heterodimeric protein molecule, The heterodimeric protein molecule comprises a first fusion protein monomer and a second fusion protein monomer, The first fusion protein monomer and the second fusion protein monomer comprise an operably linked targeting unit, heterodimerization unit, and antigenic unit, The antigenic units in each of the first fusion protein monomer and the second fusion protein monomer differ by encoding different variant antigen target proteins, The heterodimeric protein molecule comprises two monomers linked by the association of the dimerization domains, The heterodimeric protein molecule is a vaccine composition encoded by any of the DNA vaccines of [1] to [9].

[0159] 〔11〕 A pharmaceutical preparation comprising any of the DNA vaccines of [1] to [9] or the vaccine composition of

[10] and a pharmaceutically acceptable carrier.

[0160] 〔12〕 A vaccine preparation comprising the vaccine composition of

[10] and an adjuvant.

[0161] 〔13〕 For use in conferring immunity or inducing an immune response against a variant antigen target protein in a subject, comprising administering to the subject a DNA vaccine, vaccine composition, pharmaceutical preparation or vaccine preparation, preferably further comprising a second administration of the DNA vaccine, vaccine composition, pharmaceutical preparation or vaccine preparation to the subject, or further comprising administering to the subject a second DNA vaccine, vaccine composition, pharmaceutical preparation or vaccine preparation comprising one or more non-target variant antigen target protein subunits; For use in conferring immunity or inducing an immune response in a subject in need of a DNA vaccine, vaccine composition, pharmaceutical preparation or vaccine preparation; As a vaccine for preventing or treating infection by a pathogen, or As a vaccine for preventing or treating cancer, a DNA vaccine according to any one of [1] to [9], a vaccine composition according to

[10] , a pharmaceutical preparation according to

[11] , or a vaccine preparation according to

[12] .

Examples

[0162] 〔Example 1. Protection against MOPC315 myeloma in BALB / c mice by a novel heterodimer DNA vaccine〕 DNA vaccines encoding fusion proteins that target antigens to antigen-presenting cells (APCs) have a high ability to induce immune responses. It would be useful to extend this technology to vaccine molecules that can simultaneously target several different surface molecules on APCs and deliver several different antigens. In this example, the production of heterodimer vaccine molecules that can each express four different fusion moieties is described.

[0163] Two different types of heterodimers were synthesized. The first utilized the barnase-barstar protein pair, and the second utilized a modified leucine-zipper α-helix (referred to as the ACID / BASE motif). See FIGS. 1B-D. The heterodimers have a targeting motif fused to the N-terminus and an antigen fused to the C-terminus. These can be compared to a vaccibody vaccine molecule that uses the CH3 Ig domain from human IgG3 as a dimerization motif. See FIG. 1A.

[0164] Exemplary sequences of the constructs and antigenic units of the present invention are provided below.

[0165] Multiple different experiments were conducted to analyze both heterodimer formations and compare the ability of the heterodimers to elicit an immune response. The heterodimers were specifically compared to the vaccine bodies in the homodimeric form. The levels of vaccine proteins produced in vitro were similar in the in vitro ACID / BASE heterodimers ACID / BASE and vaccine body constructs. See Figure 2. Briefly, various heterodimer and homodimer plasmids were transiently transfected into Hek293E cells, the supernatants were collected and analyzed by ELISA. The levels of proteins secreted from the cells were measured by DNP-BSA, an antigen bound by both scFvM315(F) and 9A8, mAbs that recognize both λ1 and λ2 present in both anti-NIP and scFvM315. The results are shown in Figure 2. The ability of the ACID / BASE dimerization domain to form heterodimers was analyzed. See Figure 3. Again, various ACID / BASE plasmid pairs and single plasmids were transiently transfected into Hek293E cells, the supernatants were collected and analyzed by ELISA. The results indicate that the antigen and targeting unit can be equally expressed on the ACID or BASE monomers. Furthermore, the monomeric ACID and BASE fusion proteins can be secreted in vitro. A number of different antigens can be used to produce the vaccine molecules. See Figure 4 showing the results of the analysis of various ACID / BASE heterodimers in vitro. Plasmids encoding heterodimers containing antigens of tuberculosis (E and 85b), influenza (HA), myeloma antigen (F = M315), and model antigens such as OVA and mCherry were transiently transfected into Hek293E cells, the supernatants were collected and analyzed by ELISA. The ability to retain the chemotactic activity of MIP1α when used as the targeting unit of the heterodimer was analyzed. As shown in Figures 5a and 5b, supernatants from Hek293E cells transiently transfected with various vaccine molecules attract CCR1+ and CCR5+ ESb-MP cells through a transwell filter.

[0166] The vaccine molecules were also used in in vivo experiments. DNA vaccines were administered to the muscles of mice, followed by electroporation to increase DNA uptake and the production of vaccine proteins secreted into the serum. Figure 6 shows the results of the M315-specific immune response in mice immunized once i.m. with 50 μg of M315 and protein boost immunization on day 62. When compared with the normal vaccine body, the IgG1 response is slightly slower than that of the vaccine body, but IgG2a is slightly improved compared with the vaccine body vaccine. As shown in Figure 7, the immune response can be induced against MOPC315 tumor cells. Ten mice per group were inoculated with 50 μg of DNA per plasmid, and 100,000 MOPC315 tumor cells were administered as antigen 14 days later.

[0167] As can be seen from the above data, barnase-barstar heterodimers and ACID / BASE heterodimers are efficiently expressed in vitro and in vivo using fusion proteins conjugated to the N-terminus and C-terminus. Since various targeting units and antigenic units were expressed as fusion proteins in the heterodimer in vitro, both heterodimers were flexible. Finally, MIP-1α targeting induced antigen-specific IgG1 and IgG2a responses with ACID / BASE heterodimers even better than the homodimer vaccine body. Furthermore, the ACID / BASE heterodimer protected mice from MOPC315 myeloma tumors, and two MIP-1α targeting units were essential for protection.

[0168] These results indicate that heterodimer vaccine molecules provide a flexible basis for developing novel DNA vaccines with high efficacy.

[0169] 〔Sequences of exemplary constructs〕 [MIP1α-2ACID-M315 DNA sequence with intron (SEQ ID NO: 1)] MIP1α derived from nucleotides: 050 - 256 Hinge: 666 - 716 ACID - ACID with linker: 717 - 1022 Linker: 1023 - 1037 M315 antigen: 1038 - 1766 001 GGGTGACAAT GACATCCACT TTGCCTTTCT CTCCACAGGT GTGCATTCCG 051 CGCCATATGG AGCTGACACC CCGACTGCCT GCTGCTTCTC CTACAGCCGG 101 AAGATTCCAC GCCAATTCAT CGTTGACTAT TTTGAAACCA GCAGCCTTTG 151 CTCCCAGCCA GGTGTCATTT TCCTGACTAA GAGAAACCGG CAGATCTGCG 201 CTGACTCCAA AGAGACCTGG GTCCAAGAAT ACATCACTGA CCTGGAACTG 251 AACGCTGGTG AGTCGTACGC TAGCAAGCTT GGCCAGCGCA GGGAGGGAGG 301 GTGTCTGCTG GAAGCCAGGC TCAGCCCTCC TGCCTGGACG CATCCCGGCT 351 GTGCAGTCCC AGCCCAGGGC ACCAAGGCAG GCCCCGTCTG ACTCCTCACC 401 CGGAGGCCTC TGCCCGCCCC ACTCATGCTC AGGGAGAGGG TCTTCTGGCT 451 TTTTCCACCA GGCTCCGGGC AGGCACAGGC TGGATGCCCC TACCCCAGGC 501 CCTTCACACA CAGGGGCAGG TGCTGCGCTC AGAGCTGCCA AAAGCCATAT 551 CCAGGAGGAC CCTGCCCCTG ACCTAAGCCC ACCCCAAAGG CCAAACTCTC 601 TACTCACTCA GCTCAGACAC CTTCTCTCTT CCCAGATCTG AGTAACTCCC 651 AATCTTCTCT CTGCAGAGCT CAAAACCCCA CTTGGTGACA CAACTCACAC 701 ATGCCCACGG TGCCCAGGAG GTAGCAGCGG TGGAAAATTC GGCGGTTCCA 751 CTACAGCTCC ATCAGCTCAG CTCGAAAAAG AGCTCCAGGC CCTGGAGAAG 801 GAAAATGCAC AGCTGGAATG GGAGTTGCAA GCACTGGAAA AGGAACTGGC 851 TCAGGGAGGT GGTAGCGGAG GGTTAACCAA ATTCGGCGGT TCCACTACAG 901 CTCCATCAGC TCAGCTCGAA AAAGAGCTCC AGGCCCTGGA GAAGGAAAAT 951 GCACAGCTGG AATGGGAGTT GCAAGCACTG GAAAAGGAAC TGGCTCAGGG 1001 AGGTGGTAGC GGAGGGTTAA CCGGCCTCAG CGGCCTGGAT GTACAGCTTC 1051 AGGAGTCAGG ACCTGGCCTC GTGAAACCTT CTCAGTCTCT GTCTCTCACC 1101 TGCTCTGTCA CTGGCTACTC CATCACCAGT GGGTATTTCT GGAACTGGAT 1151 ACGGCAGTTT CCAGGAAACA AACTGGAATG GTTGGGCTTC ATAAAGTACG 1201 ACGGTAGCAA TGGCTACAAT CCATCTCTCA AAAATCGAGT TTCCATCACT 1251 CGTGACACAT CTGAGAACCA GTTTTTCCTG AAGTTGAATT CTGTGACTAC 1301 TGAGGACACA GCTACATATT ACTGTGCCGG AGATAATGAT CACCTCTACT 1351 ACTTTGACTA CTGGGGCCAA GGCACCACTC TCACAGTCTC CTCAGGTGGA 1401 GGCGGATCTG GCGGAGGTGG CTCTGGCGGT GGCGGATCGC AGGCTGTTGT 1451 GACTCAGGAA TCTGCACTCA CCACATCACC TGGTGGAACA GTCATACTCA 1501 CTTGTCGCTC AAGTACTGGG GCTGTTACAA CTAGTAACTA TGCCAACTGG 1551 ATACAAGAAA AACCAGATCA TTTATTCACT GGTCTAATCG GTGGTACCAG 1601 CAACCGAGCT CCAGGTGTTC CTGTCAGATT CTCAGGCTCC CTGATTGGAG 1651 ACAAGGCTGC CCTCACCATC ACAGGGGCAC AGACTGAGGA TGATGCAATG 1701 TATTTCTGTG CTCTATGGTT CAGAAACCAT TTTGTTTTCG GCGGTGGAAC 1751 CAAGGTCACT GTCCTATGAG GCCTGCAGGG CCGGTCCGTC GACTCTAGAG [MIP1α-2ACID-M315 DNA sequence with intron (SEQ ID NO: 2)] [MIP1α-2ACID-M315 amino acid sequence (SEQ ID NO: 3)] MIP1α derived from amino acid: 7 - 75 Hinge; 76 - 92 ACID - ACID with linker: 93 - 194 Linker: 195 - 199 M315 antigen: 200 - 442 1 STGVHSAPYG ADTPTACCFS YSRKIPRQFI VDYFETSSLC SQPGVIFLTK RNRQICADSK 61 ETWVQEYITD LELNAELKTP LGDTTHTCPR CPGGSSGGKF GGSTTAPSAQ LEKELQALEK 121 ENAQLEWELQ ALEKELAQGG GSGGLTKFGG STTAPSAQLE KELQALEKEN AQLEWELQAL 181 EKELAQGGGS GGLTGLSGLD VQLQESGPGL VKPSQSLSLT CSVTGYSITS GYFWNWIRQF 241 PGNKLEWLGF IKYDGSNGYN PSLKNRVSIT RDTSENQFFL KLNSVTTEDT ATYYCAGDND 301 HLYYFDYWGQ GTTLTVSSGG GGSGGGGSGG GGSQAVVTQE SALTTSPGGT VILTCRSSTG 361 AVTTSNYANW IQEKPDHLFT GLIGGTSNRA PGVPVRFSGS LIGDKAALTI TGAQTEDDAM 421 YFCALWFRNH FVFGGGTKVT VL * [MIP1α-2BASE-M315 DNA sequence with intron (SEQ ID NO: 4)] MIP1α derived from nucleotide: 050 - 256 Hinge: 666 - 716 BASE-BASE with a linker: 717 - 1022 Linker: 1023 - 1037 M315 antigen: 1038 - 1766 001 GGGTGACAAT GACATCCACT TTGCCTTTCT CTCCACAGGT GTGCATTCCG 051 CGCCATATGG AGCTGACACC CCGACTGCCT GCTGCTTCTC CTACAGCCGG 101 AAGATTCCAC GCCAATTCAT CGTTGACTAT TTTGAAACCA GCAGCCTTTG 151 CTCCCAGCCA GGTGTCATTT TCCTGACTAA GAGAAACCGG CAGATCTGCG 201 CTGACTCCAA AGAGACCTGG GTCCAAGAAT ACATCACTGA CCTGGAACTG 251 AACGCTGGTG AGTCGTACGC TAGCAAGCTT GGCCAGCGCA GGGAGGGAGG 301 GTGTCTGCTG GAAGCCAGGC TCAGCCCTCC TGCCTGGACG CATCCCGGCT 351 GTGCAGTCCC AGCCCAGGGC ACCAAGGCAG GCCCCGTCTG ACTCCTCACC 401 CGGAGGCCTC TGCCCGCCCC ACTCATGCTC AGGGAGAGGG TCTTCTGGCT 451 TTTTCCACCA GGCTCCGGGC AGGCACAGGC TGGATGCCCC TACCCCAGGC 501 CCTTCACACA CAGGGGCAGG TGCTGCGCTC AGAGCTGCCA AAAGCCATAT 551 CCAGGAGGAC CCTGCCCCTG ACCTAAGCCC ACCCCAAAGG CCAAACTCTC 601 TACTCACTCA GCTCAGACAC CTTCTCTCTT CCCAGATCTG AGTAACTCCC 651 AATCTTCTCT CTGCAGAGCT CAAAACCCCA CTTGGTGACA CAACTCACAC 701 ATGCCCACGG TGCCCAGGAG GTAGCAGCGG TGGAAAATTC GGCGGTTCCA 751 CTACAGCTCC ATCAGCTCAG TTGAAAAAGA AATTGCAAGC ACTGAAGAAA 801 AAGAACGCTC AGCTGAAGTG GAAACTTCAA GCCCTCAAGA AGAAACTCGC 851 CCAGGGAGGT GGTAGCGGAG GGTTAACCAA ATTCGGCGGT TCCACTACAG 901 CTCCATCAGC TCAGTTGAAA AAGAAATTGC AAGCACTGAA GAAAAAGAAC 951 GCTCAGCTGA AGTGGAAACT TCAAGCCCTC AAGAAGAAAC TCGCCCAGGG 1001 AGGTGGTAGC GGAGGGTTAA CCGGCCTCAG CGGCCTGGAT GTACAGCTTC 1051 AGGAGTCAGG ACCTGGCCTC GTGAAACCTT CTCAGTCTCT GTCTCTCACC 1101 TGCTCTGTCA CTGGCTACTC CATCACCAGT GGGTATTTCT GGAACTGGAT 1151 ACGGCAGTTT CCAGGAAACA AACTGGAATG GTTGGGCTTC ATAAAGTACG 1201 ACGGTAGCAA TGGCTACAAT CCATCTCTCA AAAATCGAGT TTCCATCACT 1251 CGTGACACAT CTGAGAACCA GTTTTTCCTG AAGTTGAATT CTGTGACTAC 1301 TGAGGACACA GCTACATATT ACTGTGCCGG AGATAATGAT CACCTCTACT 1351 ACTTTGACTA CTGGGGCCAA GGCACCACTC TCACAGTCTC CTCAGGTGGA 1401 GGCGGATCTG GCGGAGGTGG CTCTGGCGGT GGCGGATCGC AGGCTGTTGT 1451 GACTCAGGAA TCTGCACTCA CCACATCACC TGGTGGAACA GTCATACTCA 1501 CTTGTCGCTC AAGTACTGGG GCTGTTACAA CTAGTAACTA TGCCAACTGG 1551 ATACAAGAAA AACCAGATCA TTTATTCACT GGTCTAATCG GTGGTACCAG 1601 CAACCGAGCT CCAGGTGTTC CTGTCAGATT CTCAGGCTCC CTGATTGGAG 1651 ACAAGGCTGC CCTCACCATC ACAGGGGCAC AGACTGAGGA TGATGCAATG 1701 TATTTCTGTG CTCTATGGTT CAGAAACCAT TTTGTTTTCG GCGGTGGAAC 1751 CAAGGTCACT GTCCTATGAG GCCTGCAGGG CCGGTCCGTC GACTCTAGAG [MIP1α-2BASE-M315 DNA sequence without intron (SEQ ID NO: 5)] [MIP1α-2BASE-M315 amino acid sequence (SEQ ID NO: 6)] MIP1α derived from amino acids: 4 - 72 Hinge: 73 - 89 ACID-ACID with linker: 90 - 191 Linker: 192 - 196 M315 antigen: 197 - 439 1 VHSAPYGADT PTACCFSYSR KIPRQFIVDY FETSSLCSQP GVIFLTKRNR 61 QICADSKETW 121 VQEYITDLEL NAELKTPLGD TTHTCPRCPG GSSGGKFGGS TTAPSAQLKK 181 KLQALKKKNA 241 QLKWKLQALK KKLAQGGGSG GLTKFGGSTT APSAQLKKKL QALKKKNAQL 301 KWKLQALKKK 361 LAQGGGSGGL TGLSGLDVQL QESGPGLVKP SQSLSLTCSV TGYSITSGYF 421 WNWIRQFPGN KLEWLGFIKY DGSNGYNPSL KNRVSITRDT SENQFFLKLN SVTTEDTATY YCAGDNDHLY YFDYWGQGTT LTVSSGGGGS GGGGSGGGGS QAVVTQESAL TTSPGGTVIL TCRSSTGAVT TSNYANWIQE KPDHLFTGLI GGTSNRAPGV PVRFSGSLIG DKAALTITGA QTEDDAMYFC ALWFRNHFVF GGGTKVTVL * [Sequences of multiple myeloma antigen scFv315 used for heterodimers] SEQ ID NO: 7. Amino acid sequence: VH - linker - VL DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYFWNWIRQFPGNKLEWLGFIKYDGSNGYNPSLKNRVSITRDTSENQFFLKLNVTTEDTATYYCAGDNDHLYYFDYWGQGTTLTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPGGTVILTCRSSTGAVTTSNYANWIQEKPDHLFTGLIGGTSNRAPGVPVRFSGSLIGDKAALTITGAQTEDDAMYFCALWFRNHFVFGGGTKVTVL Sequence number: 8. DNA: VH - linker - VL GATGTACAGCTTCAGGAGTCAGGACCTGGCCTCGTGAAACCTTCTCAGTCTCTGTCTCTCACCTGCTCTGTCACTGGCTACTCCATCACCAGTGGGTATTTCTGGAACTGGATACGGCAGTTTCCAGGAAACAAACTGGAATGGTTGGGCTTCATAAAATACGACGGTAGCAATGGCTACAATCCATCTCTCAAAAATCGAGTTTCCATCACTCGTGACACATCTGAGAACCAGTTTTTCCTGAAGTTGAATTCTGTGACTACTGAGGACACAGCTACATATTACTGTGCCGGAGATAATGATCACCTCTACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGGTGGAGGCGGATCTGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGGAACAGTCATACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTATGCCAACTGGATACAAGAAAAACCAGATCATTTATTCACTGGTCTAATCGGTGGTACCAGCAACCGAGCTCCAGGTGTTCCTGTCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGATGCAATGTATTTCTGTGCTCTATGGTTCAGAAACCATTTTGTTTTCGGCGGTGGAACCAAGGTCACTGTCCTATGAG [Sequence of lymphoma antigen scFvA20 used for heterodimer] Underline the linker before VH and represent the linker between VH and VL in bold.

[0170] SEQ ID NO: 9 for the DNA sequence. SEQ ID NO: 10 for the amino acid sequence.

[0171]

Table 1

[0172] JPEG0007696169000002.jpg100169

[0173] [Amino acid sequence of A20 antigen: VHA20-VLA20] Sequence number: 11. VH-Linker-VL MVQLQQSGPDLVKPGMSVKLSCKTLGYNFSDKWIHWIKQKPGRGLEWVGRIDPSNGDTDYNADFKTPATLTVDRPSNTAYLELNNLTSGDSAVYYCSISGDYSACDYWGQGTELTVSSGGGGSGGGGSGGGGSDVVMTQTPLSLAVSLGDHVKMSCRCNQSLVNSHGDSFLHWFLQKPGQSPKLLIYKVSSRFFGVPERFSGSGSGTDFTLEISRVEAEDLGIYFCSQGAHVPWTFGGGTKLEVK [Example 2. Increase in B cell-mediated response due to the bivalency of the target DNA vaccine] Multivalent antigens can induce cross-linking between BCRs on the B cell membrane, which is thought to be essential for B cell activation. Initial B cell activation models have shown that these cross-links are necessary for activating B cells (16, 17). Other models for efficient B cell activation have been recently shown, each of which incorporates valency as an important factor (17 - 20). In both in vitro and in vivo models, multivalent antigens induce more potent B cell signaling and antibody responses, although monovalent antigens have also been shown to be able to activate B cell responses (21 - 28).

[0174] DNA vaccines have several advantages over conventional vaccines, but their immunogenicity in large animals is poor (29, 30). One efficient way to improve the immune response during DNA vaccination is to directly target antigens to APCs by genetically fusing the antigen to a single-chain variable region fragment specific for a cytokine or a molecule on the APC surface (31 - 40).

[0175] In this example, the values of the antigen valence numbers in an environment of target vaccines having different antigens are demonstrated. Monovalent and divalent DNA vaccines that target MHCII on antigen-presenting cells were synthesized. These vaccines encode an asymmetric heterodimer having two identical antigens in the divalent vaccine, or a heterodimer having two different antigens in the monovalent vaccine. Exemplary antigens used in this example are HA from two different influenza strains A / PR / 8 / 34 and A / California / 07 / 09, and single-chain Fv derived from tumor-specific immunoglobulins secreted by mouse B cell lymphomas MOPC315 and A20. The results indicate that the divalency of the antigen is an important vaccine property in the induction of an efficient B cell response.

[0176] Data are shown in FIGS. 8-12. FIGS. 8A-E provide data regarding the design and characteristics of the heterodimer vaccine molecule. Schematic diagrams of the heterodimer molecule and constructs are provided. FIG. 8A. The sequence alignment of the PR8 and Cal07 HA sequences is shown in FIG. 15. In these exemplary vaccine constructs, hemagglutinin (HA) from influenza H1N1 virus PR8 or Cal07 is a target of antigen-presenting cells having a single-chain FV specific for mouse MHC class II or the hapten NIP as non-binding control. The heterodimer is formed through acid / base interactions between two alpha helices of a leucine zipper motif rich in either acidic or basic amino acids. The targeting unit and the antigenic unit are arranged at either end of either the ACID dimerization unit or the BASE dimerization unit. The DNA cassette is expressed under the CMV promoter and contains the leader sequence of the pLNOH2 vector, designated as L. Vectors having the ACID and BASE constructs are co-transfected to express the heterodimer vaccine protein. FIG. 8B. HEK293E cells are co-transfected with the ACID and BASE constructs, and the supernatant is analyzed by sandwich ELISA. The antigen arm and the dimerization unit are detected with anti-HA (PR8) and anti-HA (Cal07) mAbs, as well as an mAb specific for the ACID / BASE motif (2H11) (26). FIG. 8C. The vaccine heterodimer in the supernatant is detected by Western blot using biotinylated 2H11. FIG. 8D. The molecular size is shown. Fibroblasts transfected with mouse MHCII (I-Eκ and I-Ed) are incubated with either a non-targeted heterodimer vaccine protein containing two HAs of PR8 or an MHCII-targeted heterodimer vaccine protein, either undiluted or at dilutions of 1-3 and 1-9 in cell culture medium. FIG. 8E. The bound vaccine protein is stained with biotinylated αHA(PR8) and detected by streptavidin-PE by flow cytometry.

[0177] Figures 9A-I show data demonstrating that the bivalency of the heterodimeric vaccine molecule increases IgG titers in mice. Balb / c mice were vaccinated intramuscularly with 10 μg of DNA plasmids (5 μg each of the ACID and BASE plasmids), followed by electroporation (n = 6 / group). See Figures 9A-F. IgG, IgG1, and IgG2A responses specific for HA(PR8) (A-C) and HA(Cal07) (D-F) were analyzed by ELISA. (G-H) Balb / c mice were vaccinated intradermally at the flank with 50 μg of DNA (25 μg each of the ACID and BASE plasmids), followed immediately by electroporation (n = 6 / group). The total IgG responses against HA(PR8) (Figure 9G) and HA(Cal07) (Figure 9H) were analyzed. Various DNA administrations of bivalent or monovalent vaccines with the HA(PR8) antigen were vaccinated into Balb / c mice by i.m. injection. See Figure 9I. At week 6, the total IgG specific for HA(PR8) was analyzed. The titer was defined as the maximum serum dilution that yielded an absorbance above background. The background absorbance was determined as twice the mean absorbance of the signal from the sera of mice vaccinated with NaCl. Mean titers ± standard error are shown. Significance of the bivalent vaccine over the monovalent vaccine is shown. * P<0.05; ** P<0.01. Mann-Whitney.

[0178] Figures 10A-E provide data showing antibody titers after vaccination with the heterodimeric anti-tumor vaccine molecule of the present invention. A heterodimeric DNA vaccine was designed to have an anti-tumor antigen. Figure 10A. The tumor antigen was a tumor-specific immunoglobulin secreted by mouse B cell lymphoma MOPC315 (annotated M315) and A20. One vaccine was generated to have a stop codon and formed a monovalent vaccine with M315. Balb / c mice were vaccinated with the heterodimeric anti-tumor vaccine. The mice were vaccinated by intramuscular injection with 100 μg of DNA plasmid (50 μg each of ACID and BASE plasmid) (n = 6 / group), followed by electroporation. IgG1 and IgG2a responses specific for the A20 (Figures 10B-C) and M315 (Figures 10D-E) tumor antigens were analyzed by ELISA. The titer was defined as the maximum serum dilution that gave an absorbance above background. The background absorbance was determined as twice the mean absorbance of the signal from the sera of mice vaccinated with NaCl. Mean titers ± standard error are shown. Significance is shown to compare the bivalent to the monovalent vaccine. * P < 0.05; ** P < 0.01; Mann-Whitney.

[0179] Figures 11A - F provide data showing that a bivalent DNA vaccine completely protects mice from homologous H1N1 influenza infection. Balb / c mice were vaccinated intramuscularly with 100 μg of DNA plasmid (50 μg each of ACID and BASE plasmids), followed by electroporation (n = 6 / group). Fourteen days later, the mice were challenged with influenza virus derived from the PR8 strain using 5×LD50 (Figures 11A and B) or 50×LD50 (Figures 11C and D). Balb / C mice were vaccinated as described above (n = 6 / group). From day 12, αCD4 and αCD8 mAb were injected intraperitoneally every two days. The control group received isotype-matched mAb after vaccination with αMHCII-Cal07 / Cal07 (indicated by the blue open circles). Fourteen days after vaccination, the mice were challenged with PR8 influenza virus (5×LD50). For each experiment, the mean weight loss ± standard error and survival rate after infection are shown. Figures 11A - E. Significance is shown to compare the bivalent vaccine with the monovalent vaccine. * P < 0.05; ** P < 0.01; *** P < 0.001 Mann - Whitney and Mantel Cox.

[0180] Figures 12A-C provide data showing that the bivalency of the heterodimeric vaccine molecule increases antigen-specific germinal centers and B cell populations in the bone marrow. Balb / c mice were vaccinated intradermally with 50 μg of DNA plasmid (25 μg each of ACID and BASE plasmids), followed immediately by electroporation (n = 3). Three weeks later, draining regional lymph nodes (iliac or inguinal) and bone marrow from the tibia were harvested. (Figs. A-B) Single cell suspensions from the lymph nodes were stained with serial dilution HA probes, gated on GC B cells (GL7+CD38lo), and binding was analyzed by flow cytometry. (Fig. A) Dilution curve of the rHA probe fitted to non-linear regression. (Fig. B) Representative example of an individual mouse with 200 nM rHA probe. (Fig. C) Bone marrow-derived cell suspensions were analyzed by B cell ELISPOT to detect PR8- or Cal07-specific B cells. * p < 0.05 and ** p < 0.01, independent two-sided Student's t-test.

[0181] Figures 13A-D provide further data showing that a heterodimer having two variants of hemagglutinin HA (e.g., PR8 and Cal07 (mixed)) showed a clear effect of bivalency on both the humoral response and protection of BALB / c mice in the antigen challenge model of PR8. Both antigens showed induction of antigen-specific antibodies in mice. The heterodimeric ACID / BASE targeting vaccine is schematically shown in Fig. 13A. Vaccination of Balb / c mice gave higher PR8-specific IgG1 against the bivalent molecule than the monovalent molecule. See Fig. 13B. Only the bivalent heterodimer induced complete protection in the PR8 antigen challenge. See Fig. 13C. Furthermore, the increasing trend of competition in the inhibition ELISA suggested that the mixed vaccine induced more cross-binding antibodies (Fig. 13D). Fig. 13D provides the results of a competitive ELISA using PR8 as the coating, where sera from vaccinated mice compete with Cal07 (Cal07 / PR8 is inhibited more than Cal07 / Cal07) or PR8 (Cal07 / PR8 is inhibited less than PR8 / PR8).

[0182] 〔Example 3. Defense against tumor formation in bone marrow〕 In this example, an experiment showing that the ACID / BASE heterodimer vaccine protects mice from the bone marrow homing MOPC.315 tumor model (MOPC.315BM) in mice will be described (Hofgaard, P. O., et al., 2012. PLoS One 7: e51892).

[0183] Figure 16 shows that the ACID / BASE heterodimer DNA vaccine induces protection against bone marrow MOPC.315.BM tumors in BALB / c mice after a single vaccination. Mice were vaccinated i.m. with 50 μg of DNA into each quadriceps muscle, followed by electroporation at the injection site. Fourteen days later (n = 10 / group, n = 5 for the NaCl group), mice were administered 104 MOPC.315.BM (bone marrow) cells intravenously as antigen. Mice that reached paralytic stage during the collection phase were euthanized. Survival curves of various vaccines compared to antigen control (MIP1α-CH3-scFvA20) and NaCl-vaccinated mice ( ** p < 0.01, Mantel-Cox analysis, non-target scFv αNIP -A / B-scFv315 compared MIP1α-targeted group) are shown in Figure 16A. Blood samples were collected 36 days after antigen administration and analyzed for tumor-specific antigen M315 by ELISA (undetectable = ND, mean shown as bar, *** p < 0.001, Mann-Whitney, two-sided) (Figure 16B).

[0184] Figure 17 shows that one targeting unit in the ACID / BASE heterodimer vaccine is sufficient for antigen-specific IgG1 and IgG2a responses in vivo. As shown in Figure 17, BALB / c mice were vaccinated i.m. with 50 μg of DNA using the A / B heterodimer vaccine. Blood was collected from the mice 36 days (A, n = 4 / group) or 14 days (B, n = 3 / group) after vaccination, and M315-specific IgG1 and IgG2a in the serum were analyzed (not detectable = ND, mean ± standard error, not significant = ns, Mann-Whitney).

[0185] In summary, the results are shown in Figures 16 and 17. The results demonstrate that the ACID / BASE heterodimer can protect against a mouse model that homes to the bone marrow (Figure 16), similar to that shown in human patients where myeloma tumors home to the bone marrow, and that one targeting in the ACID / BASE heterodimer is sufficient to induce an antigen-specific antibody response (Figure 17).

[0186] 〔Example 4. Mixed Vaccine〕 In this example, the ACID / BASE heterodimer tested as a mixed vaccine containing 18 hemagglutinin (HA) subtypes was described.

[0187]

Table 2

[0188] Figure 18 shows that a DNA vaccine having HA as an antigen is functionally expressed in vitro and in vivo. Figure 18A shows a representative example of a vaccine protein. The DNA vaccine expresses full-length HA derived from 18 different subtypes as an antigen and targets a hapten (negative control) having an scFv of an mAb against mouse APC or mouse MHCII or NIP. Heterodimerization was achieved through acid-base interactions between leucine zipper α-helices rich in either acidic amino acids (ACID) or basic amino acids (BASE). The dimerization unit is arranged between the targeting unit and the antigen. A short human Ig hinge is arranged between the dimerization unit and the targeting unit and enables the formation of a disulfide bridge between two cysteines (shown as two black lines) that stabilizes the correct orientation of ACID with respect to BASE.

[0189] Balb / c mice were vaccinated intradermally with an αMHCII-Hx bivalent dimer having HA derived from one of 18 subtypes. Serum 6 weeks after vaccination was analyzed for reactivity against recombinant HA from each HA subtype. Figure 18B shows the serum antibody titers (left panel) against the recombinant HA protein of group 1 induced by the vaccine having HA of group 1, and the titers (right panel) against the recombinant HA protein of group 2 induced by HA of group 2.

[0190] Figure 19 shows that vaccination with an HA mixed vaccine induces antibodies against heterologous HA strains. Mice were immunized intramuscularly twice (day 0 and day 35) using an HA mixed vaccine containing single HA subtype controls for 16 HA subtypes or H1 and H7 not included in the mixed vaccine. The vaccine contained 1 μg / plasmid in the mixture and 5 μg / plasmid in the H1 and H7 vaccines. Antibody titers against H1 and H7 were measured 9 weeks later (Figure 19B).

[0191] Figure 20 shows that vaccination with the HA mixture (lacking H1) confers partial protection against H1 antigen administration. Mice were vaccinated three times with the HA mixed vaccine. Two weeks after the third immunization, the mice were challenged with H1N1 influenza virus. Figure 20 shows the mean weight loss ± standard error (left panel) and survival rate (right panel).

[0192] In summary, Figure 18 shows that individual vaccines are produced, induce subtype-specific antibody responses, and do not cross-react as single vaccines. A mixed ACID / BASE vaccine containing 16 out of 18 subtypes of HA variants (including HA from groups 1 and 2 but lacking H1 and H7) showed antigen-specific antibodies against H1 and H7 (Figure 19). Furthermore, a mixed ACID / BASE vaccine containing 11 out of 12 subtypes of HA variants and lacking the H1 subtype showed protection against H1-containing influenza viruses (Figure 20).

[0193] All publications and patents mentioned in the above specification are hereby incorporated by reference in their entirety for all purposes. Various modifications and changes in the compositions, methods, and uses of the described technology that do not depart from the scope and spirit of the described technology will be apparent to those skilled in the art. Although the present technology has been described in connection with specific exemplary embodiments, it should be understood that the invention as claimed in the claims should not be unduly limited to such specific embodiments. Indeed, various modifications in the manner described for carrying out the invention that are apparent to those skilled in the pharmacological, biochemical, medical, or related arts are intended to be within the scope of the following claims.

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Claims

1. A DNA vaccine comprising a first nucleic acid construct and a second nucleic acid construct, wherein the first nucleic acid construct and the second nucleic acid construct encode a first fusion protein and a second fusion protein, wherein the first fusion protein and the second fusion protein comprise an operably linked targeting unit, heterodimerization unit and antigenic unit, wherein the antigenic units in each of the first fusion protein and the second fusion protein are different variants of hemagglutinin (HA), and when the first nucleic acid construct and the second nucleic acid construct are introduced into a cell, the first fusion protein and the second fusion protein are expressed, and a first heterodimer protein is formed through the association of the heterodimerization unit. A DNA vaccine.

2. The heterodimerization unit in one of the first nucleic acid construct and the second nucleic acid construct is an ACID heterodimerization unit, and the heterodimerization unit in the other of the first nucleic acid construct and the second nucleic acid construct is an ACID / B A BASE heterodimerization unit that interacts to form the first heterodimer protein, or when the first nucleic acid construct and the second nucleic acid construct are expressed intracellularly, the production of the heterodimer protein is substantially free of the production of a homodimer protein containing the same antigen target protein, or The DNA vaccine according to claim 1, wherein the sequence encoding the fusion protein is operably linked to a promoter.

3. The targeting units of the first fusion protein and the second fusion protein are the same, or The DNA vaccine according to claim 1, wherein the targeting units of the first fusion protein and the second fusion protein are different.

4. The DNA vaccine according to claim 1, wherein the targeting unit is an antigen-binding protein.

5. The DNA vaccine according to claim 1, wherein the vaccine comprises a variant of HA derived from at least 3, 4, 5 or 6 and up to 12 or 18 strains or serotypes of influenza virus selected from the group consisting of group 1 influenza virus and group 2 influenza virus.

6. A DNA vaccine further comprising at least a third nucleic acid construct and a fourth nucleic acid construct, wherein the third nucleic acid construct and the fourth nucleic acid construct encode a third fusion protein and a fourth fusion protein, wherein the third fusion protein and the fourth fusion protein comprise a targeting unit, a heterodimerization unit and an antigenic unit operably linked, wherein the antigenic unit in each of the third fusion protein and the fourth fusion protein is a variant of a different hemagglutinin (HA), wherein expression of the third fusion protein and the fourth fusion protein intracellularly using the first fusion protein and the second fusion protein produces a mixture of heterodimeric proteins, and when the first nucleic acid construct and the second nucleic acid construct and the at least third nucleic acid construct and fourth nucleic acid construct are expressed intracellularly, production of the heterodimeric proteins is substantially free of production of homodimeric proteins containing the same hemagglutinin (HA) variant, the DNA vaccine according to claim 1.

7. A vaccine composition comprising a heterodimeric protein molecule, wherein the heterodimeric protein molecule is encoded by the DNA vaccine according to any one of claims 1 to 6, a vaccine composition.

8. A pharmaceutical preparation for use as a vaccine, comprising the DNA vaccine according to any one of claims 1 to 6 or the vaccine composition according to claim 7 and a pharmaceutically acceptable carrier.

9. A vaccine preparation comprising the vaccine composition according to claim 7 and an adjuvant.

10. For use in conferring immunity to or inducing an immune response against a variant antigen target protein in a subject, comprising administering to the subject a DNA vaccine, a vaccine composition, a pharmaceutical preparation or a vaccine preparation; For conferring immunity or inducing an immune response in a subject in need of a DNA vaccine, a vaccine composition, a pharmaceutical preparation or a vaccine preparation; The DNA vaccine according to any one of claims 1 to 6, the vaccine composition according to claim 7, the pharmaceutical preparation according to claim 8 or the vaccine preparation according to claim 9, as a vaccine for preventing or treating infection by a pathogen.

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