JP2008515851A - VLP-antigen conjugates and their use as vaccines - Google Patents

Abstract

  The present invention relates to the fields of medicine, immunology, virology and molecular biology. The present invention is a composition comprising a virus-like particle (VLP) of RNA bacteriophage and at least one antigen, wherein the VLP is produced recombinantly in a host and is contained in the VLP. Provided is a composition in which the amount of host RNA having the following structure is up to 20% of the amount of host RNA having a secondary structure originally contained in the VLP, and the VLP and at least one antigen are bound to each other. . The present invention also provides a method for producing the composition of the present invention. The compositions of the present invention are useful for the production of vaccines for the treatment of diseases, disorders and conditions. Furthermore, the compositions of the present invention are particularly useful for efficiently eliciting strong antibody responses against antigens within the indicated ranges while reducing or eliminating undesirable T cell responses.

Description

(Background of the Invention)
The present invention relates to the fields of molecular biology, virology, immunology and medicine. The present invention is a composition comprising a virus-like particle (VLP) of RNA bacteriophage and at least one antigen, wherein the VLP is produced recombinantly in a host and contained in the VLP The amount of host RNA having secondary structure is at most 20% of the amount of host RNA having secondary structure originally contained in the VLP, and the VLP and at least one antigen bind to each other. A composition is provided.
The present invention also provides a method for producing the composition of the present invention. The compositions of the present invention are useful for the production of vaccines for the treatment of diseases, disorders and conditions. Furthermore, the compositions of the present invention are particularly useful for efficiently eliciting strong antibody responses against antigens within the indicated ranges while reducing or eliminating undesirable T cell responses.

Related Art It has been described that virus-like particles (VLPs) of RNA bacteriophages that bind antigens are useful vaccine compositions. Thus, WO 02/056905 describes VLP-antigen conjugates as vaccines for the treatment of infectious diseases to prevent or cure cancer as well as to efficiently induce self-specific immune responses. be written. Further, in the induction of a self-specific immune response, for example, for the treatment of allergies (International Publication No. 03/040164) or bone diseases (International Publication No. 03/039225), or diseases related to the renin-activated angiotensin system ( Specific VLP-antigen conjugates for the treatment and prevention of WO 03/031466) are disclosed.
Depending on the antigen and associated disease characteristics, one type of immune response usually produced by a vaccine is preferred over another. For example, it is usually desirable to induce a strong cytotoxic T cell (CTL) response against tumor cells or viral infections, but such responses, particularly in the case of vaccines containing self antigens, are self-specific T cells. (Orgogozo, JM et al., Neurology 61, 46-54 (2003); Hock, C. et al., Natural Med. 8, 1270-75 (2002); Hock C. et al., Neuron 38 , 547-554, (2003)).

Antigen-specific T cell activation is a cross-response (cross-response) between T cells and antigen-presenting cells (APCs) such as dendritic cells, B cells and macrophages that present T cell antigens in relation to MHC molecules. heavily depends on talk). One way to cause APC activation is the interaction of CD40L on Th cells and CD40 on dendritic cells (Foy, TM, et al., Annu. Rev. Immunol. 14: 591 (1996)). Interestingly, CD40L-mediated maturation of this dendritic cell appears to have an auxiliary effect on the CTL response. Indeed, it has recently been shown that dendritic cells initiate a CTL response upon Th40 CD40 induction (Ridge, JP, et al., Nature 393: 474 (1998); Bennett, SRM, et al., Nature 393: 478 ( 1998); Schoenenberger, SP, et al., Nature 393: 480 (1998)).
Another way to cause APC activation is through activation of toll-like receptors expressed on APC. To date, 10 human toll-like receptors have been identified, and their ligands represent a limited number of invariant patterns that appear to be pathogenic (Medzhitov, R. and Janeway, CA, Jr., Cell 91: 295- 298 (1997)). Examples of such patterns include double stranded RNA, unmethylated CG rich DNA (CpG) or lipopolysaccharide (LPS), specific for bacterial and viral infections, respectively.

  Systemic activation of APCs by factors that stimulate innate immunity often causes self-specific lymphocytes and autoimmunity. Activation can enhance the expression of cytokines and costimulatory molecules such as IL-12 and IFNα. This finding is consistent with the finding that administration of thyroid extract with LPS breaks tolerance and causes autoimmune thyroiditis (Weigle, W. O., Adv. Immunol. 30: 159 (1980)). Furthermore, it has recently been shown in transgenic mouse models that self-peptide alone does not cause autoimmunity unless APC is activated by another route (Garza, KM, et al., J. Exp. Med. 191: 2021 (2000)). The link between innate immunity and autoimmune disease is further understood by the finding that systemic activation of LPS, viral infection or APC delays or prevents the establishment of peripheral tolerance (Vella, AT, etc. , Immunity 2: 261 (1995); Ehl, S., et al., J. Exp. Med. 187: 763 (1998); Maxwell, JR, et al., J. Immunol. 162: 2024 (1999)). In this case, innate immunity not only enhances self-specific lymphocyte activation, but also inhibits subsequent removal.

Virus-like particles of RNA phages such as Qβ and AP205 disclosed in the prior art and used as antigen carriers for vaccination are prepared by recombinant expression from E. coli. However, VLPs obtained by subsequent purification from E. coli lysates still contain encapsulated E. coli components, mainly E. coli RNA, as well as some E. coli proteins. Many bacterial components, particularly bacterial nucleic acids, and more particularly bacterial DNA and RNA, stimulate T cell responses through the activation of toll-like receptors. As indicated, activation of the T cell response can cause severe side effects. In addition, the presence of substances such as unidentified bacterial components other than those that have been proven as active pharmaceutical ingredients in vaccines can cause side effects and risks due to such unidentified bacterial components. So undesirable.
Therefore, there is a need in the art for the development of new powerful vaccines to treat any disease. In particular, there is a need in the art for the development of new vaccines that minimize or eliminate undesirable side effects such as activation or stimulation of undesirable T cell responses while maintaining maximum therapeutic efficacy.

(Summary of Invention)
We have surprisingly found that the composition of the invention provides an immune response to the antigen contained in the vaccine, while minimizing or avoiding other undesirable side effects such as unwanted T cell responses and heat, In particular, it has been discovered that antibody responses can be strongly induced.
Stimulation of innate immunity induces IgG2a and IgG2b type antibodies. This induction destroys tissues more strongly than IgG1 (mouse). We have surprisingly found that the composition of the invention changes antibody isotype properties (human) from IgG2a and / or IgG2b or IgG1.
Furthermore, we surprisingly use the compositions of the present invention as targets in treating a variety of diseases including, but not limited to, autoimmune diseases, inflammatory diseases, obesity and drug addiction. It has been found that it is a potent vaccine that induces a strong immune response against a variety of antigens, particularly self-antigens.

  Accordingly, in a first aspect, the present invention provides (a) a virus-like particle (VLP) comprising a coat protein of RNA bacteriophage having at least one first attachment site, a mutein or a fragment thereof. The VLP is produced recombinantly in the host, and the amount of the host RNA having the secondary structure contained in the VLP is the amount of the host RNA having the secondary structure originally contained in the VLP. A composition comprising: a virus-like particle having a maximum of 20%, preferably a maximum of 10%; and (b) at least one antigen having at least one second attachment site, the at least one antigen A composition is provided wherein (b) is bound to the VLP (a) via the at least one first attachment site and the at least one second attachment site. Preferably, the composition of the present invention forms a regular and repetitive antigen sequence. Said at least one antigen may be selected from the group consisting of polypeptides, carbohydrates, steroid hormones, organic molecules or haptens. Said at least one antigen is a self-antigen or a hapten. Typically, preferably, a method is provided herein for determining the amount of RNA, preferably nucleic acid.

  Substantial reduction in the amount of host RNA, preferably host nucleic acid, in the compositions and vaccines of the present invention results in VLP or VLP-antigen conjugates, resulting in a significant reduction in the amount of Toll-like receptor ligands. In fact. Toll-like receptors are thought to be the axis that links innate and adaptive immune responses. Activation of a Toll-like receptor results in systemic activation of the innate immune system as well as APC activation, and further triggers an antigen-specific immune response. Furthermore, VLPs containing up to 20%, preferably up to 10% of the host RNA with secondary structure originally contained in the VLP, more preferably host RNA, preferably essentially free of host nucleic acids. The compositions and vaccines of the invention comprising VLPs, even more preferably VLPs essentially free of Toll-like receptors, while avoiding activation of Toll-like receptors typically avoid antigen-specific immune responses, In particular, it is surprising to induce an antigen response. Furthermore, the compositions and vaccines of the present invention induce a strong immune response against a particular antigen in the composition with minimal or no stimulation of the CTL response. Furthermore, the compositions and vaccines of the present invention reduce the production of IgG2a and / or IgG2b and change the antibody isotype from the more tissue destructive IgG2a and / or IgG2b.

In a further aspect, the present invention provides (a) a virus-like particle (VLP) comprising a coat protein of RNA bacteriophage having at least one first attachment site, a mutein or fragment thereof, wherein the VLP Is produced recombinantly in the host, wherein the VLP is essentially free of host RNA, preferably host nucleic acid; (b) at least one second attachment site having at least one second attachment site; An antigen, wherein the at least one antigen (b) binds to the VLP (a) via the at least one first attachment site and the at least one second attachment site. A composition is provided.
Furthermore, we note or typically preferred that the compositions and vaccines of the present invention are a subset of Th1 cells, as shown by the reduced IgG2a titers, particularly low IgG2a / IgG1 ratios. Have found that they induce a strong immune response against specific antigens bound to VLPs without activating significant inflammatory T cells. Activation of different subsets of T cells results in the expression of different antibody subtypes. For example, Th1 cells significantly produce IFN-γ, thereby facilitating the production of immunoglobulin IgG2a opsonin and complement-fixing antibodies, mediating macrophage activation and antibody-dependent cellular cytotoxicity. Th2 cells are characterized by IL-4 and IL-10 production and are useful for B cell maturation and IgG1 antibody production. Thus, the IgG2a / IgG1 ratio is an excellent accepted parameter for measuring the extent of Th1 cell activity relative to Th2 cells. In addition, a reduction in IgG2a and / or IgG2b is very useful when the complement fixing properties and opsonins of the antibody subclass are undesirable, for example in the case of vaccines developed for the induction of antibodies against self-antigens.

  In another preferred embodiment of the invention, the invention further comprises at least one polyanionic polymer bound to the VLP. In an even more preferred embodiment of the present invention, the at least one polyanionic polymer is encapsulated or packaged in a VLP. Typically, the presence of at least one polyanionic polymer further increases the antibody titer to the antigen, particularly the IgG1 titer, while maintaining a low IgG2a / IgG1 ratio and a low CTL response. Further, the presence of at least one polyanionic polymer that binds to, or preferably is packaged or packaged in, the VLP is typically an RNA bacteriophage coat protein, mutein or fragment thereof. Is useful and beneficial for the restructuring (reassembly) of the DNA into the VLP as well as its stability. In a preferred embodiment, the polyanionic polymer is a polyanionic polypeptide, more preferably the polyanionic polymer is polyglutamic acid and / or polyaspartic acid.

  In a further aspect, the present invention provides a method for modulating each of the VLPs of the compositions and RNA bacteriophage-antigen conjugates of the present invention. In a preferred embodiment, the present invention provides a method for preparing each of the VLPs of the compositions and RNA bacteriophage-antigen conjugates of the present invention, comprising: (a) a virus-like particle having at least one first attachment site ( VLP) is produced recombinantly by the host, wherein the VLP contains an RNA bacteriophage coat protein, mutein or fragment thereof; (b) the RNA bacteriophage coat protein, mutein or (C) purifying the coat protein, mutein or fragment thereof; (d) purifying the purified RNA bacteriophage coat protein, mutein or fragment thereof into a virus-like particle; The virus-like particles at this time (E) binding at least one antigen having at least one second attachment site to the VLP obtained from step (d); essentially lacking host RNA, preferably host nucleic acid; Providing a method. In a preferred embodiment, the step of reconstituting the purified coat protein is performed in the presence of at least one polyanionic polymer.

In another aspect, the present invention provides a vaccine composition comprising a composition of the invention, preferably further comprising a buffer. The vaccine is administered to a patient with or without at least one adjuvant. In a further aspect, the present invention provides a method of immunization comprising administering said vaccine to an animal or human.
In a further aspect, the present invention provides a method for treating a disease comprising administering the composition to an animal or a human. In a preferred embodiment, the present invention provides methods for the treatment of diseases such as, but not limited to, inflammatory diseases, chronic autoimmune diseases, obesity or drug addiction.
In yet another aspect, the present invention provides a pharmaceutical composition comprising a composition of the present invention and an acceptable pharmaceutical carrier.
In a still further aspect, the vaccine composition of the present invention is essentially free of bacterial RNA as well as other bacterial components. This results in a clearer composition and vaccine, respectively. This not only results in a safer vaccine, but also meets good manufacturing practice (GMP) standards.

(Detailed description of the invention)
Definition:
Antibody isotype: Unless otherwise stated, the antibody isotype nomenclature used in this application was the mouse nomenclature. Those skilled in the art understand the corresponding human nomenclature. For example, mouse antibody isotype IgG2a is human antibody isotype IgG1.
Antigen: As used herein, the term “antigen” refers to a molecule capable of binding to an antibody or T cell receptor (TCR) when presented by an MHC molecule. The term “antigen”, as used herein, also includes T cell epitopes. Furthermore, the antigen can be recognized by the immune system and / or can induce a humoral and / or cellular immune response that results in activation of B and / or T lymphocytes. However, this may require that, in at least some cases, the antigen contains or is linked to a Th cell epitope and present in an adjuvant. An antigen can have one or more epitopes or antigenic sites (B and T epitopes). As used herein, specific binding refers to the possibility that an antigen reacts with its corresponding antibody or TCR, preferably in a very selective manner, and is induced by other antigens. It is intended to indicate that it does not react with a number of other antibodies or TCRs. The antigen used here may be a mixture of several separate antigens.

  First attachment site: As used herein, the term “first attachment site” refers to a site that is naturally occurring in a VLP or artificially added to a VLP and to which a second attachment site binds. The first attachment site is a protein, polypeptide, amino acid, peptide, sugar, polynucleotide, natural or synthetic polymer, secondary metabolite or compound (biotin, fluorescein, retinol, digoxigenin, metal ion, phenylmethylsulfonyl fluoride) Or a chemically reactive group such as an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, a guanidyl group, a histidyl group, or a combination thereof. A preferred embodiment of the chemically reactive group that is the first attachment site is the amino group of an amino acid such as lysine. The first attachment site is typically located on the surface of the VLP, preferably on the outer surface of the VLP. A number of first attachment sites are present on the surface of the virus-like particle, preferably on the outer surface, typically in a repetitive shape. In a preferred embodiment, the first attachment site associates (binds) with the VLP via at least one covalent bond, preferably via at least one peptide bond. In a further preferred embodiment, the first attachment site is naturally occurring in the VLP. Alternatively, in a preferred embodiment, the first attachment site is artificially added to the VLP.

  Second attachment site: As used herein, the term “second attachment site” is a component that occurs naturally in the antigen of the present invention or is artificially added to the antigen of the present invention and to which the first attachment site binds. Point to. The second attachment site of the present invention is a protein, polypeptide, peptide, amino acid, sugar, polynucleotide, natural or synthetic polymer, secondary metabolite or compound (biotin, fluorescein, retinol, digoxigenin, metal ion, phenylmethylsulfonyl Fluoride), or a chemically reactive group such as an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, a guanidyl group, a histidyl group, or a combination thereof. A preferred embodiment of the chemically reactive group that is the first attachment site is preferably a sulfhydryl group of an amino acid such as cysteine. Thus, the term “at least one antigen having at least one second attachment site” refers to a construct comprising an antigen of the invention and at least one second attachment site. However, typically and preferably, such constructs further comprise a “linker”, particularly in the case of a second attachment site that does not occur naturally within the antigen. In another preferred embodiment, the second attachment site associates (binds) with an antigen of the present invention via at least one covalent bond, preferably via at least one peptide bond. In a further embodiment, the second attachment site occurs naturally within the antigen of the invention. In yet another preferred embodiment, the second attachment site is artificially added to the antigen of the present invention, preferably via a linker containing cysteine, by protein fusion.

  Bound: As used herein, the term “bound” refers to covalent, eg, chemical coupling, or non-covalent, eg, ionic, hydrophobic, hydrogen bonding, etc. Refers to a bond that is The covalent bond may be, for example, an ester, ether, phosphate ester, amide, peptide, imide, carbon-sulfur bond, carbon-phosphorus bond, and the like. The term also includes encapsulation or partial encapsulation of the substance. The term “bound” is broader and includes “coupled”, “fused”, “enclosed”, “packaged”, and “attached”. The term is also included. For example, a polyanionic polymer such as polyglutamic acid is typically and preferably not actually covalently bonded, but is typically and preferably encapsulated or packaged in a VLP.

Coat protein: In this application, the term “capsid protein” used interchangeably with the term “coat protein” refers to a viral protein that can be encapsulated within a viral capsid or VLP. Typically and preferably, the term “coat protein” refers to a coat protein encoded by the genome of an RNA bacteriophage or a variant of an RNA bacteriophage. More preferably, by way of example, the term “coat protein of AP205” refers to SEQ ID NO: 29 or an amino acid sequence in which the first methionine is cleaved from SEQ ID NO: 29. More preferably, by way of example, “Qβ coat protein” refers to SEQ ID NO: 10 (“Qβ VLP”) and SEQ ID NO: 11 (A1) with or without N-terminal methionine (SEQ ID NO: 67). The capsid of bacteriophage Qβ mainly consists of Qβ VLP and a small amount of A1 protein.
Epitope: As used herein, the term “epitope” refers to a continuous or discontinuous portion of an antigenic or immunologically active polypeptide of an animal, preferably a mammal, and most preferably a human. Epitopes are recognized by antibodies or T cells via their T cell receptors in the context of MHC molecules.

  Essentially lacking host RNA, preferably host nucleic acid: As used herein, the term “essentially lacking host RNA, preferably host nucleic acid” refers to host RNA contained in a VLP, preferably Means the amount of host nucleic acid, which amount is typically preferably less than 30 μg per mg VLP, preferably less than 20 μg, more preferably less than 10 μg, even more preferably less than 8 μg, even more preferably 6 μg Less, even more preferably less than 4 μg, most preferably less than 2 μg. A host used within the above range means a host in which VLP is produced recombinantly, and the host is preferably a bacterium, even more preferably E. coli. Conventional methods for measuring the amount of RNA, preferably nucleic acid, are well known to those skilled in the art. An exemplary and suitable method for measuring the amount of RNA, preferably nucleic acid according to the present invention is described in Example 17 for conventional Qβ VLPs and Qβ VLPs of the present invention. Typically, preferably the same, homologous or similar conditions are used to determine the amount of RNA, preferably nucleic acid, for a composition of the invention comprising a VLP other than Qβ. The required change of conditions is within the knowledge of those skilled in the art. Typically, the numerical value of the quantity measured can be understood as a value having a deviation of ± 10% of the indicated numerical value, preferably a deviation of ± 5%.

Immunostimulatory nucleic acid: As used herein, the term “immunostimulatory nucleic acid” refers to a nucleic acid that can induce and / or enhance an immune response.
Immunostimulatory substance: As used herein, the term “immunostimulatory substance” induces an immune response, typically and preferably an immune response specific for an antigen contained in the composition of the invention. And / or substances that can be enhanced.
Linked: As used herein, the term “linked” preferably means a chemical interaction in which at least a first attachment site and at least one second attachment site are linked together, if possible. Means. Chemical interactions include covalent and non-covalent interactions. Typical examples of non-covalent interactions are ionic interactions, hydrophobic interactions or hydrogen bonds, whereas covalent interactions are covalent bonds such as esters, ethers, phosphate esters, amides. , Peptides, carbon-phosphorus bonds, carbon-sulfur bonds, such as those based on thioether or imide bonds. In certain preferred embodiments, the first attachment site and the second attachment site are attached via at least one covalent bond, preferably at least one non-peptide bond, and even more preferably only through non-peptide bonds. However, the term “binding” as used herein not only encompasses the direct binding of at least one first attachment site and at least one second attachment site, but is also preferably an intermediate molecule, and thereby Typically and preferably, it involves indirect attachment of at least one first attachment site and at least one second attachment site via at least one, preferably one heterobifunctional crosslinker.

  Linker: As used herein, a “linker” connects a second attachment site and an antigen of the present invention, already contains a second attachment site, consists essentially of a second attachment site, or is a second attachment site Consists of. Preferably, a “linker” as used herein already includes a second attachment site, typically and preferably, but not as a single amino acid residue, preferably as a cysteine residue. In addition, the “linker” used herein is referred to as “amino acid linker” particularly when the linker of the present invention contains at least one amino acid residue. Accordingly, the terms “linker” and “amino acid linker” are used interchangeably herein. However, this term is not meant to indicate that such an amino acid linker consists only of amino acid residues, even if an amino acid linker consisting of amino acid residues is a preferred embodiment of the present invention. The amino acid residues of the linker are preferably composed of naturally occurring amino acids or non-natural amino acids known in the art, all L forms or all D forms, or mixtures thereof. Accordingly, amino acid linkers comprising molecules having sulfhydryl groups or cysteine residues are preferred embodiments of the linkers of the invention, and such molecules are also included within the invention. In addition, linkers useful in the present invention are molecules comprising C1-C6 alkyl-, cycloalkyl, such as cyclopentyl or cyclohexyl, cycloalkenyl, aryl or heteroaryl molecules. In addition, linkers comprising C1-C6 alkyl-, cycloalkyl (C5, C6), aryl, or heteroaryl moieties and additional amino acids can also be used as linkers for the present invention and are within the scope of the present invention. It is. The association (bonding) between the antigen of the present invention and the linker is preferably by at least one covalent bond, and more preferably by at least one peptide bond. If the second attachment site is not naturally present in the antigen of the invention, the linker binds to at least one second attachment site, such as cysteine, preferably by at least one covalent bond, more preferably by at least one peptide bond. .

Regular and repetitive antigen arrays: As used herein, the term “regular and repetitive antigen arrays” generally refers to antigens in relation to virus-like particles, typically preferably very It refers to a repetitive pattern of antigens characterized by regular and uniform spatial arrangement. In one embodiment of the invention, the repeating pattern is a geometric pattern. The VLP of the RNA phage is preferably 1-30 nanometer apart, preferably 2-15 nanometer apart, more preferably 2-10 nanometer apart, even more preferably 2-8 nanometer apart, More preferably, it has a quasi-crystalline, strictly repetitive sequence of antigens with a spacing of 1.6 to 7 nanometers.
Packaged: As used herein, the term “packaged” refers to the state of a polyanionic polymer in relation to a VLP. As used herein, the term “packaging” includes bonds that can be covalent, eg, chemical coupling, or non-covalent, eg, ionic, hydrophobic, hydrogen bonding, and the like. In a preferred embodiment, the term “packaging” refers to the encapsulation or partial encapsulation of polyanionic polymers by VLPs. Thus, polyanionic polymers can be encapsulated by VLPs without actually binding, in particular covalently bonding. In a preferred embodiment, at least one polyanionic polymer is packaged within the VLP, most preferably in a non-covalent manner.

  Polyanionic polymer: As used herein, the term “polyanionic polymer” refers to a relatively high mass molecule comprising a negatively charged repetitive group and its structure. In practice or conceptually consists essentially of multiple repetitions of units derived from relatively low mass molecules. The polyanionic polymer is one having a molecular weight of at least 2000 daltons, preferably at least 3000 daltons, and even more preferably at least 5000 daltons. As used herein, the term “polyanionic macromolecule” typically and preferably refers to a molecule that cannot activate a toll-like receptor. Thus, the term “polyanionic macromolecule” typically and preferably excludes Toll-like receptor ligands, and even more preferably further immunostimulatory substances such as Toll-like receptor ligands, immunostimulatory nucleic acids and lipopolysaccharides. Excluding saccharide (LPS). More preferably, the term “polyanionic polymer” as used herein refers to a molecule that is unable to induce cytokine production. Even more preferably, the term “polyanionic polymer” excludes immunostimulatory substances.

Polyaspartic acid: As used herein, the term “polyaspartic acid” means at least 50%, preferably at least 70%, preferably at least 90%, more preferably at least 90% of the total number of amino acids comprising the polypeptide. 95%, more preferably at least 99%, more preferably 100% refers to a polypeptide that is aspartic acid. As a result, the aspartic acid molecule is all L or all D or mixtures thereof.
Polyglutamic acid: As used herein, the term “polyglutamic acid” refers to at least 50%, preferably at least 70%, preferably at least 90%, more preferably at least 95% of the total number of amino acids comprising the polypeptide. More preferably at least 99%, more preferably 100% is a polypeptide that is glutamic acid. Consequently, the glutamic acid molecule is all L or all D or a mixture thereof.
Poly (GluAsp): As used herein, the term “poly (GluAsp)” refers to at least 50%, preferably at least 70%, preferably at least 90%, more preferably of the total number of amino acids comprising the polypeptide. Refers to a polypeptide in which at least 95%, more preferably at least 99%, more preferably 100% is the sum of glutamic acid and aspartic acid. As a result, the glutamic acid and aspartic acid molecules are all L or all D or mixtures thereof.

Polypeptide: As used herein, the term “polypeptide” refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also called peptide bonds). This represents a molecular chain of amino acids and does not refer to a product of a specific length. Thus, peptides, dipeptides, tripeptides, oligopeptides and proteins are included within the definition of polypeptide. For example, post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, etc. are also included.
The term “recombinant VLP” as used herein refers to a VLP obtained by a method comprising at least one step of recombinant DNA technology. The term “recombinantly produced VLP” as used herein refers to a VLP obtained by a method comprising at least one step of recombinant DNA technology. Thus, the terms “recombinant VLP” and “recombinantly produced VLP” are used interchangeably herein and have the same meaning.

RNA having secondary structure, preferably nucleic acid: As used herein, the term “RNA having secondary structure, preferably nucleic acid” refers to RNA, preferably nucleic acid, intervening with ethidium bromide. , Refers to what can be visualized by the fluorescence of ethidium bromide under ultraviolet light. Representative and preferred methods of inserting ethidium bromide into a host RNA having the secondary structure of the present invention, preferably host nucleic acid, are described in Examples 2 and 3 of the present invention using Qβ VLPs. Typically and preferably, the same, similar or similar conditions are used to insert ethidium bromide into a host RNA having a secondary structure contained in a VLP other than Qβ, preferably the host nucleic acid. Changes in the conditions ultimately required are within the knowledge of those skilled in the art.
Host RNA, preferably host nucleic acid: As used herein, the term “host RNA, preferably host nucleic acid” refers to RNA or preferably nucleic acid that is naturally synthesized by the host. However, RNA, preferably nucleic acid, is typically and preferably chemically and / or physically changed during the method of reducing or removing RNA, preferably nucleic acid, according to the method of the invention. Can happen. For example, the size of RNA, preferably nucleic acid, can be shortened or its secondary structure can change. However, the resulting RNA or nucleic acid is also considered host RNA or host nucleic acid.

  The amount of host RNA having a secondary structure, preferably a nucleic acid: As used herein, the term “amount of host RNA having a secondary structure, preferably the amount of a nucleic acid” refers to an RNA intervening with ethidium bromide, preferably Refers to nucleic acid, which can be visualized by fluorescence of ethidium bromide under ultraviolet light. Representative and suitable methods for measuring the amount of host RNA, preferably nucleic acid, and the amount of host RNA having a secondary structure contained in the VLP, preferably nucleic acid, that can sandwich the ethidium bromide of the present invention are: , Measuring the amount of ethidium bromide intercalated in RNA, preferably nucleic acid, as described in Examples 2 and 3 of the present invention using Qβ VLPs. Typically and preferably, the same, homogeneous or similar conditions are used to determine the amount of host RNA having a secondary structure contained in a VLP other than Qβ, preferably the host nucleic acid. Changes in the conditions ultimately required are within the knowledge of those skilled in the art.

  A host RNA having a secondary structure originally contained (encapsulated) in a VLP, preferably the amount of nucleic acid: as used herein, “a host RNA having a secondary structure originally contained (encapsulated) in a VLP, The term "preferably the amount of nucleic acid" typically and preferably reduces or eliminates the amount of host RNA, preferably nucleic acid, having the same VLP secondary structure made by the method of the invention. Refers to the amount of host RNA, preferably nucleic acid, having a secondary structure contained in the VLP after it has been recombinantly produced by the host. “Same VLP” as used in this context refers to VLPs that typically and preferably occur in the same expression batch. As used herein, the term “amount of host RNA, preferably nucleic acid having a secondary structure that is initially contained (encapsulated) in a VLP” is typically and preferably made by the method of the present invention. VLPs after purification of the resulting VLPs produced recombinantly by the host before reducing or eliminating the amount of host RNA, preferably nucleic acids, typically and preferably having the same VLP secondary structure Refers to the amount of host RNA, preferably nucleic acid, having a secondary structure. As indicated, “same VLP” as used in this context refers to a VLP that typically and preferably results from the same batch of expression, preferably the same batch of purification. Recombinantly produced VLPs are typically and preferably purified from host cell lysates. Methods for the purification of VLPs from host cell lysates are disclosed in the art, for example WO 02/056905, WO 04/007538, preferably Example 1 of this application for Qβ VLPs. Is disclosed. Typically and preferably, the reduction of the host RNA, preferably nucleic acid, having the secondary structure of the present invention is reduced by the amount of host RNA, preferably nucleic acid, having the secondary structure originally contained within the VLP, and then The amount of host RNA, preferably nucleic acid, having a secondary structure contained within the VLP of the present invention (ie, a VLP typically and preferably obtained by adapting the method of the present invention) is continued, preferably simultaneously. Measured by subtracting the appropriate amount of recombinantly expressed VLP after purification. As described above, representative and suitable methods for determining the amount of host RNA, preferably nucleic acid, having secondary structure are described in Examples 2 and 3 of the present invention with Qβ VLP. Typically and preferably, the same, homogeneous or similar conditions are used to determine the amount of host RNA having a secondary structure contained in a VLP other than Qβ, preferably the host nucleic acid. Changes in the conditions ultimately required are within the knowledge of those skilled in the art.

  Self-antigen: As used herein, the term “self-antigen” refers to (i) RNA or a polypeptide-derived product encoded by host DNA, encoded by a polypeptide or protein or host DNA, (ii) ) a polypeptide or protein having a high homology to (i), the homology of which is at least 90%, preferably at least 92%, and even more preferably at least 95%, even more preferably at least 97% (I) a polypeptide or protein that induces in vivo the production of an antibody that specifically binds to the protein or polypeptide of (i), and (iii) an ortholog of the autoantigen defined in (i) Means. Typically and preferably, said ortholog induces in vivo production of an antibody that specifically binds to the polypeptide or protein of (i). The term “ortholog” refers to a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide from a different species. Sequence differences between orthologs are the result of speciation. The term “autoantigen” as used herein further includes chemical modifications as defined above, including but not limited to glycosylation, acetylation, phosphorylation of the autoantigen. Furthermore, the term “self-antigen” as used herein preferably refers to a polypeptide resulting from a combination of two or several self-antigens.

Autoantigen fragment: As used herein, the term “self-antigen fragment” is a polypeptide comprising, or consisting of, a fragment of an autoantigen, preferably at least 4, preferably at least 5, preferably At least 6, at least 7, at least 8, or more preferably at least 12 or at least 15 amino acids in length, and typically and preferably specifically binds to the polypeptide or protein of (i) in the definition of autoantigen A polypeptide that induces the production of an antibody in vivo. The term “autoantigen fragment” as used herein further includes chemical modifications as defined above, including but not limited to glycosylation, acetylation, phosphorylation of “autoantigen fragment”.
Autoantigen variant: As used herein, the term “autoantigen variant” is a polypeptide that is homologous to the polypeptide or protein of (i) in the definition of autoantigen, and its homology. An antibody that specifically binds to the polypeptide or protein of (i), having a sex at least 75%, preferably at least 80%, even more preferably at least 87%, and typically and preferably in the definition of a self-antigen Refers to a polypeptide that induces the production of The term “autoantigen variant” as used herein further includes chemical modifications as defined above, including but not limited to glycosylation, acetylation, phosphorylation of “autoantigen variant”. Including.

  Toll-like receptor (TLR) ligands: Toll-like receptors (TLRs) are similarly expressed in myelomonocytic and endothelial and epithelial cells and various organ systems. Toll-like receptors are transmembrane proteins, all of which have a common extracellular leucine-rich domain and a conserved cytoplasmic domain. The cytoplasmic domain of TLR is homologous to the IL-1 and IL-18 receptors and contains a Toll / IL-1 receptor (TIR) homology domain common to these receptors. Toll-like receptors share a common activation pathway mediated through the TIR signaling domain, thus activating nuclear translocation and the pro-inflammatory transcription factor NF-kB (Abreu MT and Arditi MJ Pediatrics (2004), 421-9). To date, 10 different TLR molecules have been cloned from the human genome (Zarember KA et al., J. Immunol. (2002), 168: 554-61) and 11 TLRs have been cloned in mice (Zhang et al., Science , (2004), 303: 1522-6).

  As used herein, the term “Toll-like receptor ligand” or “TLR ligand” refers to any ligand capable of activating at least one TLR (eg, Beutler, B. 2002, Curr. Opin. Hematol., 9, 2-10, Schwarz et al., 2003, Eur. J. Immunol., 33, 1465-1470). The TLR ligand of the present invention activates, but is not limited to, at least one Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or TLR11. For example, peptidoglycan (PGN) or lipoteichoic acid (LTA) typically and preferably activates TLR2 (Aliprantis et al., Science (1999), 285: 736-9; Underhill, et al., Nature, (1999), 401 : 811-5); double stranded RNA such as poly (I: C) typically and preferably activates TLR3 (Alexopoulou et al., Nature (2001), 413: 732-8); lipopolysaccharide (LPS ) Typically and preferably activates TLR4 (Poltorak, et al., Science (1998), 282: 2085-8); flagellin typically and preferably activates TLR5 (Hayashi et al. Nature (2001), 410). : 1099-103); single-stranded RNA such as bacterial RNA and certain synthetics such as imidazoquinoline typically and preferably activate TLR7 and TLR8 (Diebold S. et al. Science 303: 1529; Heil, FH. Et al. Science 303: 1526); bacterial DNA, especially DN containing CpG motifs A typically and preferably activates TLR9 (Schnare et al. Curr. Biol. (2000), 10: 1139-42; Hemmi H et al. Nature (2000), 408: 740-5). The papers listed here are incorporated here by reference. A summary of TLR ligands is listed in the Abreu article table and is incorporated herein by reference (Abreu MT and Arditi MJ, Pediatrics (2004), 421-9), and references on TLR11 and its ligands are available. Zhang et al., Science, (2004), 303: 1522-6. Referring to these incorporated articles together with general knowledge of those skilled in the art, it is common practice to test whether a molecule is a TLR ligand according to the present invention and whether the TLR ligand activates at least one TLR. Is within the range.

One common, representative and preferred method of measuring the activation of Toll-like receptors of the invention is to transfect a cell line with a construct capable of expressing at least one Toll-like receptor, It is not to express. An NF-kB activation reporter gene (eg, luciferase) can be transfected into the cell line or included in the genome of the cell line. After adding the molecule to be tested to the cell culture, the activity of the reporter gene can be quantified and compared to cells that have not added the molecule to be tested. For TLR1 and TLR6 activity studies, co-expression of TLR2 is said to be essential. It will be apparent to those skilled in the art that some TLR-ligands that are unstable, such as RNA with secondary structure, need to be stabilized in order to be tested. One method for stabilization is to encapsulate RNA with secondary structure in liposomes or materials (eg DOTAP) used for transfection.
References to TLR sequences can be found in the database Swisssprot as accession numbers TLR1_human, TLR2_human, TLR3_human, TLR4_human, TLR5_human, TLR6_human, TLR7_human, TLR8_human, TLR9_human. TLR10_human can be found as GenBank accession number AAQ88667 or AAK26744. Therefore, the accession numbers of the mouse sequences are TLR1_mouse, TLR2_mouse, TLR3_mouse, TLR4_mouse, TLR5_mouse, TLR6_mouse, TLR7_mouse, TLR8_mouse, TLR9_mouse, and TLR10_mouse in the Swisssprot database. Mouse TLR11 is GenBank accession number AY531552. One skilled in the art can prepare assays corresponding to TLR receptors from other mammalian species.

A typical and preferred example of such a test for the present invention is as follows: 3 × 10 ⁶ HEK293 cells were loaded with 1 μg of TLR expression plasmid and 20 ng of NF-kB luciferase reporter plasmid at 960 μF at 200 volts. Perform electroporation. Keep the total amount of plasmid DNA at 15 μg per electroporation by adding the appropriate empty expression vector. 10 ⁵ cells per well are seeded and incubated overnight, then stimulated with the ligand to be tested for an additional 7-10 hours. The concentration range of known TLR ligands is typically 25 μg / ml RNA40-42 and DOTAP complex (to promote RNA internalization into cells), 1 μM CpG-ODN2006, 10 μM R-848, 50 μg. / Ml poly (I: C), or 1 μg / ml Pam3Cys (Heil, FH. Et al. Science 303: 1526). Lysed cells are lysed using a reporter lysis buffer (Promega, Mannheim, Germany) and lysed luciferase using a luminometer, typically and preferably a Berthold luminometer (Wildbad, Germany) according to the manufacturer's instructions. Assess activity. This is within the knowledge of those skilled in the art and thus applies the above-described experiment for the testing of any ligand.
If the induced luciferase activity is then statistically significantly higher than the threshold measured in the negative control activity (identical experiment and identical experimental conditions without ligand addition tested), the ligand is It is considered to activate TLR. The threshold in this case is defined by the average of the negative control luciferase activity in 6 independent experiments and the standard deviation of luciferase activity from 3 6 experiments. Then, typically and preferably, the ligand is deemed to be “statistically significant” to activate the TLR if the luciferase activity of the ligand is above the threshold measured as described above. Preferably, a ligand "statistically significantly" activates a TLR when the luciferase activity of the ligand is at least 2-fold, preferably 3-fold, more preferably 5-fold higher than the threshold measured as described above. It is regarded.

  Virus-like particle of RNA phage: As used herein, the term “virus-like particle of RNA phage” comprises, preferably consists essentially of, or consists of an RNA phage coat protein, mutein or fragment thereof. Refers to a virus-like particle. Furthermore, RNA phage virus-like particles that resemble the structure of RNA phage are non-replicating or non-infectious, and are typically and preferably non-replicating and non-infectious. Typically and preferably, the “virus-like particle of RNA phage” is at least one gene encoding the replication mechanism of RNA phage, preferably all genes, and typically and more preferably, whether the virus adheres to the host. It further refers to a virus-like particle of an RNA phage that lacks at least one gene, preferably all of the genes that encode the protein for entry or the protein involved therein. However, this definition also includes an RNA phage virus-like particle in which the aforementioned gene or group of genes is present but inactive, so that the RNA phage virus-like particle is non-replicating and / or non-infectious Is included. In addition, the term “RNA phage virus-like particle” is broadly defined as an RNA phage virus particle that cannot be infected and / or replicated because the genome has been inactivated by chemical or genetic methods. Included in the definition. A preferred VLP obtained from RNA phage is a symmetric icosahedron and consists of 180 subunits. Within the scope of the present disclosure, the terms “subunit” and “monomer” are used interchangeably interchangeably in this context. In this application, the terms “RNA phage” and “RNA-bacteriophage” are used interchangeably.

Amino acid homology of polypeptides can be routinely determined using known computer programs such as Bestfit. Use best fit or any other sequence alignment program, preferably using best fit, to determine whether a particular sequence is 95% homologous, for example, to the referenced amino acid sequence The ratio of homology to the full length of the amino acid sequence is calculated, and parameters are set so that a gap is inserted in the homology of 5% or less of the total number of amino acid residues in the reference sequence. The above-described method for measuring the percentage of homology between polypeptides is suitable for all proteins, polypeptides or fragments thereof disclosed in the present invention.
One, a, or an: When the terms “one”, “a”, or “an” are used in this disclosure, they are “at least one” or “one or more” unless otherwise indicated. "Means.

The invention comprises (a) a virus-like particle (VLP) comprising an RNA-bacteriophage coat protein having at least one first attachment site, a variant (mutein) or fragment thereof, wherein the VLP is recombined by the host. The amount of host RNA produced, preferably having a secondary structure contained in the VLP, preferably the amount of host nucleic acid is at most 20% of the amount of host RNA having a secondary structure originally contained in the VLP, preferably the host nucleic acid. Preferably 10%, more preferably 5%, more preferably 3%, even more preferably 1%; (b) comprising at least one antigen having at least one second attachment site; A composition in which one antigen (b) is bound to the VLP (a) via the at least one first attachment site and the at least one second attachment site is provided. To do. In a preferred embodiment, the VLPs of the invention are essentially free of host RNA, preferably host nucleic acid.
In one aspect, the invention provides (a) a virus-like particle (VLP) comprising an RNA-bacteriophage coat protein, variant or fragment thereof having at least one first attachment site, wherein the VLP is recombined by the host. Wherein the VLP is essentially free of host RNA, preferably host nucleic acid; (b) comprises at least one antigen having at least one second attachment site; Provided is a composition wherein b) is bound to the VLP (a) via the at least one first attachment site and the at least one second attachment site.

In one aspect, the invention provides (a) a virus-like particle (VLP) comprising an RNA-bacteriophage coat protein, variant or fragment thereof having at least one first attachment site, wherein the VLP is recombined by the host. The VLP has a secondary structure of 60 μg or less, preferably 50 μg or less, more preferably 40 μg or less, preferably 27 μg or less, more preferably 20 μg or less, particularly 9 μg or less, most preferably 5 μg or less. A host RNA, preferably comprising a host nucleic acid; (b) comprising at least one antigen having at least one second attachment site; and wherein said at least one antigen (b) comprises said at least one first attachment. A composition is provided that binds to the VLP (a) via a site and the at least one second attachment site. In a preferred embodiment, the VLPs of the invention are essentially free of host RNA, preferably host nucleic acid.
The virus-like particle of the present invention is a recombinant VLP. Typically and preferably, the recombinant VLP is cloned into an expression vector that encodes a viral coat protein or variant or fragment thereof that retains the ability to form a VLP and is adapted to a compatible host, such as a bacterium. It is typically and preferably produced by expressing the resulting construct in an E. coli, yeast, insect or mammalian expression system.

A preferred example is disclosed in WO 02/056905, which is hereby incorporated by reference. A detailed description for preparing VLP particles from Qβ is disclosed in particular in Example 18 of WO 02/056905. VLP-antigen conjugates (conjugates) described in the prior art, such as WO 02/056905, are obtained and generated through expression in the manner described above, so that a certain amount of host in the VLP Note that it contains RNA. On the other hand, VLPs or VLP-antigen conjugates of the invention are prepared by the method of the invention or further processed to eliminate, reduce or remove host RNA, preferably host nucleic acids.
In a preferred embodiment, the virus-like particle comprises, consists essentially of or consists of RNA-bacteriophage coat proteins, and / or variants, and / or fragments thereof. In a still further preferred embodiment, the composition comprises virus-like particles of RNA-bacteriophage having at least one first attachment site. In a further preferred embodiment, the RNA-bacteriophage comprises: a) bacteriophage Qβ; b) bacteriophage R17; c) bacteriophage fr; d) bacteriophage GA; e) bacteriophage SP; f) bacteriophage MS2; g B) bacteriophage M11; h) bacteriophage MX1; i) bacteriophage NL95; j) bacteriophage f2; k) bacteriophage PP7, and l) bacteriophage AP205. In a further preferred embodiment, the RNA phage coat protein comprises SEQ ID NO: 10; a mixture of SEQ ID NO: 10 and SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 15 and SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22 and SEQ ID NO: 29, Or having an amino acid sequence selected from the group consisting of them.

The term “coat protein fragment” as used herein is able to aggregate to form a virus-like structure of an RNA phage, preferably at least 70%, preferably at least 80% of the length of the wild-type coat protein. , More preferably defined as a polypeptide that is at least 90%. Preferably, the fragment is obtained from at least one internal deletion, or at least one truncation at the N and / or C terminus, or at least one combination thereof. The coat protein fragments can assemble to form virus-like particles of RNA phage and have at least 80%, preferably 90%, more preferably 95% amino acid sequence identity with the coat protein fragments defined above. It further comprises a polypeptide having.
The term “mutant coat protein” or “coat protein variant” used interchangeably in this invention means that the wild type sequence and at least 80%, preferably at least 85%, 90%, 95%, 97% or A polypeptide having an amino acid sequence derived from a wild-type coat protein that is 99% identical and preferably retains the ability to assemble to form a VLP. Typical and preferred examples of mutations include one or more amino acid truncations, internal deletions, additions or substitutions relative to the wild type coat protein, and thus preferably against the wild type coat protein. A maximum of 10, more preferably a maximum of 6, even more preferably a maximum of 4, 3, 2 and 1 amino acid mutations are included. The “mutant coat protein” preferably includes fragments of a wild type coat protein that can be assembled to form a VLP. Furthermore, the term “mutant coat protein” as used herein is preferably at least 80%, preferably at least 85%, 90%, 95%, 97% or 99% identical to the corresponding wild type coat protein. It includes a polypeptide having an amino acid sequence, or preferably fragments thereof that can be assembled to form a VLP.

Formation of VLPs by a collection of coat proteins, variants or fragments thereof, as will be understood by those skilled in the art, allows expression of coat proteins, or variants or fragments thereof in E. coli, and optionally cell lysates. By purifying the capsid by gel filtration and analyzing the capsid formation by immunodiffusion assay (Oak Talony test) or electron microscopy (EM) (Kozlovska, TM et al., Gene 137: 133-37 (1993)) Can be tested. Immunodiffusion assays and EM may be performed directly on cell lysates.
In a further preferred embodiment of the present invention, the VLP comprises a mosaic VLP comprising or consisting of one or more sequences, preferably two sequences, of a coat protein of RNA phage and / or variants and / or fragments thereof. It is. Preferably, the mosaic VLP comprises or consists of two different coat proteins of RNA phage, the two coat proteins comprising SEQ ID NO: 10 and SEQ ID NO: 11, or SEQ ID NO: 15 and SEQ ID NO: 16. It has the amino acid sequence of In a further preferred embodiment, the mosaic VLP comprises or consists of two different coat proteins of RNA phage, the two coat proteins comprising C-terminal truncations of SEQ ID NO: 10 and SEQ ID NO: 11, or It has the C-terminal truncation amino acid sequence of SEQ ID NO: 15 and SEQ ID NO: 16. As used herein, the term “C-terminal truncation of SEQ ID NO: 11” refers to 1, 2, 3,... 50,. , ... means a sequence resulting from cleavage of 195 or 196 amino acids.

In a further preferred embodiment of the invention, the virus-like particles and the composition of the invention comprise: a) bacteriophage AP205; b) bacteriophage fr; c) bacteriophage R17; d) bacteriophage f2; e) bacteriophage SP; f) bacteriophage MS2; g) bacteriophage M11; h) bacteriophage MX1; i) bacteriophage NL95; and j) bacteriophage PP7 coat protein and / or fragment thereof, And / or comprise, consist essentially of, or consist of variants thereof.
In a further preferred embodiment of the invention, the virus-like particle and the composition of the invention comprise RNA-bacteriophage Qβ, or RNA-bacteriophage fr, or RNA-bacteriophage AP205, or RNA-bacteriophage GA coat protein, And / or comprises, consists essentially of or consists of fragments thereof. In a further preferred embodiment of the invention said RNA-bacteriophage is not MS2.

The capsid or virus-like particle of RNA-phage Qβ exhibits an icosahedral phage-like capsid structure of 25 nm in diameter and T = 3 pseudosymmetric. The capsid is linked to covalent pentamers and hexamers by disulfide bridges (Golmohammadi, R. et al., Structure 4: 543-5554 (1996)), leading to an extremely stable Qβ capsid, 180 of the coat protein. A copy of However, capsids or VLPs made from recombinant Qβ coat proteins may contain subunits that are not or incompletely bound to other subunits within the capsid via disulfide bonds. . However, typically about 80% or more of the subunits are linked to each other through disulfide bridges in the VLP. Qβ capsid proteins also exhibit unusual resistance to organic solvents and denaturing agents.
WO 2004/007538, in particular Example 1 and Example 2, describes a method for obtaining a VLP containing or consisting of AP205 coat protein, and in particular a method for its expression and purification. Yes. WO 2004/007538, and in particular its Example 1 and Example 2, are hereby incorporated by reference. However, it is noted that the VLP-antigen conjugate described in WO 2004/007538 contains a predetermined amount of host RNA obtained and expressed through expression in the VLP. On the other hand, the VLP-antigen conjugate or VLP of the present invention comprising a coat protein of RNA bacteriophage AP205, a variant thereof or a fragment thereof is used to eliminate, reduce or eliminate host RNA, preferably host nucleic acid. Prepared or further processed by the method of the present invention.

In addition, RNA phage coat proteins have also been shown to self-assemble upon expression in bacterial hosts (Kastelein, RA et al., Gene 23: 245-254 (1983), Kozlovskaya, TM et al., Dokl. Akad. Nauk SSSR 287: 452-455 (1986), Adhin, MR et al., Virology 170: 238-242 (1989), Priano, C et al., J. Mol. Biol. 249: 283-297 (1995)). In particular, GA (Ni, CZ et al., Protein Sci. 5: 2485-2493 (1996), Tars, K et al., J. Mol. Biol. 271: 759-773 (1997)) and fr (Pushko P et al., Prot. Eng 6: 883-891 (1993), Liljas, L et al., J. Mol. Biol. 244: 279-290 (1994)), the biological and biochemical properties are disclosed.
In a preferred embodiment, the RNA phage mutant coat protein is modified by removal of at least one or at least two lysine residues by substitution or deletion. In other embodiments, the RNA phage mutant coat protein is modified by addition of at least one, or at least two lysine residues, by substitution or insertion. In one highly preferred embodiment, the mutant coat protein is of RNA phage Qβ, wherein at least one or at least two lysine residues have been removed by substitution or deletion. In another highly preferred embodiment, the mutant coat protein is of RNA phage Qβ to which at least one or at least two lysine residues have been added by substitution or insertion. In a further preferred embodiment, the mutant coat protein of RNA phage Qβ has an amino acid sequence selected from any one of SEQ ID NOs: 23-27. Deletion, substitution or addition of at least one lysine residue, in order to adjust the degree of coupling, ie the amount of the antigen of the invention per subunit of the VLP of RNA-phage, in particular to suit the vaccine requirements Can be changed. The construction, expression and purification of the above-mentioned Qβ coat protein, mutant Qβ coat protein VLP and capsid are described in WO 02/056905, respectively. Reference is made in particular to Example 18 of the above-mentioned application. It should be noted that VLP-antigen conjugates described in the prior art, such as WO 02/056905, are obtained and produced through expression and therefore contain a certain amount of host RNA in the VLP. . On the other hand, VLP-antigen conjugates containing VLPs or Qβ mutant coat proteins of the invention are prepared by the methods of the invention and further processed to eliminate, reduce or remove host RNA, preferably host nucleic acids. Yes.

In a further preferred embodiment, the compositions and vaccines of the present invention have an antigen density of 0.05 to 4.0. The term “antigen density” as used herein is intended to mean the average number of antigens bound per subunit, preferably per coat protein of the VLP of RNA phage. Thus, this value is calculated as the average of all VLP monomers or subunits in the composition or vaccine of the invention. In a further preferred embodiment of the invention where the antigen has a molecular weight of 8 KDa or more, the antigen density is preferably 0.1 to 1.5. In a further preferred embodiment, wherein the antigen preferably consists of 5-30 amino acids, the antigen density is preferably 0.5-4.
In one preferred embodiment, the VLP comprises, consists essentially of or consists of a mutant coat protein of RNA-phage Qβ, fr, AP205 or GA, and / or fragments thereof.

In one preferred embodiment of the invention, the virus-like particle comprises, consists essentially of or consists of a coat protein of Qβ, a variant or fragment thereof, wherein the coat protein comprises the aforementioned It comprises, consists essentially of, or consists of either a variant and a corresponding A1 protein.
An assembly-eligible variant of AP205VLP, including AP205 coat protein, in which amino acid 5 is replaced with proline for threonine or amino acid 14 for asparagine for aspartic acid, can also be used in the practice of the present invention. It becomes another preferred embodiment.

The crystal structures of several RNA bacteriophages have been determined (Golmohammadi, R et al., Structure 4: 543-554 (1996)). Such information can be used to identify surface exposed residues, and thus RNA-phage coat proteins can be modified such that one or more reactive amino acid residues can be inserted by insertion or substitution. . As a result, modified forms of bacteriophage coat protein can also be used for the present invention. Another advantage of VLPs derived from RNA phage is their high expression yield in bacteria, especially E. coli, which allows the production of large quantities of material at an affordable cost.
In another preferred embodiment of the present invention, the composition further comprises at least one polyanionic polymer that binds to the VLP. Preferably, the polyanionic polymer is accommodated in the VLP. In a still further preferred embodiment, the at least one polyanionic polymer is (a) a polyanionic polypeptide; (b) a polyanionic saccharide, preferably not LPS; (c) a polyanionic organic polymer, and ( d) selected from the group consisting of nucleic acids, preferably not Toll-like receptor ligands, preferably not immunostimulatory nucleic acids.

In a further preferred embodiment, the at least one polyanionic polymer is a nucleic acid, wherein the nucleic acid is not a Toll-like receptor ligand. In a further preferred embodiment, the nucleic acid comprises or consists of more than 20 nucleotides. In a further preferred embodiment, the nucleic acid is tRNA. tRNA can be purchased from chemical companies or extracted from living organisms. Preferred tRNAs for this invention are, for example, wheat germ tRNA or yeast tRNA. In another preferred embodiment, the nucleic acid is DNA, where the DNA does not contain a CpG motif and does not stimulate TLR9.
In a preferred embodiment, the polyanionic polypeptide comprises (a) polyglutamic acid; (b) polyaspartic acid; (c) poly (GluAsp), and (d) any chemically modified of (a) to (c). Selected from the group consisting of things. Examples of chemical modifications include, but are not limited to, glucosylation, acetylation, and phosphorylation.

In another preferred embodiment of the present invention, the polyanionic saccharide comprises (a) anionic dextrans; (b) phosphocellulose; (c) polyglucoronic acid; (d) polygalacturonic acid; Selected from the group consisting of:) polysialic acid; (f) hyaluronic acid, and (g) glycosaminoglycans. In a further preferred embodiment, the anionic dextran is from the group consisting of (a) dextran sulfate; (b) carboxylmethyldextran; (c) sulfopropyldextran; (d) methylsulfonate dextran; and (e) dextran phosphate. Selected. In another more preferred embodiment, the glycosaminoglycan is selected from the group consisting of (a) heparin; (b) heparin sulfate; (c) dermatan sulfate; (d) chondroitin sulfate; and (e) keratan sulfate. .
In another preferred embodiment of the present invention, the polyanionic organic polymer is selected from the group consisting of (a) polyvinyl sulfate and (b) polyacrylates.

In certain preferred embodiments of the invention, the molecular weight of the at least one polyanionic polymer is from about 2000 to about 200000 daltons, and the lowest molecular weight is preferably at least about 3000 daltons, more preferably at least about 5000 daltons, and even more preferably Is at least about 7000 daltons, and the highest molecular weight is preferably at most about 200000 daltons, more preferably at most about 180000 daltons, more preferably at most about 160000 daltons, and even more preferably at most about 150,000 daltons .
The preferred molecular weight range varies depending on the type of polyanionic polymer. For example, in a polyanionic polypeptide, particularly polyglutamic acid and polyaspartic acid, the preferred molecular weight is 5000 daltons to 150,000 daltons. Thereby, the lowest molecular weight is preferably at least about 5000 daltons, more preferably at least about 10,000 daltons, and even more preferably at least about 30000 daltons. The highest molecular weight is preferably at most about 150,000 daltons, more preferably at most about 120,000 daltons, and even more preferably at most about 100,000 daltons.

In a highly preferred embodiment, the composition of the present invention contains at least one polyanionic polymer, wherein the polyanionic polymer is preferably at least one polyanionic polypeptide, and more preferably. The polyanionic polymer is at least one polyglutamic acid and / or polyaspartic acid.
The polyanionic polymer can be conveniently purchased from a chemical company. For example, polyglutamic acid and polyaspartic acid have a wide range of molecular weights (polyglutamic acid: 750-1500, 1500-3000, 3000-15000, 15000-50000, 50000-100000; polyaspartic acid: 5000-15000, 15000-50000). Covering, available from Sigma, New Jersey, USA. The molecular weight is determined based on the viscosity (Idelson, M. & Blout, ER (1958) J. Am. Chem. Soc. 80, p.4631) and the LALLS method, ie the low angle laser light scattering method.

Polygalacturonic acid (MW, 25000-50000) is from Fluka, Buchs, Swizerland, Dextran sulfate (MW, 5000, 8000 and 10000) is from Sigma, Dextran sulfate (MW, 100,000) is from Fluka. Can be purchased. Molecular weight is determined by LALLS, viscosity, or gel filtration methods. Organic polymers such as polyvinyl sulfate (MW, ˜170000) are purchased from Aldrich.
In a further aspect, the present invention provides the following steps: (a) recombinantly producing virus-like particles (VLPs) having at least one first attachment site by a host, wherein the VLPs are RNA-bacteriophages. (B) incubating the VLP with a solution containing metal ions capable of hydrolyzing host RNA, preferably host nucleic acid; (c) (a) or (b) The composition of the present invention comprising binding at least one antigen having at least one second attachment site to the VLP obtained from step (b), preferably from the step (b). A method for the preparation of bacteriophage VLP-antigen conjugates. In a preferred embodiment, the metal ions are selected from zinc (Zn) ions, copper (Cu) ions and iron (Fe) ions. More preferably, the VLP incubation is followed by a purification step of at least one VLP, preferably by dialysis, to remove the digested nucleic acid.

In another embodiment, the present invention provides the following steps: (a) Recombinantly producing a virus-like particle (VLP) having at least one first attachment site by a host, wherein the VLP is RNA-bacterio A phage coat protein, variant or fragment thereof; (b) incubating the VLP with RNase; (c) the VLP obtained from step (a) or (b), preferably from step (b) Provided is a composition of the present invention and a method for preparing a VLP-antigen conjugate of RNA-bacteriophage, comprising binding at least one antigen having at least one second attachment site to the resulting VLP . In a preferred embodiment, the RNase is RNaseA. In another preferred embodiment, the incubation of the VLP with the RNase, preferably RNase A, is performed in a low ionic strength buffer, wherein the low ionic strength buffer is preferably less than 50 mM, more preferably less than 40 mM, More preferably, it has a concentration of 30 mM or less, and a more preferable low ionic strength buffer has a concentration of 30 mM. More preferably, the incubation of said VLP step is followed by a purification step of at least one VLP, preferably by dialysis, in order to remove the digested nucleic acid.
In a further embodiment, the present invention provides the following steps: (a) recombinantly producing virus-like particles (VLPs) having at least one first attachment site by a host, wherein the VLPs are RNA-bacteriophage. (B) degrading the virus-like particle into the coat protein, variant or fragment of the RNA-bacteriophage; (c) the coat protein, variant thereof (D) allowing the purified coat protein, variant or fragment thereof of the RNA-bacteriophage to be reconstituted into a virus-like particle; wherein the virus-like particle is a host RNA, preferably Is essentially free of host nucleic acids; and (e) at least one antigen having at least one second attachment site in the VLP obtained from step (d). It comprises binding, provides a process for preparing a composition, and RNA- bacteriophage VLP- antigen conjugates of the present invention. In a further preferred embodiment, reconstitution of the purified coat protein is achieved by the presence and / or addition of at least one polyanionic polymer. The method of the invention is particularly advantageous because it allows for the release of essentially all host nucleic acids, especially through a degradation step.

In a preferred embodiment of the invention, the degradation of the virus-like particles is preferably achieved under denaturing conditions. Preferred denaturing conditions are high concentrations of salt, for example 0.5M to 1M magnesium chloride, or 5M or more urea. More preferably, the step of purifying the coat protein, variant or fragment thereof comprises, in particular, cation exchange chromatography and / or size exclusion chromatography in order to separate the host nucleic acid from the coat protein, variant or fragment thereof. Is achieved.
In a further preferred embodiment, the present invention comprises the following steps: (a) recombinantly producing an RNA-bacteriophage coat protein, variant or fragment by a host; (b) the coat protein, variant or (C) forming a virus-like particle having at least one first attachment site, wherein the virus-like particle is essentially free of host RNA, preferably host nucleic acid; and ( d) a composition of the present invention comprising binding at least one antigen having at least one second attachment site to the VLP obtained from step (c), and an RNA-bacteriophage VLP-antigen Methods for preparing conjugates are provided. In a further preferred embodiment, reconstitution of the purified coat protein is achieved by the presence and / or addition of at least one polyanionic polymer.

Again, in a further aspect, the invention provides a composition of the invention and a method for producing a VLP-antigen conjugate of RNA-bacteriophage, wherein the VLP and the antigen are via at least one peptide bond. Are connected. The method comprises the following steps: (a) recombinantly producing a fusion protein containing an antigen and an RNA-bacteriophage coat protein, variant or fragment thereof by a host; (b) purifying the fusion protein. (C) forming a virus-like particle, wherein the virus-like particle comprises essentially no host RNA, preferably no host nucleic acid. In a further preferred embodiment, reconstitution of the purified fusion protein is achieved by the presence and / or addition of at least one polyanionic polymer.
An alternative method is (a) recombinantly producing virus-like particles (VLP) by a host, wherein the VLP contains at least one antigen and an RNA-bacteriophage coat protein, variant or fragment thereof. Degrading the VLP into the fusion protein; purifying the fusion protein; reconstructing the purified fusion protein into a VLP, wherein the virus-like particle is a host RNA, preferably a host nucleic acid Containing a process essentially free of In a further preferred embodiment, reconstitution of the purified fusion protein is achieved by the presence and / or addition of at least one polyanionic polymer. In a preferred embodiment, the VLP is a VLP of RNA-bacteriophage AP205.

In a highly preferred embodiment of the invention, the composition of the invention essentially comprises the host nucleic acid of RNA phage Qβ, AP205, fr or GA, more preferably RNA phage Qβ or GA, most preferably RNA phage Qβ. And the composition further comprises at least one polyanionic molecule, preferably polyglutamic acid and / or polyaspartic acid. In another preferred embodiment of the invention, the composition further comprises a VLP of RNA phage AP205 or fr that is further free of at least one polyanionic polymer and is essentially free of host nucleic acid.
In a further preferred embodiment of the invention, the at least one antigen is selected from the group consisting of proteins, polypeptides, carbohydrates, steroid hormones, organic molecules and haptens.

In one preferred embodiment of the invention, the at least one antigen is a hapten. Preferred haptens are hormones, drugs and toxic compounds. More preferred are drugs, especially drugs that are dependent and abused, respectively, and even narcotics for recreation. Representative examples of such antigens include opioid and morphine derivatives such as codeine, fentanyl, heroin, morphium, opium; relaxants such as diazepam; stimulants such as amphetamine, cocaine, MDMA (methylenedioxymethamphetamine), methamphetamine, Methylphenidate and nicotine; hallucinogens such as PCP, LSD, mescaline, and sirocibin; cannabinoids such as hashish and marijuana; and desipramine / imipramine class drugs, and nortriptyline / amitriptyline class drugs. In the treatment of nicotine addiction, metabolites including nornicotine and cotinine, and nicotine derivatives such as O-succinyl-3′-hydroxymethyl-nicotine may be targeted.
Detailed teachings of conjugating nicotine or nicotine derivatives, or other drugs that are abused, such as cocaine, to VLPs are disclosed in WO 2004/009116, in particular line 28 on page 61 to line 13 on page 66. Yes. Thus, the general and specific disclosures of WO 2004/009116 are hereby incorporated by reference.

Thus, in one preferred embodiment, the at least one antigen is an organic molecule selected from addictive drugs, wherein preferably the addictive drug is nicotine or cocaine, especially nicotine.
In a preferred embodiment of the invention, the at least one antigen is a self antigen or a fragment or variant thereof. Preferably, the self-antigen is a protein or fragment thereof suitable for inducing an immune response against the self-antigen. Thus, the present invention provides vaccine compositions suitable for use in methods for preventing and / or attenuating diseases or conditions caused or exacerbated by “self” gene products (eg, tumor necrosis factor). Thus, vaccine compositions of the present invention include compositions that result in the production of antibodies that prevent and / or attenuate diseases or conditions caused or exacerbated by "self" gene products. Examples of such diseases or conditions include graft-versus-host disease, IgE-mediated allergic reaction, anaphylaxis, adult respiratory distress syndrome, Crohn's disease, allergic asthma, acute lymphocytic leukemia (ALL), non-Hodgkin lymphoma ( NHL), Graves' disease, systemic lupus erythematosus (SLE), inflammatory autoimmune disease, myasthenia gravis, immunoproliferative disease lymphadenoma (IPL), vascular immunoproliferative lymphadenoma (AIL), immunoblastic Includes lymphadenopathy (IBL), rheumatoid arthritis, diabetes, psoriasis, myocarditis, multiple sclerosis, Alzheimer's disease, and osteoporosis.

In a highly preferred embodiment of the present invention, the at least one antigen of the present invention comprises lymphotoxins (eg, lymphotoxin α (LTα), lymphotoxin β (LTβ)), lymphotoxin receptor, nuclear factor kB ligand receptor activator. (RANKL), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGF-R), interleukin-5, interleukin-17, interleukin-13, IL-23p19, ghrelin, CCL21, CXCL12, SDF- 1, M-CSF, MCP-1, endoglin, GnRH, TRH, eotaxin, bradykinin, BLC, tumor necrosis factor α, amyloid beta peptide (Aβ _1-42 ), Aβ _1-6 , angiotensin, gastrin, progastrin, CETP, CCR5, C5a, CXCR4, Des-A Selected g- bradykinin, or from the group consisting of fragments or variants of the above antigens. When used against the antigens described above, the term “fragment” of the antigen refers to (i) the sequence of the corresponding antigen, or in some cases, the self-antigen, the human sequence of the wild-type antigen deposited in GeneBank, or At least 4, 5, preferably at least 6, 7, 8, 9, 10, 11, 12, 17 of the human sequence of the corresponding ortholog from any other animal, preferably the wild type antigen deposited in GeneBank. Any polypeptide comprising, or preferably consisting of, 18, 19, 20, 25 or 30 contiguous amino acids; and (ii) having an amino acid sequence identical to any one of the polypeptides of (i) Any polypeptide should be referred to, wherein the amino acid sequence identity is 65% or more, preferably 80% or more, more preferably 90% or more, and more preferably 95%. That is all. Furthermore, the term “fragment” of an antigen listed above should include at least one antigenic site. Preferably, as used herein, the term “fragment” of the antigens listed above refers to no more than 100 amino acids, preferably no more than 80 amino acids, preferably no more than 60 amino acids, more preferably no more than 40 amino acids. Again, it should mean a polypeptide having a length of more preferably 30 amino acids or less, and even more preferably 20 amino acids or less. More preferably, as used herein and when referring to an autoantigen, the term “fragment” of an antigen listed above should refer to a polypeptide when provided in the present invention, and The production of antibodies that specifically bind to the listed corresponding antigens should be inducible in vivo. Further, as used herein, the term “fragment” of an antigen listed above refers to a polypeptide obtained by truncation or internal deletion of at least one, preferably two, more preferably one of the corresponding antigens. Should preferably mean. Methods for determining the antigenic site (s) of a protein are known to those skilled in the art. Some of these methods are detailed in US Provisional Application No. 60/669322, page 26, first paragraph to page 27, fourth paragraph, the specific disclosures of which are incorporated herein by reference. Is done. It should be noted that these methods are generally applied to antigens of other polypeptides and are therefore not limited to IL-23p19 as disclosed in US Provisional Application No. 60/669322.
When used with the antigens described above, the term “variant” of that antigen refers to the antigens listed above, or in the case of autoantigens, the human sequence of the wild-type antigen deposited in GeneBank or any other 70% or more, preferably 80% or more, more preferably 90% or more, more preferably 95%, relative to the sequence of the corresponding ortholog from the animal, preferably the human sequence of the wild-type antigen deposited in GeneBank Most preferably should refer to any polypeptide comprising or preferably consisting of any natural or genetically engineered polypeptide or protein having 97% or more amino acid sequence identity. Preferred methods for producing protein variants are by genetic engineering, preferably insertions, substitutions, deletions or combinations thereof. As provided in the present invention, protein variants should be capable of in vivo production of antibodies that specifically bind to the corresponding antigens listed above.

In this application, the antibody is 10 ⁶ M ⁻¹ or more, preferably 10 ⁷ M ⁻¹ or more, more preferably 10 ⁸ M ⁻¹ or more, most preferably 10 ⁹ M ⁻¹ or more. The binding affinity (Ka) is defined as that which specifically binds to the antigen, antigen protein, antigen fragment or antigen variant of the present invention if it binds. Antibody affinity can be readily determined by those skilled in the art, typically and preferably by Scatchard analysis.
In one preferred embodiment, the at least one antigen is a GnRH-peptide (Luteinizing Hormone Releasing Hormone, or Gonadotropin Releasing Hormone, also known as LHRH). VLP-GnRH conjugates useful for vaccine manufacture are disclosed in PCT / EP2005 / 053858, which is hereby incorporated by reference in its entirety.

As used herein, the term “GnRH-peptide” means at least one, preferably one mammalian GnRH, and in particular at least one of SEQ ID NO: 1 or SEQ ID NO: 28, preferably SEQ ID NO: 1. A peptide comprising, consisting essentially of or consisting of one amino acid sequence, preferably one amino acid sequence, or a fragment or variant thereof. In some embodiments, the GnRH peptide comprises an N-terminal puroglutamic acid (pGlu or pE). In other embodiments, the GnRH peptide comprises a C-terminal glycinamide (G-NH2). In another embodiment of the invention, the GnRH peptide comprises more than one GnRH peptide or fragment thereof, eg, two, three or more GnRH peptides or fragments thereof, as in SEQ ID NOs: 30-32. Included in tandem. The tendem-GnRH peptide of the present invention further includes a peptide in which GnRH sequences are linked to each other via a spacer. The nature of the spacer group can vary greatly from one or more amino acids to shorter or longer hydrocarbon chains and other compound groups or molecules.
In a preferred embodiment, the GnRH peptide is linked to an amino acid linker comprising or consisting of cysteine. In a further preferred embodiment, the amino acid linker is a cysteine fused to either the N- or C-terminus of the GnRH peptide. In a further preferred embodiment, cysteine is either N- or C-terminal of SEQ ID NO: 1 (becomes SEQ ID NO: 4 or 5), preferably N-terminal of SEQ ID NO: 1 (SEQ ID NO: 4). Is fused. In another preferred embodiment, the linker CGG is fused to the N-terminus of the GnRH peptide, preferably the N-terminus of SEQ ID NO: 1 or SEQ ID NO: 28, more preferably the N-terminus of SEQ ID NO: 1. Number: 2 In yet another preferred embodiment, the linker GGC is fused to the C-terminus of the GnRH peptide, preferably the C-terminus of SEQ ID NO: 1 or SEQ ID NO: 28, more preferably the C-terminus of SEQ ID NO: 1. It becomes sequence number: 3.

This composition of the invention containing GnRH as at least one antigen can be administered to mammals such as pigs to prevent boar contamination of meat. Compositions containing GnRH can be administered to animals, such as dogs, cats, sheep, cows, to control their reproductive behavior and / or reduce their reproductivity. This modified VLP containing GnRH can be administered to a human suffering from a gonadal steroid hormone dependent cancer.
In a preferred embodiment, the at least one antigen of the invention comprises the amino acid sequence DAEFRHDSGYEVHHQKLVFFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 86), or an amyloid beta peptide (Aβ _1-42 ) having a fragment thereof, such as the amino acid sequence DAEFRH (SEQ ID NO: 87). Aβ _1-6 . Accumulation of amyloid beta peptide can cause or exacerbate Alzheimer's disease, so the compositions of the present invention provide a method of treating or alleviating a disease state. For coupling of various Aβ fragments to various VLPs, such as Qβ or fr, and their vaccination to mice, Example 13 of WO 02/056905, incorporated herein by reference, 15, 17 and 54. Fusion of viral coat protein and Aβ _1-6 fragment is disclosed in WO 04/016282, Examples 7, 8, 9, 10, 11; further coupling of Aβ _1-6 to VLPs Are disclosed in Examples 13, 18 and 20; immunization of animals with VLP-Aβ _1-6 and the resulting vaccine effect is described in Examples 12, 14 of WO 04/016282. 16, 17, 19 and 20.

In another preferred embodiment of the invention, the at least one antigen is ghrelin or a fragment or variant thereof. Ghrelin is a major regulator of eating behavior. Peripheral administration of ghrelin increases food intake and leads to weight gain (Tschop et al., Nature 407: 908-12). Thus, immunization against ghrelin of the present invention provides a method for treating obesity. Preferred ghrelin or ghrelin peptides are disclosed on pages 68-74 of WO 04/009124. Ghrelin may be selected from other mammalian species than humans, such as dogs and cats. Examples 8, 9, 13, 15 of WO 04/009124 disclose several methods for coupling ghrelin to VLPs and are hereby incorporated by reference. In a further preferred embodiment, the at least one antigen is a ghrelin fragment comprising or consisting of the amino acid sequence of SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79 or SEQ ID NO: 82 or SEQ ID NO: 85. In another more preferred embodiment, the at least one antigen is a ghrelin fragment comprising or consisting of SEQ ID NO: 83, wherein said composition of the invention is preferably administered to a dog; SEQ ID NO: 84 In this case, the composition of the present invention is preferably administered to a cat.
In another preferred embodiment of the invention, the at least one antigen is an IL-23p19 protein or IL-23p19 fragment, as described in PCT / EP2005 / 004980, which is hereby incorporated by reference in its entirety. It is. In one preferred embodiment, the IL-23 protein comprises, consists essentially of or consists of SEQ ID NO: 6 or SEQ ID NO: 7. In one preferred embodiment, the IL-23 fragment is any one of the sequences of (a) SEQ ID NO: 8-9, and (b) SEQ ID NO: 33-44; (c) (a) and (b). It comprises, consists essentially of or consists of any sequence selected from the group consisting of fragments or variants.

In another preferred embodiment of the invention, the at least one antigen is an angiotensin peptide or a fragment thereof. As used herein, the term “angiotensin peptide” includes any peptide or fragment thereof comprising an angiotensinogen, angiotensin I or angiotensin II sequence. The sequences are as follows: Angiotensinogen: DRVYIHPFHLVIHN (SEQ ID NO: 108); Angiotensin I: DRVYIHPFHL (SEQ ID NO: 109); Angiotensin II: DRVYIHPF (SEQ ID NO: 110). Typically, one or more additional amino acids are added to either the C- or N-terminus of the angiotensin peptide sequence. These additional amino acids are particularly beneficial for oriented and regular binding to VLPs. A further preferred embodiment is disclosed in WO 03/031466, which is hereby incorporated by reference. Particularly preferred angiotensin fragment sequences having a second attachment site are: a) CGGDRVYIHPF (SEQ ID NO: 111); b) CGGDRVYIHPFHL (SEQ ID NO: 112); c) DRVYIHPHFLGGC (SEQ ID NO: 113); d) CDRVYIHPFHL (SEQ ID NO: 114); e) CHPFHL (SEQ ID NO: 115); f) CGPFHL (SEQ ID NO: 116); g) CYIHPF (SEQ ID NO: 117); h) CGIHPF (SEQ ID NO: 118); i) CGGHPF (SEQ ID NO: 119); j) DRVYIGGC (SEQ ID NO: 120); k) DRVYGGC (SEQ ID NO: 121); and l) DRVGGC (SEQ ID NO: 122). Thus, the compositions of the present invention provide a method of treating or alleviating a hypertensive condition. A detailed description of the preparation of the composition and its use is disclosed in WO 03/031466, the entire application of which is hereby incorporated by reference.
In another preferred embodiment of the invention, the at least one antigen is RANKL (osteoclast differentiation factor), or a fragment or variant thereof. RANKL is a 245 amino acid transmembrane protein belonging to the TNF-superfamily. A portion of the extracellular region (178aa) can be cleaved by a TACE-like protease (Lum et al., J Biol Chem. 274: 13613 (1999)). The amino acid sequence of the extracellular portion of human RANKL is shown in SEQ ID NO: 221 (TrEMBL: O14788), and the amino acid sequence of the human isoform is SEQ ID NO: 222 of WO 02/056905. The sequence and isoform of the extracellular portion of murine RANKL is shown in SEQ ID NO: 223 (TrEMBL: O35235) and SEQ ID NO: 224 (TrEMBL: Q9JJK8 and TrEMBL: Q9JJK9) of WO 02/056905. ing.

RANKL has been shown to be an essential factor in osteoclast formation. Inhibition of the interaction between RANKL and its receptor RANK leads to suppression of osteoclast formation, thus providing a means to stop excessive bone resorption as seen in osteoporosis and other medical conditions.
A human-RANKL construct with an N-terminal amino acid linker containing a cysteine residue fused to the extracellular portion of RANKL is a highly preferred embodiment of the present invention. Further information on the physiological function of RANKL and methods for expressing it is disclosed on pages 62-64 of WO 02/056905, and the human-RANKL construct is disclosed in WO 02/056905. Example 6, which is hereby incorporated by reference. Further preferred embodiments of the RANKL protein, fragment or RANKL peptide variant are disclosed in WO 03/039225 at paragraphs 223-235, incorporated herein by reference.

In another embodiment of the invention, the at least one antigen is interleukin-17 (IL-17) or a fragment or variant thereof. Human IL-17 is a 32-kDa disulfide-linked homodimeric protein with variable glycosylation (Yao, Z. et al., J. Immunol. 155: 5483-5486 (1995)). The amino acid sequences of human and mouse IL-17 are given in SEQ ID NO: 228 (Accession #: AAC50341) and SEQ ID NO: 229 (Accession #: AAA37490) of WO 02/056905, respectively.
Clinical trials have shown that IL-17 is involved in many inflammatory diseases. High levels of IL-17 have been reported in patients with rheumatoid arthritis (Ziolkowska M. et al., J Immunol. 164: 2832-8 (2000)). Interleukin 17 has an effect on proteoglycan degradation in the knee joint of mice (Dudler J. et al., Ann Rheum Dis. 59: 529-32 (2000)) and contributes to synovial matrix destruction (Chabaud M. Et al., Cytokine. 12: 1092-9 (2000)). Increased levels of IL-17 mRNA have been found in mononuclear cells from patients with multiple sclerosis (Matusevicius, D. et al., Mult. Scler. 5: 101-104 (1999)). In patients suffering from painful systemic lupus erythematosus, elevated serum levels of IL-17 are observed (Wong CK et al., Lupus 9: 589-93 (2000)). Furthermore, IL-17 mRNA levels are increased in T cells isolated from damaged psoriatic skin (Teunissen, MB et al. And J. Invest. Dermatol. 111: 645-649 (1998)). . The involvement of IL-17 in rejection of kidney grafts has also been shown (Fossiez F. et al., Int. Rev. Immunol. 16: 541-51 (1998)). The above findings suggest that IL-17 plays a central role in the initiation and maintenance of inflammatory responses (Jovanovic, DV et al., J. Immunol. 160: 3513-3521 (1998)). Human and mouse IL-17 sequences were provided in WO 02/056905, SEQ ID NO: 228 (AAC50341) and SEQ ID NO: 229 (AAA37490). A method for expressing IL-17 is described on page 69 of WO 02/056905, hereby incorporated by reference.

In another preferred embodiment of the invention, the at least one antigen is interleukin-13 (IL-13) or a fragment or variant thereof. The amino acid sequence of human IL-13 precursor is shown in SEQ ID NO: 230 of WO 02/056905, and the amino acid sequence of processed human IL-13 is shown in SEQ ID NO: 231. The first 20 amino acids of the precursor protein correspond to the signal peptide and not in the processed protein.
IL-13 is a helper T cell-inducible cytokine (IL-4, IL-5, etc.) that has recently been associated with allergic airway responses (asthma). Upregulation of IL-13 and IL-13 receptor has been found in many tumor types (Hodgkin lymphoma). Interleukin 13 is secreted from Hodgkin and Reed-Sternberg cells and promotes its growth (Kapp U et al., J Exp Med. 189: 1939-46 (1999)). Thus, immunization against IL-13 provides a method for treating other conditions described above, such as asthma or Hodgkin lymphoma. A method for expressing IL-13 is described on page 70 of WO 02/056905, and the IL-13 construct is disclosed in Example 9 of WO 02/056905. Incorporated herein by reference.

In yet another embodiment of the invention, the at least one antigen is interleukin-5 (IL-5) or a fragment or variant thereof. IL-5 is a lineage-specific cytokine for eosinophilopoiesis, and plays an important role in diseases associated with an increase in the number of eosinophils such as asthma. The sequence of the precursor and processed human IL-5 is provided in SEQ ID NO: 233 and SEQ ID NO: 234 of WO 02/056905, respectively, and the processed mouse amino acid sequence is It is shown in SEQ ID NO: 235 of No. 02/056905.
The biological function of IL-5 has been shown in several studies (Coffman RL et al., Science 245: 308-10 (1989); Kopf et al., Immunity 4: 15-24 (1996)). It presents a beneficial effect of inhibiting the function of IL-5 in sphere-mediated diseases. Thus, inhibiting the action of IL-5 provides a method for treating asthma and other diseases associated with eosinophils.

In another preferred embodiment of the invention, the at least one antigen is CCL-21 or a fragment or variant thereof. In a related preferred embodiment, the antigenicity is CXCL12, also referred to as SDF-1. The chemokine receptors CCR7 and CXCR4 are upregulated in breast cancer cells, and CCL21 and CXCL12, their respective ligands, have been shown to be highly expressed in organs that represent the first destination of breast cancer metastasis (Muller et al. (Nature 410: 50-6 (2001))). Thus, immunization against each of CCL21 and CCL12 provides a method for treating metastatic spread in cancer, particularly breast cancer. Furthermore, the CCL12 / CXCR4 chemokine-receptor pair has been shown to increase the effectiveness of homing of primary hematopoietic progenitor cells to the bone marrow. Furthermore, CXCR4 and SDF-1 appear to influence the distribution of chronic lymphocytic leukemia cells. Thus, immunization against CXCL12 provides a method for treating chronic lymphocytic leukemia. Furthermore, CCL12-CXCR4 interaction plays a central role in the accumulation of CD4 + T cells in the rheumatoid arthritis synovium (Nanki et al., 2000). Thus, immunization against SDF-1 provides a method for treating rheumatoid arthritis.
The human and mouse sequences of CCL21 are found in WO 02/056905, SEQ ID NO: 236 (Swissprot: SY21_human) and SEQ ID NO: 237 (Swissprot: SY21_mouse), respectively. The human and mouse sequences of CCL12 are found in WO 02/056905, SEQ ID NO: 238 (Swissprot: SDF1_human) and SEQ ID NO: 239 (Swissprot: SDF1_mouse), respectively.

In yet another embodiment of the invention, the at least one antigen is a B-lymphocyte chemoattractant (BLC, CXCL13) or a fragment or variant thereof. The sequences of human and mouse BLC are shown in SEQ ID NO: 240 (accession number: NP_006410) and SEQ ID NO: 241 (accession number: NP_061354) of WO 02/056905, respectively. The signal peptide is the first 22 or 21 amino acids for human and mouse, respectively. Compositions of the invention that contain BLC preferably comprise the mature form of the protein.
Additional physiological functions of BLC and methods for preparing the compositions are disclosed in WO 02/056905, pages 74-75, incorporated herein by reference. Immunization against BLC provides a method for the treatment of autoimmune diseases that involve lymphoid neogenesis, such as rheumatoid synovitis and rheumatoid arthritis or type I diabetes.

In another specific embodiment, at least one antigen of the invention is eotaxin or a fragment or variant thereof. IL-5 is thought to be responsible for the migration of eosinophils from the bone marrow to the blood, whereas eotaxin is for local migration within the tissue (Humbles et al., J. Exp. Med 186: 601-12 (1997)). In WO 02/056905, which is hereby incorporated by reference, the sequence of human eotaxin-1 is shown in SEQ ID NO: 242 (amino acids 1-23 correspond to signal peptides) and human eotaxin-2 The sequence of is shown in SEQ ID NO: 243 (amino acids 1-26 corresponds to a signal peptide), and the sequence of human eotaxin-3 is shown in SEQ ID NO: 244 (amino acids 1-23 corresponds to a signal peptide). The sequence of mouse eotaxin-1 is shown in SEQ ID NO: 245 (amino acids 1-23 corresponds to the signal peptide), and the sequence of mouse eotaxin-2 is SEQ ID NO: 246 (amino acids 1- 2 23 corresponds to a signal peptide). Further, physiological and biochemical information of eotaxin and its expression and construction in the present invention are disclosed in WO 02/056905, pages 75 to 76, and are incorporated herein by reference.
In yet another specific embodiment of the invention, the at least one antigen is macrophage colony stimulating factor (M-CSF or CSF-1) or a fragment or variant thereof. Increased expression levels of M-CSF and its receptors are associated with poor prognosis in certain epithelial cancers such as breast cancer, uterine cancer and ovarian cancer. The structural data of the solubilized form of M-CSF is available (Crystal structure: Pandit et al., Science 258: 1358-62 (1992)). The human sequence is shown in SEQ ID NO: 247 (Accession Number: NP_000748) of WO 02/056905. Further preferred antigens of the invention comprise an N-terminal fragment consisting of residues 33-181 or 33-185 of SEQ ID NO: 247, corresponding to a solubilized form of the receptor. The mouse sequence (Accession number: NP_031804) is shown in SEQ ID NO: 248 of WO 02/056905. Further biological information of M-CSF and its expression and construction of the compositions of the present invention are disclosed on pages 77-78 of WO 02/056905, incorporated herein by reference.

In another preferred embodiment of the invention, the at least one antigen is a TNF-superfamily member or a fragment or variant thereof. As used herein, the term “TNF-superfamily member” refers to a protein containing a TNF-like domain. As used herein, “TNF-superfamily member” refers to all forms of TNF known in humans, cats, dogs, mice, rats, common wild animals, generally mammals, as well as other animals. -Including super family members. TNF-superfamily members contain a spherical TNF-like extracellular domain of about 150 residues, which is a conserved domain database CDD (Marchler-Bauer A, et al., (2003), “CDD: a curated Entrez database of conserved domain alignments ", Nucleic Acids Res. 31: 383-387), classified as cd00184, pfam00229 or smart00207. As used herein, TNF-superfamily members are: TNFα, LTα, LTα / β, FasL, CD40L, TRAIL, RANKL, CD30L, 4-1BBL, OX40L, GITRL and BAFF, CD27L, TWEAK, APRIL, TL1A, Includes EDA and any other polypeptide from which a TNF-like domain can be identified.
In a further preferred embodiment, at least one antigen of the invention is a TNF-peptide. As used herein, the term “TNF-peptide” refers to an amino acid sequence homologous to that corresponding to amino acid residues 3-8 of the consensus sequence for the conserved domain pfam00229 (SEQ ID NO: 45), preferably the conserved domain pfam00229. It means a peptide sequence containing a peptide sequence homologous to amino acid residues 1 to 8 of the consensus sequence for (SEQ ID NO: 45), more preferably a peptide sequence homologous to amino acid residues 1 to 13 of the consensus sequence. When the TNF-peptide is a peptide from human or mouse TNFα, the TNF-peptide is 6-18 amino acid residues long, preferably 6-16 amino acid residues long, more preferably 6-14 It consists of a peptide having an amino acid residue length. Homologous peptides are peptides derived from TNF-superfamily members of animals, including humans, particularly mammalian TNF-superfamily members such as mouse or human RANKL, or mouse or human TNFα, Represents the corresponding amino acid residue. These homologous peptides can be identified by those skilled in the art in a manner that aligns the TNF-superfamily members of other animals with the TNF-superfamily consensus sequence (SEQ ID NO: 45). As explained above, the TNF-peptide includes a peptide sequence corresponding to the amino acid sequence described above of the consensus sequence. Other TNF-peptides of a particular homologous region having a consensus sequence (eg, amino acid residues 3-8 of the consensus sequence) differ from polypeptides that are TNF-superfamily members. Preferably, however, some of the other TNF-peptides having the consensus sequence identified above have a TNF-superfamily member, preferably a mammalian TNF-superfamily member, more preferably human TNF-super. It is at least 70% identical, preferably at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to a polypeptide that is a family member.

In a further preferred embodiment, at least one antigen of the invention is a human TNFα peptide (4-23) having the sequence SSRTPSDKPVAHVVANPQAE (SEQ ID NO: 88). In another more preferred embodiment of the invention, the antigen is a mouse TNFα peptide (4-23) having the sequence SSQNSSDKPVAHVVANHQVE (SEQ ID NO: 89). In another preferred embodiment, the antigen is amino acid residues 22-32 of mature mouse TNFα: VEEQLEWLSQR (SEQ ID NO: 90). In yet another preferred embodiment, the antigen is amino acid residues 22-32 of human TNF-α: AEGQLQWLNRR (SEQ ID NO: 91).
In a preferred embodiment, the composition of the invention containing mouse TNFα peptide (4-23) is an autoimmune disease or a bone-related disease such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, diabetes Used in the manufacture of a medicament for treating autoimmune thyroid disease, autoimmune hepatitis, psoriasis, and psoriatic arthritis, wherein the bone-related diseases are osteoporosis, periondontis, periprosthetic osteolysis, Selected from the group consisting of bone metasis, bone cancer pain, Paget's disease, multiple myeloma, Sjorgen's syndrome, and primary biliary cirrhosis.

In one preferred embodiment of the invention, the autoantigen is VEGF or VEGFR disclosed on pages 55-56 of WO 02/056905, or a fragment or variant thereof; 79 of WO 02/056905. Lymphotoxin β disclosed on page 80; may be lymphotoxin β and lymphotoxin α and lymphotoxin β receptor on page 93 of WO 02/056905. That information is incorporated herein by reference.
In a preferred embodiment, the at least one antigen is CXCR4 or a fragment or variant thereof. The chemokine receptor CXCR4, also known as LESTR or Fusin, belongs to the family of seven transmembrane domain G-protein coupled receptors (Federsppiel et al. (1993), Genomics 16: 707). The only known ligand for CXCR4 is SDF-1 (Pelchen-Mattews et al. (1999) Immunol. Rev. 168: 33). CXCR4 and SDF-1 are involved in many areas of human physiology such as hematopoiesis, T-cell activation and migration to inflammatory sites, and T-cell homing, angiogenesis, brain development, and embryogenesis. (Murdoch, (2000) Immunol. Rev. 177: 175).

Recently, CXCR4 has been identified as a co-receptor for HIV (Feng et al. (1996) Science 272: 872). Thus, HIV strains that require CXCR4 for insertion are classified as X4 strains. SDF-1 has been shown to block HIV-1 insertion (Oberlin et al. (1996), Nature 382: 833; Bleul et al. (1996) Nature 382: 829).
In a preferred embodiment of the invention, the at least one antigen comprises or consists of a fragment of the CXCR4 extracellular domain. The CXCR4 extracellular domain fragment has at least 6, 7, preferably 8, 9, 10 amino acids, and the CCR5 extracellular domain fragment is 30, preferably 20, more preferably 15, even more preferably less than 12. It has the amino acid.

In a preferred embodiment, the at least one antigen comprises or consists of the N-terminal extracellular domain of CXCR4. In a further preferred embodiment, the N-terminal extracellular domain of CXCR4 comprises or consists of SEQ ID NO: 66. In a preferred embodiment, the at least one antigen comprises or consists of a fragment of the CXCR4 extracellular domain ECL2. Preferably, said fragment has at least 6, preferably 7 amino acids. In a further preferred embodiment, the at least one antigen comprises or consists of a fragment of CXCR4 extracellular domain ECL2 having the amino acid sequence of SEQ ID NO: 65.
In one preferred embodiment of the invention, at least one antigen is CCR5 or a fragment or variant thereof. The HIV R5 strain uses cell surface molecules CD4 and CCR5 for binding and insertion into macrophages and CD4 + T cells. CCR5 is a seven-transmembrane type with four extracellular domains: 3 loops exposed to the extracellular space and N-terminal sequences, termed ECL-1, ECL-2, ECL-3, and PNt It is a receptor. The natural CCR5 ligand, RANTES, MIP-1α, MIP-1β and their analogs can block virus-co-receptor interactions and also cause internalization of CCR5 (Lederman et al., 2004, Science 306 , p485). CCR5-specific autoantibodies have been found in 12.5% of women who have been repeatedly exposed to HIV but remain uninfected (Lopalco et al., 2000, J. Immunology 164, 3426). These antibodies have been shown to bind to the first extracellular loop of CCR5 (ECL-1) and to inhibit R5-tropic HIV infection of peripheral blood mononuclear cells (PBMC). Allogeneic immunization in women results in CCR5-specific antibodies that can inhibit R5-HIV infection in vitro (Wang et al., 2002, Clin. Exp. Immunol. 129, 493).

Monoclonal α-CCR5 antibodies can prevent HIV infection (Olson et al., 1999, J. Virol. 73, 4145; Wu and LaRosa et al., 1997, J. Exp. Med. 186, 1373). Antibodies that bind to cyclic peptides corresponding to the small extracellular loop ECL-2 (Arg168-Thr177) suppress infection by HIV-1 R5 (Misumi et al., 2001, J. Virol. 75, 11614). Antibodies generated by immunizing monkeys with linear CCR5 peptides (N-terminal, ECL-1 or ECL-2 sequences) have viral inhibitory effects in vitro (Lehner et al., 2001, J. Immunology 166, 7446). The N-terminal domain of CCR5 was presented in papillomavirus-like particles and immunized monkeys. The viral load was low, rapidly decreasing and eventually became undetectable in all 5 monkeys examined (Chackerian et al., 2004, J. Virol. 78, 4037), but 6 control macaques In half, plasma-associated virus was further reduced to undetectable levels.
In one preferred embodiment of the invention, the at least one antigen comprises or consists of a fragment of the CCR5 extracellular domain. The fragment of the CCR5 extracellular domain has at least 6 or 7, preferably at least 8, 9, or 10 amino acids, and the fragment of the CCR5 extracellular domain is less than 35, preferably less than 30, preferably less than 20, more preferably Has less than 15, more preferably less than 12 amino acids.

In a preferred embodiment, the CCR5 extracellular domain fragment comprises or consists of ECL2A. As is generally understood in the art, ECL2A preferably starts with the first amino acid of ECL2 and preferably stops at the threonine immediately preceding the cysteine of ECL2. In a further preferred embodiment, ECL2A comprises or consists of SEQ ID NO: 62. In one preferred embodiment, ECL2A is cyclized. In a further preferred embodiment, the cyclized ECL2A comprises or consists of SEQ ID NO: 62. In a further preferred embodiment, ECL-2A is a cyclic peptide as in SEQ ID NO: 61, wherein the peptide is cyclized by both C and G residues.
In a preferred embodiment, the antigen of the invention comprises or consists of the CCR5 extracellular domain PNt. In a further preferred embodiment, the PNt domain comprises or preferably consists of SEQ ID NO: 63.

In a preferred embodiment of the invention, the at least one antigen is gastrin and / or progastrin. Gastrin (G17) is a group of classic intestinal peptide hormones contained in fairly small amounts in the colon and pancreas (Koh, Regulatory Peptides. 93, 37-44 (2000)). Gastrin is processed from the precursor progastrin (G34). Both gastrin and progestin exist in a C-terminal glycine-extended form and a C-terminal phenylalanine amidated form.
Gastin is well known for its ability to stimulate gastric acid secretion (Pharmacol Ther. 98, 109-127 (2003)). As gastrin, the related hormone cholecystokinin (CCK) having a C-terminal tetrapeptide amide is synthesized in the duodenum and causes pancreatic enzyme secretion. Amidated G17 binds to CCK-2 receptor, while CCK binds to both CCK-1 receptor and CCK-2 receptor (Steel. IDrugs. 5, 689-695 (2002)). The receptor for gaycine-elongated gastrin remains unclear. Recent data suggest that gastrin may promote gastrointestinal tract growth (Watson. Aliment Pharmacol Ther. 14, 1231-1247 (2000)). In contrast, non-amidated gastrin stimulates colonic mucosal growth, promotes early stages in colorectal cancer formation and is elevated in the circulation and tumors of patients suffering from colorectal cancer (Watson. Aliment Pharmacol Ther. 14, 1231-1247 (2000)).

In a preferred embodiment, the at least one antigen comprises or preferably consists of G17 (SEQ ID NO: 50). In a further preferred embodiment, the at least one antigen comprises or consists of G17 (SEQ ID NO: 51) with an additional glycine at the C-terminus. In a still further preferred embodiment, the at least one antigen comprises or preferably consists of G17 in which the last amino acid F is amidated. In one preferred embodiment, the at least one antigen comprises or consists of progastrin G34 (SEQ ID NO: 52). In a further preferred embodiment, the at least one antigen comprises or consists of progastrin G34 (SEQ ID NO: 53) with an additional glycine at the C-terminus. In a still further preferred embodiment, the at least one antigen comprises or preferably consists of progastrin G34 in which the last amino acid F is amidated.
In one preferred embodiment, the at least one antigen comprises a G17 1-9 fragment (SEQ ID NO: 49) having a linker sequence preferably fused to the C-terminus, more preferably a linker sequence SSPPPPC fused to the C-terminus. Or consist of it.

In one highly preferred embodiment, the at least one antigen fused to the linker comprises or consists of an amino acid sequence such as SEQ ID NO: 54.
Note that as part of the gastrin sequence, the E at position 1 of the sequence EGPWLEEEE can be E, pyroE or Q. When an additional amino acid is fused to the N-terminus of EGPWLEEE, E at position 1 of the sequence EGPWLEEE can be E or preferably Q.

In one preferred embodiment of the invention, the at least one antigen is C5a or a fragment or variant thereof. The 74-amino acid, 4-helix bundle glycoprotein C5a (Fernandez and Hugli, J. Biol. Chem. 253, 6955-6964, 1978) is localized to cell lines, particularly neutrophils, endothelial cells and macrophages. It is a factor that induces a variety of effects to induce inflammatory inflammation and resist microbial infection (Ward P., Nat. Rev. Immunol. 4: 133, 2004). Similarly, however, excessive C5a in sepsis leads to severe functional loss in neutrophils (Czermak et al., Nat. Med. 5: 788, 1999; Huber-Lang et al., J. Immunol. 166: 1193, 2001). C5a with increased activity is also associated with a number of primary and / or chronic inflammatory diseases such as rheumatoid arthritis (Jose P. Ann Rheum. Dis. 49: 747, 1990), psoriasis (Takematsu H., Arch. Dermatol). 129: 74, 1993), adult respiratory distress syndrome (Langlois P., Heart Lung 18:71, 1989), reperfusion injury (Homeister, J. Annu. Rev. Pharmacol. Toxicol. 34:17, 1994), lupus It is also associated with nephritis and pemphigoid.
In one preferred embodiment, the at least one antigen comprises or consists of C5a. In a further preferred embodiment, the C5a protein has the amino acid sequence of SEQ ID NO: 57. In a preferred embodiment, at least one antigen comprises or consists of a C5a fragment. In a further preferred embodiment, the C5a fragment has an amino acid sequence as SEQ ID NO: 59.

In a preferred embodiment of the invention, the at least one antigen is CETP or a fragment or variant thereof. Cholesteryl-ester transfer protein (CETP) is a cholesterol between high density lipoprotein (HDL) particles and apolipoprotein B rich particles such as very low density lipoprotein (VLDL) particles or low density lipoprotein (LDL) particles. It is a plasma glycoprotein that mediates the exchange of esters (CE) and triglycerides (TG). CETP further transfers phospholipids (PL). The human CETP cDNA encodes a Mr53000 protein with 476 amino acids, which results in a Mr74000 glycoprotein by glycosylation.
HDL is considered anti-atherogenic when an inverse correlation is observed between HDL-cholesterol levels and coronary heart disease (CHD) (Barter PJ and Rye K.-A. (1996) Atherosclerosis 121: 1-12). In contrast, LDL and VLDL are atherogenesis-inducing lipoproteins. CETP deficiency in humans is associated with increased HDL-c and decreased LDL-c and is typically anti-atherogenic. Patients with metabolic syndrome have low HDL-c, concentrated fractions of low density LDL particles, mild to moderate hypertriglyceridemia, mild hypertension, trunk obesity, and insulin resistance, especially There is a risk of CHD. In addition, patients suffering from non-insulin dependent diabetes or familial combined hyperlipidemia also have low HDL-c and are at risk for CHD (Barter PJ and Rye K.-A. (1996). ) Atherosclerosis 121: 1-12).

In one preferred embodiment, the at least one antigen comprises or consists of a CETP fragment having the amino acid sequence of SEQ ID NO: 69.
In a preferred embodiment of the invention, the at least one antigen comprises or consists of bradykinin or a fragment or variant thereof. Bradykinin (BK, KRPPGFSPFR, SEQ ID NO: 80) is a major vasodilatory peptide and plays an important role in local regulation of blood pressure, blood flow and vascular permeability (Margolius HS et al., Hypertension, 1995). In addition, several other biological activities of bradykinin have been described, including smooth muscle contraction and relaxation, induction of pain and hyperalgesia, and mediation of inflammatory responses. Bradykinin exerts its effect via the B2-receptor.

In a preferred embodiment of the invention, the at least one antigen comprises or consists of des-Arg9-bradykinin, a fragment or variant thereof. des-Arg9-BK (KRPPGFSPF, SEQ ID NO: 81) has both overlapping and different functions with bradykinin. Evidence shows that des-Arg9-BK occurs quickly after tissue injury and regulates most events observed during the inflammatory process, including vasodilation, increased vascular permeability, plasma exudation, cell migration, pain and hyperalgesia (Calixto JB et al., Pain 2000). Des-Arg9-BK exerts its effect via the B1-receptor.
BK and Des-Arg9-BK have been reported to play a role in several inflammatory diseases. For example, upregulation of B2 and B1-receptors in synovial fibroblasts and increased serum kinin production was observed in antigen-induced chronic or rheumatoid arthritis (RA) (Cruwys SC et al., Br J Pharmacol, 1994; Cassim B. et al., Immunopharmacology 1997). Experimental evidence suggests that both BK and des-Arg9-BK play a role in the development of asthma (Christiansen SC et al., Am. Rev. Dis. 1992). BK and Des-Arg9-BK have a role in primary and chronic inflammatory diseases, particularly arthritis, and airway inflammation induced by allergens or particulate allergens such as viruses.

The present invention further includes compositions comprising mimotopes of the antigens described herein. Further information about using mimotopes in the vaccine compositions of the present invention is disclosed on pages 83-85 of WO 02/056905, incorporated herein by reference.
At least one antigen can be prepared by purification from a natural source or more preferably in a bacterial expression system, most preferably in an E. coli system, preferably by recombinant expression. For purification purposes, the antigens of the invention are usually expressed as a fusion protein with a tag, such as a histidine tag, Flag tag, myc tag or antibody constant region (Fc region). Typically, although not required, an enterokinase cleavage site is between the tag and the antigen so that the tag can be cleaved. In another preferred embodiment, at least one antigen having 50 or fewer amino acids is chemically synthesized.

Prior art, for example, International Publication No. WO 02/056905, International Publication No. 03/031446, International Publication No. 03/039225, International Publication No. 03/040164, International Publication No. 04/009116, International Publication No. 04/009124. No., WO 04/016282, US Pat. No. 60 / 589,322, WO 03/031466, WO 2004/007538, and other incorporated references according to the present invention. It should be noted that VLP-antigen conjugates are derived from expression and result, and therefore still contain a certain amount of host RNA in the VLP. On the other hand, in the present invention, the VLP-antigen conjugate of the present invention has a reduced or essentially eliminated amount of host RNA, preferably host nucleic acid.
In one embodiment of the invention, the VLP of the invention and the at least one antigen of the invention are fused via at least one first and at least one second attachment site, ie via at least one peptide bond. is doing. Such a fusion can occur here, typically by genetic engineering, for example by fusion of at least one antigen of the invention with viral coat proteins, building blocks of virus-like particles.

A gene encoding an antigen of the present invention, preferably less than 100 amino acids, preferably less than 80 amino acids, more preferably less than 60 amino acids, even more preferably less than 40, most preferably less than 20, amino acids Is linked to the gene encoding the coat protein, either internally, or preferably at the N- or C-terminus, in an identical reading frame. Fusion also occurs by inserting the antigen sequence into a variant of the VLP subunit, where a portion of the subunit sequence has been deleted, which is also referred to as a truncated variant. A truncation variant may have a deletion at the N- or C-terminus of the sequence of the VLP coat protein, or internally. Preferably, the fusion protein retains the ability to form VLPs that can be examined by electron microscopy.
A flanking amino acid residue is added to increase the distance between the coat protein and the foreign epitope. Glycine and serine residues are particularly preferred amino acids used in flanking sequences. Such flanking sequences provide additional flexibility, reduce the effects that may destabilize the fusion of foreign sequences in the sequence of the VLP coat protein, and hinder assembly due to the presence of foreign epitopes. Can be reduced.

In one preferred embodiment, the modified VLP is a mosaic VLP, wherein preferably the mosaic VLP comprises or consists of at least one fusion protein and at least one viral coat protein.
In a preferred embodiment, at least one antigen of the present invention, preferably an antigen consisting of less than 50 amino acids, is fused to a C-terminal truncated form of many viral coat proteins, such as the A1 protein of Qβ (Kozlovska, TM et al. Intervirology 39: 9-15 (1996)), or between positions 72 and 73 of CP extension. For example, Kozlovska et al. (Intervirology, 39: 9-15 (1996)) describe a QβA1 protein fusion in which the epitope is fused at the C-terminus of the QβCP extension truncated at position 19. As another example, an antigen can be inserted between amino acids 2 and 3 of fr CP, leading to an antigen-fr CP fusion protein (Pushko P et al., Prot. Eng. 6: 883-891 (1993)). Furthermore, the antigen can be fused to the N-terminal raised β-hairpin of the coat protein of RNA phage MS-2 (WO 92/13081).

In a preferred embodiment of the invention, at least one antigen is fused to the N- or C-terminus of the AP205 bacteriophage coat protein, variant or fragment thereof. In a further preferred embodiment, at least one antigen is fused via a spacer to the N- or C-terminus of the coat protein, variant or fragment of AP205. In general, flexible spacers are preferred. The design of the spacer between the first polypeptide and the second polypeptide is accomplished by recombinant DNA technology. In one particular embodiment of the invention, the amino acid sequence of the spacer is: (a) GSGG (SEQ ID NO: 92); (b) GSG (SEQ ID NO: 93); (c) GTAGGGSG (SEQ ID NO: 94); (d) It is selected from the group consisting of GSGTAGGGGS (SEQ ID NO: 95).
In one preferred embodiment of the invention, the composition has at least one first attachment site bound to at least one antigen having at least one second attachment site via at least one covalent bond. It comprises or consists essentially of virus-like particles, and the covalent bond is preferably a non-peptide bond. The inherently highly repetitive and organized structure of RNA phage VLPs advantageously contributes to the ability to display the antigens of the present invention, preferably in a highly ordered repetitive array. This is further ensured by an oriented and defined bond, as disclosed in the present invention.

In a preferred embodiment of the invention, the first attachment site comprises or is preferably an amino group, preferably the amino group of a lysine residue. In another preferred embodiment of the invention, the second attachment site comprises or is preferably a sulfhydryl group, preferably a cysteine sulfhydryl group. In another preferred embodiment of the invention, the second attachment site comprises or is preferably a maleimide group which binds to, preferably covalently binds to at least one antigen.
US Pat. No. 5,698,424 describes a modified coat protein of bacteriophage MS-2 capable of forming a capsid, wherein the coat protein inserts a cysteine residue into the N-terminal hairpin region, leaving a non-cysteine amino acid residue. The group is modified by replacing each cysteine residue located outside the N-terminal hairpin region. Subsequently, the inserted cysteine residue directly binds to a desired molecular species and is presented as an epitope or an antigen protein.

However, we note that the presence of free cysteine residues exposed on the capsid can lead to the oligomerization of the capsid through the formation of disulfide bridges. Furthermore, the binding of capsids to antigenic proteins by disulfide bonds is unstable, especially for sulfhydryl-containing molecules, and is less stable in serum than thioether attachment (Martin FJ. And Papahadjopoulos D. (1982) Irreversible Coupling of Immunoglobulin Fragments to Preformed Vesicles. J. Biol. Chem. 257: 286-288).
Thus, in a further highly preferred embodiment, the binding between the VLP and at least one antigen does not include a disulfide bond. More preferably, the at least one second attachment comprises or is a sulfhydryl group. Furthermore, and in a highly preferred embodiment of the invention, the binding between the VLP and at least one antigen does not include a sulfur-sulfur bond. In a further highly preferred embodiment, said at least one first attachment site is not or does not comprise a cysteine sulfhydryl group. In a still further highly preferred embodiment, the at least one first attachment site is not or does not contain a sulfhydryl group.

In a highly preferred embodiment of the invention, the first attachment site comprises or preferably is an amino group, preferably the amino group of lysine, and the second attachment site is a sulfhydryl group, preferably It contains or is preferably the sulfhydryl group of cysteine.
In one preferred embodiment of the invention, at least one antigen is typically and preferably conjugated to the VLP by chemical cross-linking using a heterobifunctional cross-linking agent. In a preferred embodiment, the heterobifunctional crosslinker is native to the antigen of the present invention with a preferred first attachment site, i.e., preferably a functional group capable of reacting with the amino group of the lysine residue (s) of the VLP. Or is added artificially and optionally utilized for the reaction by reduction, preferably a second attachment site, i.e. preferably a further functional group which can react with the sulfhydryl group of the cysteine (s) residue. Including. Several heterobifunctional crosslinkers are known in the art. These include SMPH (Pierce), sulfo-MBS, sulfo-EMCS, sulfo-GMBS, sulfo-SIAB, sulfo-SMPB, sulfo-SMCC, SVSB, SIA, and the preferred crosslinkers, such as Pierce Other cross-linking agents available from Pierce Chemical Company are included. All the crosslinking agents described above form amide bonds after reaction with amino groups and sulfhydryl groups and thioether bonds. Another class of crosslinkers suitable for the practice of the present invention is characterized by the introduction of disulfide bonds between the antigen and the VLP during coupling. Preferred crosslinking agents belonging to this class include, for example, SPDP and sulfo-LC-SPDP (Pierce).

In a preferred embodiment of the present invention, the composition of the present invention further contains a linker. Thus, in some embodiments, manipulation of the second attachment site in the antigen is accomplished by association with a linker comprising or consisting of a suitable amino acid as the second attachment site in accordance with the disclosure of the present invention. . Thus, in a preferred embodiment of the invention, the linker is associated with the antigen by at least one covalent bond, preferably typically at least one peptide bond. Preferably, the linker comprises or consists of a second attachment site. In a further preferred embodiment, the linker comprises a sulfhydryl group of a cysteine residue. In another preferred embodiment, the linker is a cysteine residue.
The choice of linker will depend on the nature of the antigen, its biochemical properties such as pi, charge distribution, and glycosylation. In general, flexible amino acid linkers are preferred. In a further preferred embodiment of the invention, the linker consists of amino acids, more preferably the linker consists of at most 25, preferably at most 20, more preferably at most 15 amino acids. In another preferred embodiment of the invention, the amino acid linker comprises less than 10 amino acids. Preferred embodiments of the linker are: (a) CGG (SEQ ID NO: 96); (b) N-terminal gamma 1-linker (eg CGDKTHTSPP, SEQ ID NO: 97); (c) N-terminal gamma 3-linker (eg CGGPKPSTPPGSSGGAP, SEQ ID NO: 48); (d) Ig hinge region; (e) N-terminal glycine linker (eg, GCGGGG, SEQ ID NO: 98); (f) n = 0-12, k = 0-5 ( G) kC (G) n; (g) N-terminal glycine-serine linker (n = 1-3 (GGGGGS) n with one cysteine) )); (H) n = 0-3, k = 0-5, m = 0-10, l = 0-2 (G) kC (G) m (S) l (GGGGGS) n (eg, array Number: 100, n = 1, k = 1, l = 1 and m = 1) (i) GGC; (k) GGC-NH2 (l) C-terminal gamma 1-linker (eg DKTHTSPPCG, SEQ ID NO: 101); (m) C-terminal gamma 3-linker (eg PKPSTPPGGSSGGAPGCG, SEQ ID NO: 102); (n) C-terminal glycine linker (GGGGCG (O) n = 0-12 and k = 0-5 (G) nC (G) k; (p) C-terminal glycine-serine linker (n with one cysteine) = 1-3 (SGGGG) n (for example, SEQ ID NO: 104, corresponding to the embodiment of n = 1)); (q) n = 0-3, k = 0-5, m = 0-10, l = 0-2 and o = 0-8 (G) m (S) l (GGGGGS) n (G) oC (G) k (e.g. SEQ ID NO: 105, n = 1, k = 1, l = 1, (corresponding to an embodiment with o = 1 and m = 1). In a further preferred embodiment, the linker is fused to the N-terminus of the antigen. In another preferred embodiment of the invention, the linker is fused to the C-terminus of the antigen.

Preferred linkers according to this invention are glycine linkers (G) n further comprising a cysteine residue as the second attachment site, such as N-terminal glycine linker (GCGGGG) and C-terminal glycine linker (GGGGCG). Further preferred embodiments include a C-terminal glycine-lysine linker (GGKKGC, SEQ ID NO: 106) and an N-terminal glycine-lysine linker (CGKKGG, SEQ ID NO: 107), GGCC, GGC or GGC at the C-terminus of the peptide. The -NH2 ("NH2" represents amidation) linker, or at its N-terminus, is a CGG linker. In general, a glycine residue is inserted between the cysteine used as the second attachment site and the large amino acid to avoid potential steric hindrance of the larger amino acid during the coupling reaction.
In another embodiment of the invention, the linker binds to the antigen of the invention by chemical interaction, preferably at least one covalent bond that is not a peptide bond.

Other methods for linking antigens to VLPs include using carbodiimide EDC and NHS to crosslink to VLPs. The antigen of the invention may first be thiolated, for example via a reaction using SATA, SATP or iminothiolane. In further methods, homobifunctional crosslinkers such as glutaraldehyde, DSG, BM [PEO] 4, BS3 (Pierce), or other known functional groups that react with amino or carboxyl groups of VLPs Homobifunctional crosslinkers are used to bind the antigen to the VLP.
In a preferred embodiment, the first attachment site comprises a sulfhydryl group, more preferably a sulfhydryl group that is naturally or artificially added to a coat protein that constitutes or consists essentially of a VLP, or Preferably it is this group. The second attachment site is chemically associated with the antigen, preferably covalently attached to the antigen, more preferably covalently attached to the amino group of the antigen, and more preferably the amino acid of the N-terminal amino acid of the antigen. Such as SMPH (succinimidyl-6- [β-maleimidopropionamido] hexanoate) or MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), covalently linked to the group by the NHS-ester group of the linker, This is the maleimide group of the linker. In one preferred embodiment, antigens, preferably antigens of 70 or less, more preferably 50 or less, even more preferably 30 or less amino acids, are preferably chemically synthesized, and the maleimide group is preferably the amino acid of the N-terminal amino acid. Bonded to the group. The first attachment site and the second attachment site are linked via a thio-ether bond.

In one preferred embodiment of the composition, the first attachment site comprises or preferably is an amino group, preferably the amino group of a lysine residue, and the second attachment site comprises a maleimide group. Or preferably the group. The amino group of lysine, which is the first binding site, may be naturally occurring or may be artificially added to the coat protein. Preferably, the second attachment site is a linker group as detailed in the above paragraph. The amino group of the VLP is derivatized to a sulfhydryl group with a heterobifunctional crosslinker such as N-succinimidyl-S-acetylthioacetate (SATA) or 2-iminothiolane, which then reacts with the maleimide group of the linker.
Preferred linkers having at least one maleimide group are, for example, SMPH, sulfo-MBS. Further preferred linkers are sulfo-EMCS, sulfo-GMBS, sulfo-SIAB, sulfo-SMPB, sulfo-SMCC, SVSB, SIA, and other crosslinkers available from, for example, Pierce Chemical Co., which are reactive with sulfhydryl groups. And one functional group reactive with an amino group.

In another embodiment of the present invention, the VLP and at least one antigen of the present invention are linked by chemical interaction, wherein at least one of these chemical interactions is not a covalent bond. Binding of VLP to the antigen can be achieved by biotinylation of the VLP and expression of the antigen as a streptavidin-fusion protein. Further, both the antigen and VLP are biotinylated as described in, for example, WO 00/23955. Other binding pairs such as ligand-receptor, antigen-antibody can be used as coupling reagents in the same manner as biotin-avidin.
In one aspect, the present invention provides a vaccine composition comprising the composition of the present invention. Preferably the vaccine composition further comprises a suitable buffer. The antigen that binds to the VLP in the vaccine composition may be derived from an animal, preferably a mammal or a human. In preferred embodiments, the antigen may be derived from a human, cow, dog, cat, mouse, rat, pig, or horse.

In one preferred embodiment, the vaccine composition further comprises at least one adjuvant. Here, administration of at least one adjuvant can be done before, simultaneously with, or after administration of the composition of the present invention. Specific examples of at least one adjuvant are aluminum salts, monophosphoryl lipid A (MPL), incomplete Freund's adjuvant (IFA). Adjuvants induce the formation of local antigen depots.
In another preferred embodiment, the vaccine composition of the invention lacks an adjuvant. An advantageous property of the present invention is that the composition is highly immunogenic, even without an adjuvant. Furthermore, the absence of an adjuvant minimizes the occurrence of unwanted inflammatory T cell responses that present a vaccination safety issue against self-antigens. Thus, administration of a vaccine of the present invention to a patient will preferably be made before, simultaneously with, or after administration of the vaccine without administering at least one adjuvant to the same patient.

The present invention discloses an immunization method comprising administering a vaccine of the present invention to an animal or a human. The animal is preferably a mammal, such as a cat, sheep, pig, horse, cow, dog, rat, mouse, especially a human. Vaccines can be administered to animals or humans by a variety of methods known in the art, but are generally administered by injection, infusion, inhalation, oral administration, or other suitable physical method. . The conjugate can also be administered intramuscularly, intravenously, transmucosally, transdermally, intranasally, intraperitoneally, or subcutaneously. Conjugate components for administration include sterile water (eg, physiological saline) or non-aqueous solutions and suspensions. Specific examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable organic esters such as ethyl oleate. Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption.
Vaccines of the invention can be said to be “pharmaceutically acceptable” if their administration is acceptable to the recipient individual. Further, the vaccines of the invention will be administered in a “therapeutically effective amount” (ie, an amount that produces the desired physiological effect). The nature and type of immune response is not a limiting factor in this disclosure. While not intending to limit the invention by describing the following mechanism, the vaccine of the invention binds to the antigen of the invention and thus reduces its concentration and / or its physiological or pathological Induce antibodies that interfere with functional function.

In one embodiment, the present invention provides pharmaceutical compositions and acceptable pharmaceutical carriers. When the vaccine of the present invention is administered to an individual, it may be in a form that includes salts, buffers, adjuvants, or other substances desirable to improve the efficacy of the conjugate. Specific examples of materials suitable for use in preparing pharmaceutical compositions are provided in a number of sources, including REMINGTON'S PHARMACEUTICAL SCIENCES (Osol, Ed. A, Mack Publishing Co. (1990)).
The present invention provides a method of using the composition of the present invention for treating and / or alleviating a disease or condition, wherein at least one antigen of the present invention has an important pathological function in animals or humans. Make.
In another aspect, the present invention provides the use of a composition of the invention in the manufacture of a medicament for the treatment of a disease in an animal or human, wherein at least one antigen of the invention is an important disease Performs a physical function.

  For convenience, the terms “conventional VLP” and “inventive VLP” and the more specific terms “conventional Qβ VLP”, “inventive Qβ VLP”, “conventional AP205 VLP” and “inventive AP205 VLP” Are used in particular in the items of the examples and in the short explanatory text of the figures. The term “conventional VLP” and the more specific terms “conventional Qβ VLP”, “conventional AP205 VLP” and the like used in the items of the examples are WO02 / 056905 and WO04 / 007538. Or, particularly as described in Example 1 of this application, refers to a VLP obtained by recombinant expression from E. coli and subsequent purification. The term “VLP of the present invention” and more specific terms “Qβ VLP of the present invention”, “AP205 VLP of the present invention”, etc. used in the section of the examples are VLPs according to the present invention, in particular the methods of the present invention. Refers to the VLP obtained by Further, for convenience, the term “reconstructed (reconstructed) VLP” as well as more specific terms “reconstructed (reconstructed) Qβ VLP”, “reconstructed (reconstructed) AP205 VLP”, etc. Is typically and preferably used in this example section to refer to a VLP obtained by the method of the invention as claimed in claim 35, 37, 39 or 41. Furthermore, “RNase-treated VLP” as well as the more specific terms “RNase-treated Qβ VLP”, “RNase-treated AP205 VLP” and the like are typically and preferably determined by the method of the present invention according to claim 43. Used in this example section to refer to the resulting VLP. Furthermore, the term “metal ion-treated VLP” as well as the more specific terms “metal ion-treated Qβ VLP”, “metal ion-treated AP205 VLP” and the like are typically and preferably the book of claim 44. Used in this example section to refer to VLPs obtained by the inventive method.

Example 1 Expression of Qβ coat protein and resulting purification of conventional Qβ VLP E. coli JM109 was transformed with a plasmid expressing Qβ coat protein. Clones transformed with a plasmid expressing Qβ coat protein were inoculated into LB liquid medium containing 20 μg / ml ampicillin. The seeded culture was cultured at 37 ° C. for 16-24 hours without shaking. The culture was then diluted 1: 100 in 100-300 ml fresh LB medium containing 20 μg / ml ampicillin and cultured overnight at 37 ° C. without shaking. The resulting secondary inoculum was diluted 1:50 in a flask with M9 medium containing 1% casamino acid and 0.2% glucose and incubated overnight at 37 ° C. with shaking. Cells were centrifuged to pellet and stored frozen.

Purification solutions and buffers for purification procedures:
1. Lysis buffer LB
50 mM Tris-HCl pH 8.0 with 5 mM EDTA at a concentration of 5 μg / ml without lysozyme and DNase, 0.1% Triton X100 and freshly prepared PMSF.
2. Ammonium sulfate saturated with water (SAS)
3. Buffer NET
20 mM Tris-HCl pH 7.8 containing 5 mM EDTA and 150 mM NaCl.
4. PEG
NET, 40% (w / v) polyethylene glycol 6000
Disruption and lysis Frozen cells were suspended in LB at a concentration of 2 ml / g cells. The mixture was treated 5 times with 22 kH ultrasound for 15 seconds. The solution was chilled by placing on ice for 1 minute between each treatment. The lysate was then centrifuged for 1 hour at 14000 rpm using a Janecki K60 rotor. Unless otherwise stated, all centrifugation steps described below were performed using the same rotor. The supernatant was stored at 4 ° C. and the cell debris was washed twice with LB. After centrifugation, the lysate supernatant and the washed fraction were pooled.

Fractionation A saturated ammonium sulfate solution was added dropwise to the above pooled lysate with stirring. The capacity of the SAS was adjusted to 1/5 of the total volume and saturated by 20%. The solution was left overnight and centrifuged the next day for 20 minutes at 14000 rpm. The pellet was washed with a small amount of 20% ammonium sulfate and centrifuged again. The resulting supernatants were pooled and SAS was dropped into 40% saturation. The solution was left overnight and centrifuged the next day for 20 minutes at 14000 rpm. The obtained pellet was dissolved in a NET buffer.
Chromatography VLP protein or capsid dissolved in NET buffer was applied to a Sepharose CL-4B column. Three peaks were obtained by chromatography. The first peak mainly contained membranes and membrane fragments and was not collected. The second peak contains VLP and the third peak contains other E. coli proteins.
Peak fractions were pooled and the NaCl concentration was adjusted to a final concentration of 0.65M. An amount of PEG solution corresponding to half of the pooled peak fractions was added dropwise with stirring. The solution was left overnight without stirring. The capsid protein was precipitated by centrifugation at 14000 rpm for 20 minutes. It was then dissolved in a minimal amount of NET and loaded onto a Sepharose CL-4B column. Peak fractions were pooled and precipitated with 60% saturated (w / v) ammonium sulfate. After centrifugation and dissolution in NET buffer, the capsid protein was loaded onto a Sepharose CL-6B column for rechromatography.
Dialysis and drying The peak fractions obtained above were pooled, dialyzed on a large scale with sterile water, lyophilized and stored.

Example 2 Hydrolysis of RNA Encapsulated in Conventional Qβ VLP by RNase A Conventional Qβ VLP containing 0.2 × HBS (4 mM HEPES, 30 mM NaCl pH 7.4) at a concentration of 1.0 mg / ml Digested with RNase A (Qiagen AG, Switzerland) added at a final concentration of 300 μg / ml. Samples were incubated for 3 hours at 37 ° C. in a thermomixer at 650 rpm. Samples were then dialyzed overnight with a 300,000 molecular weight cut-off membrane using 0.2 HBS buffer with one buffer change. Appropriate proportions of conventional Qβ VLPs from the same processing unit (batch) were not treated with RNase A and were saved for later determination of RNA, preferably nucleic acid levels.

In order to determine the amount of RNA, preferably nucleic acid remaining in the secondary structure in the VLP, the equivalent amount (weight) of RNase A treated Qβ VLP and conventional Qβ VLP not treated with RNase A was Preferably on a 1% agarose gel containing ethidium bromide (typically preferably at a concentration of 0.5 μg / ml) that enters the secondary structure of the nucleic acid. Equal volumes of VLPs were not treated with RNase A, typically by Bradford assay according to a standard protocol, preferably a product (BIO-RAD) using Albumin (PIERCE) as a standard. The protein concentrations of the Qβ VLP and RNase A treated Qβ VLP were determined by measuring in the same experiment. As used herein, “equivalent” refers to conventional VLPs in which the amount of RNase-treated VLP, preferably RNase A-treated VLP, and even more preferably RNase A-treated Qβ VLP, run on a gel. , Preferably greater than 5%, preferably greater than 3%, even more preferably greater than 1%, compared to conventional Qβ VLPs. After preferably electrophoresis for 1 hour, typically preferably at 80V, Sambrook, J. et al., Eds., Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and especially as described in its chapters 6.15 and 6.19, typically the gel was preferably exposed to UV light. The amount of RNA was indicated by the fluorescence intensity of ethidium bromide incorporated into the RNA. Conventional Qβ VLPs that were not treated with RNase A showed strong fluorescence intensity whereas RNase A-treated VLPs had very low intensity. Depending on the amount of fluorescence intensity by a densitometer (Kodak 1D Scientific Imaging System), RNA having a secondary structure, preferably nucleic acid, contained in RNase A-treated VLP is not treated with RNase A. Conventional Qβ VLP Was shown to be less than 10% of the amount of RNA, preferably nucleic acid, with secondary structure contained (values of 3-9% by various experiments) (FIG. 1).
The amount of host RNA contained in a conventional Qβ VLP has secondary structure remaining in the RNase A treated Qβ VLP, as measured by the method described in Example 17, at 300 μg / VLPmg. The amount of RNA, preferably nucleic acid, was less than 30 μg / VLPmg (or equivalent to 9-27 μg / VLPmg in various experiments).

Example 3 Nonenzymatic hydrolysis of RNA content of VLPs ZnSO ₄ dependent degradation of nucleic acid content of VLPs:
Conventional Qβ VLP (1.0 mg / ml in 0.2 × HBS buffer) was incubated for 24 hours at 60 ° C. in the presence of 2.5 mM ZnSO ₄ . The resulting sample was dialyzed overnight with one buffer change using a 300,000 molecular weight cut-off membrane with 0.2 HBS buffer. An appropriate proportion of conventional Qβ VLP from the same processing unit (batch) was not treated with ZnSO ₄ and was saved for later determination of RNA, preferably nucleic acid levels.

In order to determine the amount of RNA, preferably nucleic acid remaining in the VLP in the secondary structure, the equivalent amount (weight) of ZnSO ₄ treated Qβ VLP and conventional Qβ VLP not treated with ZnSO ₄ was measured using RNA, preferably Was run on a 1% agarose gel containing ethidium bromide (typically preferably at a concentration of 0.5 μg / ml) that enters the secondary structure of the nucleic acid. An equal volume of VLPs was not treated with ZnSO ₄ by the Bradford assay according to the standard protocol, typically with the product (BIO-RAD), preferably using Albumin (PIERCE) as a standard. The protein concentration of Qβ VLP and ZnSO ₄ treated Qβ VLP was determined by measuring in the same experiment. As used herein, “equivalent” means that the amount of ZnSO ₄ treated VLPs, preferably ZnSO ₄ treated Qβ VLPs, flowed through the gel is 5% higher than the conventional VLPs, preferably the conventional Qβ VLPs flowed through the gels. Means greater, preferably greater than 3%, even more preferably greater than 1%. After preferably electrophoresis for 1 hour, typically preferably at 80V, Sambrook, J. et al., Eds., Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and especially as described in its chapters 6.15 and 6.19, typically the gel was preferably exposed to UV light. The amount of RNA was indicated by the fluorescence intensity of ethidium bromide incorporated into the RNA. The conventional Qβ VLP not treated with ZnSO ₄ showed strong fluorescence intensity, whereas the intensity of ZnSO ₄ treated VLP was very small. Depending on the amount of fluorescence intensity with a densitometer (Kodak 1D Scientific Imaging System), RNA with secondary structure, preferably nucleic acid, bound to ZnSO ₄ treated VLP bound to conventional Qβ VLP not treated with ZnSO ₄ It was shown to be 9% of the amount of RNA, preferably nucleic acid, having secondary structure.
RNA with secondary structure remaining in ZnSO ₄ treated Qβ VLP, as measured by the method described in Example 17, the amount of host RNA contained in conventional Qβ VLP is 300 μg / VLP mg Of 27 μg / VLPmg.

Example 4 Preparation of Qβ VLPs of the Invention by Degradation / Reconstruction in the Presence of Different Polyanionic Polymers Resulting in Reconstructed Qβ VLP
(A) Degradation of conventional Qβ VLP PBS containing 20 mg of conventional Qβ VLP purified from E. coli lysate (2.5 mg / ml, measured by Bradford analysis) (20 mM phosphate, 150 mM NaCl, pH 7.5) Was reduced with 10 mM DTT and placed at room temperature for 15 minutes with stirring. Magnesium chloride was then added to a final concentration of 0.7M and incubation continued at room temperature for 15 minutes with agitation to obtain a precipitate of encapsulated host cell RNA. To remove the precipitated RNA from the solution, the solution was centrifuged at 4000 rpm, 4 ° C. for 10 minutes (Eppendorf 5810 R, using a fixed angle rotor A-4-62 for all the following steps). The supernatant containing the released dimeric Qβ coat protein was used for the chromatographic purification step.

(B) Purification of Qβ coat protein by cation exchange chromatography and size exclusion chromatography The supernatant of the degradation reaction containing dimeric coat protein, host cell protein and residual host cell RNA was diluted 1:15 with water, The conductivity of 10 ms / cm or less was adjusted and applied to an SP-Sepharose FF column (xk16 / 20, 6 ml, Amersham Bioscience). The column was pre-equilibrated with 20 mM sodium phosphate buffer pH 7. The bound coat protein was eluted by a 20 mM sodium phosphate / 500 mM sodium chloride gradient and the protein was recovered in a fraction volume of approximately 25 ml. Chromatography was performed at room temperature at a flow rate of 5 ml / min and the absorbance at 260 nm and 280 nm was monitored.
In the second step, the isolated Qβ coat protein (fraction eluted from the cation exchange column) was run on a Sephacryl S-100HR column (xk26 / 60, 320 ml, Amersham Bioscience) (2 rows) and 20 mm phosphorus Equilibrated with sodium acid / 250 mm sodium chloride, pH 6.5. Chromatography was performed at room temperature with a flow rate of 2.5 ml / min, and absorbance at 260 nm and 280 nm was monitored. 5 ml fractions were collected.

(C1) Reconstruction of Qβ VLP by dialysis Purified Qβ coat protein (2.2 mg / ml in 20 mM sodium phosphate pH 6.5), 1 polyanionic polymer ( ₂ mg / ml in H ₂ O) , Urea (7.2 M in H ₂ O) and DTT (0.5 M in H ₂ O), respectively, with a final concentration of 1.4 mg / ml coat protein and 0.14 mg / ml polyanionic polymer, respectively Mixed with 1M urea and 2.5 mM DTT. The mixture (1 ml each) was dialyzed for 2 days at 5 ° C. against 20 mM Tris HCl, 150 mM NaCl pH 8 using a 3.5 kDa cutoff membrane. The polyanionic polymers were as follows: polygalacturonic acid (25000-50000, Fluka), dextran sulfate (MW 5000 and 10000, Sigma), poly-L-aspartic acid (MW 11000 and 33400, Sigma), Poly-L-glutamic acid (MW 3000, 13600 and 84600, Sigma), and tRNA from baker's yeast and wheat germ.
(C2) Reconstitution of Qβ VLPs by diafiltration (diafiltration) 33 ml of purified Qβ coat protein (1.5 mg / ml in 20 mM sodium phosphate pH 6.5, 250 mM NaCl) was added to H ₂ O and Mixed with urea (7.2 M in H ₂ O), NaCl (5 M in H ₂ O) and poly-L-glutamic acid ( ₂ mg / ml in H ₂ O, MW: 84600). The volume of the mixture was 50 ml and the final concentration of the components was 1 mg / ml coat protein, 300 mM NaCl, 1.0 M urea and 0.2 mg / ml poly-L-glutamic acid. The mixture was then diafiltered at room temperature with 500 ml of 20 mM Tris HCl pH 8, 50 mM NaCl. This diafiltration was performed in a tangential flow filtration apparatus using a Pellicon XL thin film cartridge (Biomax 5K, Millipore) at a cross flow rate of 10 ml / min and a permeation flow rate of 2.5 ml / min.

Analysis of reconstructed Qβ-VLP
(D1) Formation of disulfide bonds in Qβ-VLP As described in Example 4 (C1) and (C2), reconstituted Qβ-VLP in the presence of a different polyanionic polymer was converted to non-reduced SDS- Analysis by PAGE and comparison with conventional Qβ VLP. Since the reconstructed Qβ-VLP shows the disulfide-bonded pentameric and hexameric bands of the coat protein, as in the conventional Qβ VLP, the reconstructed Qβ VLP has an appropriately structured coat protein unit. It was suggested that it is coordinated to (Fig. 2).
(D2) Hydrodynamic size of Qβ VLP reconstructed in the presence of different polyanionic polymers by analytical size exclusion chromatography, as described in Examples 4 (C1) and (C2) Reconstructed Qβ-VLP samples in the presence of polyanionic polymers were analyzed by analytical size exclusion chromatography and compared to conventional Qβ VLPs. The reconstructed Qβ VLP showed a peak that migrated with the same residence time as the peak representing the conventional Qβ VLP, suggesting that the overall size and structure of the reconstructed VLP is the same as the conventional VLP.

Example 5 In Vitro Construction (Assembly) of AP205 VLP
(A) Purification of AP205 coat protein Degradation: 20 ml of AP205 VLP solution (1.6 mg / ml in PBS, purified from E. coli extract) is mixed with 0.2 ml of 0.5 M DTT and incubated at room temperature for 30 minutes did. 5 ml of 5M NaCl was added and the mixture was then incubated at 60 ° C. for 15 minutes to obtain a precipitate of DTT reduced coat protein. The turbid mixture was centrifuged (Rotor Sorvall SS34, 10000 g, 10 min, 20 ° C.), the supernatant was discarded and the pellet was seeded in 20 ml of 1 M urea / 20 mM sodium citrate pH 3.2. After stirring for 30 minutes at room temperature, the dispersion was adjusted to pH 6.5 by adding 1.5 M Na ₂ HPO ₄ and then centrifuged to obtain a supernatant containing the dimeric coat protein (rotor Sorvall SS34 10,000 g, 10 minutes, 20 ° C.).
Cation exchange chromatography: The supernatant (see above) was diluted with 20 ml of water to adjust the conductivity to about 5 mS / cm. The resulting solution was loaded onto a column of 6 ml SP Sepharose FF (Amersham Bioscience) pre-equilibrated with 20 mM sodium phosphate pH 6.5 buffer. After flushing, the column was washed with 48 ml of 20 mM sodium phosphate pH 6.5 buffer, after which the bound coat protein was eluted with a 20-fold column volume proportional gradient to 1M NaCl. Main peak fractions were pooled and analyzed by SDS-PAGE and UV spectroscopy. According to SDS-PAGE, the isolated coat protein was essentially free of other protein contamination. According to UV spectroscopy, the protein concentration is 0.6 mg / ml (total amount 12 mg) and 1A280 units represents 1.01 mg / ml of AP205 coat protein. Furthermore, the value of A280 (0.5999) is 2 versus the value of A260 (0.291), suggesting that the preparation is essentially devoid of nucleic acids.

(B) Construction of AP205 VLP Construction under conditions without any polyanionic polymer: Protein fraction eluted above is diafiltered and protein concentration of 1 mg / ml at 20 mM sodium phosphate pH 6.5 Concentrated by TFF. 500 ml of the solution was mixed with 50 ml of 5M NaCl solution and incubated for 48 hours at room temperature. The formation of reconstituted VLPs in the mixture was demonstrated on non-reducing SDS-PAGE and on size exclusion HPLC (FIG. 5A). A TSKgel G5000 PWXL column (Tosoh Bioscience) equilibrated with 20 mM sodium phosphate, 150 mM NaCl pH 7.2 was used for HPLC analysis.
Construction in the presence of polyglutamic acid: 375 ml of purified AP205 coat protein (1 mg / ml in 20 mM sodium phosphate pH 6.5), 50 ml NaCl stock solution (5 M in H ₂ O), 50 ml polyglutamic acid stock Mixed with solution ( ₂ mg / ml in H ₂ O, MW: 86400, Sigma) and 25 ml H ₂ O. The mixture was incubated at room temperature for 48 hours. The formation of reconstituted VLPs in the mixture was shown by non-reducing SDS-PAGE and size exclusion HPLC (FIG. 5B). The coat protein in the mixture was almost completely incorporated into the VLP, indicating that it was constructed more efficiently and more efficiently than the AP205 coat protein constructed under the absence of any polyanionic polymer. (FIG. 5A).

Example 6 Preparation of reconstructed frVLP or reconstructed GA in the presence of polyanionic polymer Recombinant production of conventional fr or GA VLP in E. coli under the same experimental conditions as disclosed in Examples 1 and 4 Then, these VLPs were purified and decomposed, and the resulting coat protein of the corresponding RNA phage was purified. The coat protein was then reconstructed in the reconstructed frVLP or reconstructed GA VLP under the same experimental conditions.

Example 7 Coupling of Nicotine Derivatives to Conventional Qβ VLPs and Qβ VLPs of the Invention A nicotine derivative suitable for binding to VLPs was synthesized according to Langone et al. (1982, supra). Trans-4'-carboxycotinine is commercially available. The methyl ester of trans-4′-carboxycotinine was produced by reacting trans-4′-carboxycotinine with methanolic sulfate. The solution was neutralized with sodium bicarbonate, extracted with chloroform, concentrated on a rotary dryer, and recrystallized from ether-acetone. The methyl ester was then reduced with ether lithium aluminum hydride to produce trans-3'-hydroxymethylnicotine. Then, succinic anhydride was added to benzene to produce O′-succinyl-hydroxymethylnicotine. The solution was concentrated on a rotary dryer. EDC (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide) and N-hydroxysuccin yielding N-hydroxysuccinimide ester of O′-succinyl-hydroxymethylnicotine (hereinafter abbreviated as “Suc-Nic”) The carboxyl group is activated by the addition of imide (NHS).
The Qβ VLP of the present invention (including RNase A treated Qβ VLP), which is a reconstituted Qβ VLP in the presence of a conventional Qβ VLP and a different polyanionic polymer as shown in FIG. 3, is Hepes-buffered saline. Dialyzed against HBS (50 mM Hepes, 150 mM NaCl, pH 8.0). The nicotine derivative Suc-Nic was dissolved in HBS at a concentration of 121 mM. 1 ×, 5 ×, 50 ×, 100 × and 500 × molar excess of different Qβ VLP solutions (0.14 mM) were added and incubated for 2 hours at room temperature on a shaker. The reaction solution was then dialyzed against HBS, pH 8.0 (10000 Dalton cutoff), rapidly frozen in liquid nitrogen and stored at -80 ° C. The nicotine derivative suc-nic was reacted with lysine on the surface of Qβ under the formation of an amide bond. The resulting covalent complexes are referred to herein as “conventional Qβ VLP-Nic” and “inventive Qβ VLP-Nic”, respectively.

Example 8 Qβ VLP-Nic of the Invention Inducing Specific Antibody Response
(A) Immunization of mice 7-8 week old female Balb / c mice were vaccinated twice with 60 μg each of the conventional Qβ VLP-Nic and the Qβ VLP-Nic vaccine of the present invention. The nicotine derivative at this time was Suc-Nic. This vaccine was diluted with 200 μl of sterile PBS and administered subcutaneously to the left and right groin. Mice were boosted 14 days after the initial immunization. Sera were collected on day 14 (before booster immunization) and day 21 (7 days after booster immunization). Serum nicotine-specific antibodies were measured by ELISA.

(B) ELISA
A microtiter plate (Maxisorp, Nunc) was coated overnight with PBS (pH 7.3-7.7) containing nicotine (BSA-NicB01) bound to 5 μg / ml BSA. After washing (0.05% Tween20 / PBS) and blocking with PBS containing 2% BSA, serum diluted variously with 2% BSA / 1% FCS / PBS was added. For detection of IgE, serum was depleted from IgG by incubating with protein G beads (Pharmacia). After incubating for 2 hours at room temperature, the plates were washed and washed with mouse IgG (goat anti-mouse IgG (H + L), Jackson ImmunoResearch), IgG1 (rabbit anti-mouse IgG1, Zymed), IgG2a (rat anti-mouse IgG2a, Pharmingen) and IgE. HRP-labeled antibody specific for (goat anti-mouse IgE) was added. After 1 hour incubation, the plates were washed and citrate buffer containing the color substrate OPD (Fluka) was added according to the manufacturer's instructions. After 5 minutes, the color reaction was stopped with 5% H ₂ SO ₄ . The optical density at 450 nm was read with an ELISA reader (Benchmark, Biorad). The ELISA titer was determined as a reciprocal dilution of serum showing half-maximal optical density signal (OD50%) in the ELISA.
All Qβ VLP-Nic vaccines of the present invention induced nicotine-specific antibodies to levels comparable to antibody titers induced by conventional Qβ VLP-Nic vaccines. (Figure 3). ELISA titers of serum pools of 3 mice per group were measured for total IgG response on days 14 and 21. As shown in FIG. 3, the IgG titer of Qβ VLP-Nic of the vaccine of the present invention on the 21st day was the same as that of the conventional Qβ VLP-Nic vaccine.

FIG. 4 shows the anti-nicotine titers of subclass IgG1 and IgG2a at day 21 as measured by ELISA. Conventional Qβ VLP-Nic induced a Th1 type of immune response with high IgG2a titers. The ratio of IgG2a / IgG1 titers was approximately 0.5. RNase-treated Qβ VLP-Nic did not affect IgG1 titers, but significantly reduced IgG2a titers, resulting in a ratio of 0.006. Also, the overall antibody titer was reduced compared to the conventional VLP. Compared to conventional Qβ VLP-Nic, the reconstituted Qβ VLP-Nic vaccine induced a high titer of IgG1 but a low IgG2a. This is indicated by an IgG2a / IgG1 titer ratio of less than 0.035, suggesting that the Th2-type response is greater than before. However, despite the more Th2 response, IgE was not detected in serum vaccinated with reconstituted Qβ VLP-Nic. Similarly, IgE was not detected in serum vaccinated with conventional Qβ VLP-Nic.
This data indicated that reconstituted Qβ VLP-Nic retains high immunogenicity, but the immune response has shifted to more Th2-type response changes without inducing IgE production.

Example 9 Coupling of GnRH peptide to reconstructed Qβ VLP GnRH (SEQ ID NO: 1) extended by cysteine as attachment site for coupling or by two glycine residues and a cysteine residue as attachment site The following peptide analogs comprising amino acids 1-10 of) were chemically synthesized:
CGG-GnRH CGGEHWSYGLRPG-NH2 (SEQ ID NO: 2)
GnRH-GGC pEHWSYGLRPGGGC (SEQ ID NO: 3)
C-GnRH CEHWSYGLRPG-NH2 (SEQ ID NO: 4)
GnRH-C pEHWSYGLRPGC (SEQ ID NO: 5)
The peptide was coupled to reconstituted Qβ VLP as described below.
Recombinantly produced reconstituted Qβ VLP (2 mg / ml) is derivatized for 30 minutes in 50 mM NaCl, 20 mM Hepes pH 7.2 containing a 20-fold molar excess of SMPH (100 mM, Pierce in DMSO) at 25 ° C. This coupled CGG-GnRH (SEQ ID NO: 2) with high efficiency (> 90% of monomers have at least one peptide).
Then, in order to remove unreacted SMPH, it was dialyzed twice for 2 hours against 20 mM Hepes pH 7.2 at 4 ° C. using a 10.000 MWCO dialysis tube. A 7-fold molar excess of GnRH peptide (5 mM in DMSO) was added and reacted in a thermomixer at 25 ° C. for 2 hours. The reaction was dialyzed overnight against 20 mM Hepes pH 7.2 to remove uncoupled peptides.

  Peptides CGG-GnRH (SEQ ID NO: 2) and GnRH were derivatized by placing reconstituted Qβ VLP (1 mg / ml) in 20 mM Hepes pH 7.2 containing 18-fold molar excess SMPH at 25 ° C. for 30 minutes. -GGC (SEQ ID NO: 3) was coupled with moderate efficiency (82% of monomers have at least one peptide). The reaction was then dialyzed against 20 mM Hepes pH 7.2 and coupled with a 10-fold molar excess of GnRH peptide (10 mM in DMSO) by incubating at 25 ° C. for 2 hours on a thermoshaker. Reconstructed Qβ VLP (2.8 mg / ml) was derivatized in 20 mM Hepes pH 7.2 containing 20-fold molar excess of SMPH (50 mM in DMSO) at 25 ° C. for 30 minutes and then to 20 mM Hepes pH 7.2. The peptides C-GnRH (SEQ ID NO: 4) and GnRH-C (SEQ ID NO: 5) were coupled by dialysis overnight. Subsequently, high (> 90%) and low (60-71%) efficiencies were obtained by incubating 7- and 2.5-fold molar excess of peptide (5 mM in DMSO) on a thermoshaker at 25 ° C. for 2 hours, respectively. Coupled with. The reaction was dialyzed overnight against 20 mM Hepes pH 7.2 to remove uncoupled peptides. The Qβ-GnRH coupling product was centrifuged and the supernatant was analyzed on an SDS-PAGE gel under reducing conditions. The Qβ-GnRH coupling products are obtained according to the respective peptides (SEQ ID NOs: 2, 3, 4 and 5) used for the coupling, Qβ-CGG-GnRH, GnRH-GGC-Qβ, Qβ-C-GnRH and GnRH-C. -Called Qβ.

Example 10 Coupling of human IL-23 p19 to VLPs of the invention Reconstructed Qβ virus-like particles (2 g / l), AP205 (2 g / l), fr (2 g / l) and GA (2 g / l), RNase A treated Qβ virus-like particles (2 g / l), AP205 (2 g / l), fr (2 g / l) and GA (2 g / l), ZnSO ₄ treated Qβ virus-like particles (2 g / l), AP205 ( 2 g / l), fr (2 g / l) and GA (2 g / l) were derivatized with 0.714 mM SMPH (Pierce, Perbio Science) for 30 minutes at 25 ° C., then with 20 mM Hepes pH 8, 150 mM NaCl Dialyzed. Qβ particles (0.5 g / l) derivatized with human IL-23 p19 protein (SEQ ID NO: 7, 0.28 g / l) were mixed with 1 mM EDTA and 10 μM, 30 μM or 90 μM TCEP (Pierce, Perbio Science) Incubated for 2 hours at 25 ° C. in the presence. Coupling products were analyzed by SDS-PAGE. The antigen concentration of the vaccine was measured by densitometric analysis.

Example 11 Fusion of Aβ1-6 peptide to C-terminus of QβA1 protein cleaved at CP extension position 19 Primer that anneals to 5 ′ end of QβA1 gene, primer that anneals to 3 ′ end of A1 gene, sequence DAEFRH Alternatively, those additionally containing a sequence encoding DAEFGH Aβ1-6 peptide were used in a PCR reaction using pQβ10 as a template. The PCR product was cloned into pQβ10 (Kozlovska ™ et al., Gene 137: 133-37 (1993)) and VLPs comprising the fusion protein were expressed and purified under the same conditions as described in Example 1. .
Purified Qβ VLP-Aβ1-6 is degraded as described in Example 4. Purified Qβ-Aβ1-6 fusion protein (2.2 mM / ml in 20 mM sodium phosphate pH 6.5), 1 polyanionic polymer ( ₂ mg / ml in H ₂ O), urea (H ₂ 7.2 M in O) and DTT (0.5 M in H ₂ O), final concentration of 1.4 mg / ml fusion protein, 0.14 mg / ml of each polyanionic polymer, 1 M urea and Mixed to 2.5 mM DTT. The mixture (1 ml each) was dialyzed for 2 days at 5 ° C. against 20 mM Tris HCl, 150 mM NaCl pH 8 using a 3.5 kDa cutoff membrane. The polyanionic polymers are as follows: polygalacturonic acid (25000-50000, Fluka), poly-L-glutamic acid (MW 3000, 13600 and 84600, Sigma) or tRNA derived from baker's yeast or wheat germ.

Example 12 Insertion of Aβ1-6 peptide between position 2 and position 3 of fr coat protein Complementary primer encoding the sequence of the Aβ1-6 peptide of sequence DAEFRH or DAEFGH, in-frame insertion with Bsp119I compatible ends Those containing additional nucleotides that allow for insertion into the Bsp119I site of the pFrd8 vector by standard molecular biology techniques (Pushko, P. et al., Prot. Eng. 6: 883-91 (1993) ). Alternatively, after digestion with Bsp119I, the pFrd8 vector overhang was inserted with Klenow, and after Klenow treatment, an oligonucleotide encoding the sequence of the Aβ1-6 peptide and an additional nucleotide encoding the in-frame were ligated. Clones with the insert in the right coordinate were analyzed by sequencing. Except for performing chromatography steps using Sepharose CL-4B or Sephacryl S-400 (Pharmacia) columns, as described in Pushko, P. et al., Prot. Eng. 6: 883-91 (1993) Expression and purification of the chimeric fusion protein were performed in E. coli JM109 or E. coli K802. Similar to the procedure described for Qβ in Example 1, the cell lysate was precipitated with ammonium sulfate and purified by two successive gel filtration purification steps.
Purified chimeric fr VLP-Aβ1-6 is to be degraded as described in Example 4. Purified frAβ1-6 fusion protein (2.2 mM / ml containing 20 mM sodium phosphate pH 6.5), 1 polyanionic polymer ( ₂ mg / ml in H ₂ O), urea (in H ₂ O) 7.2M) and DTT (0.5M in H ₂ O), final concentration of 1.4 mg / ml fusion protein, 0.14 mg / ml of each polyanionic polymer, 1M urea and 2. Mixed to 5 mM DTT. The mixture (1 ml each) was dialyzed for 2 days at 5 ° C. against 20 mM Tris HCl, 150 mM NaCl pH 8 using a 3.5 kDa cut-off membrane. The polyanionic polymers are as follows: polygalacturonic acid (25000-50000, Fluka), dextran sulfate (MW 5000 and 10000, Sigma), poly-L-aspartic acid (MW 11000 and 33400, Sigma).

Example 13 Coupling of Angiotensin I and Angiotensin II Derived Peptides to Reconstructed AP205 VLP and GAVLP and Immunization of Mice with Resulting Conjugates Conjugate production The following angiotensin peptide components were chemically synthesized:
CGGDRVYIHPF (“Angio 1”), CGGDRVYIHPFHL (“Angio 2”), DRVYIHPFHLGGC (“Angio 3”), CDRVYIHPFHL (“Angio 4”), CHPFHL (“Angio 5”), CGPFHL (“Angio 6”), CYIHPF ( “Angio 7”), CGIHPF (“Angio 8”), CGGHPF (“Angio 9”), DRVYIGGC (“Angio 13”), DRVYGGC (“Angio 14”) and DRVGGC (“Angio 15”). These were used for chemical coupling to reconstituted AP205 or GAVLP as described below.
Peptides Angio 1 to Angio 4: Reconstituted 5 mg solution of 20 mM Hepes, 150 mM NaCl pH 7.4 containing 2 mg / ml AP205 or GA VLP, 507 μl of aqueous solution containing 13 mg / ml Sulfo-MBS (Pierce) and shaking The mixture was reacted at 25 ° C. for 30 minutes on a shaker. Thereafter, the reaction solution was dialyzed twice with 2 L of 20 mM Hepes, 150 mM NaCl pH 7.4 at 4 ° C. for 2 hours. 665 μl of the dialyzed reaction mixture was then reacted with 2.8 μl of each corresponding 100 mM peptide stock solution (in DMSO) for 2 hours at 25 ° C. on a shaker. The reaction mixture was then dialyzed twice against 2 l of 20 mM Hepes, 150 mM NaCl pH 7.4 at 4 ° C. for 2 hours.
Peptides Angio 5-9 and Angio 13-15: 3 ml solution of 20 mM Hepes, 150 mM NaCl pH 7.2 containing 2 mg / ml AP205 or GA VLP reconstituted with 100 mM SMPH (succinimidyl-6- (β-maleimidopropionamide) Hexanoate, Pierce) was reacted with 86 μl of DMSO solution on a shaker shaker for 50 minutes at 25 ° C. The reaction solution was then reacted with 2 L of 20 mM Hepes, 150 mM NaCl pH 7.2 at 4 ° C. for 2 hours. Then, 514 μl of the dialyzed reaction mixture was reacted with 3.6 μl of each corresponding 100 mM peptide stock solution (in DMSO) for 4 hours at 25 ° C. on a shaker shaker. The reaction mixture was washed with 2 l of 20 mM Hepes, 150 mM NaCl pH 7.2 at 4 ° C. for 2 hours. It was dialyzed twice.

B. Immunization Female Balb / c mice were vaccinated with one of nine angiotensin peptide derivatives coupled to reconstituted AP205 or GA VLPs without added adjuvant. 50 μg (Angio 1-4 vaccine) or 20 μg (Angio 5-9 vaccine) of the total protein of each sample was diluted to 200 μl with PBS and injected subcutaneously on day 0 and day 14 (abdominal 2 100 μl each). On day 21, mice were bled from the retro-orbital and serum was analyzed using an angiotensin-specific ELISA.
The angiotensin peptide human and mouse sequences should correspond equally to each other. Thus, immunization of humans or mice with a vaccine or conjugate, respectively, containing an angiotensin peptide molecule as an antigenic determinant of the invention is a vaccination against a self antigen.

Example 14 Elimination of CD8 + T cell response of reconstituted Qβ VLP in the presence of polyglutamic acid C57BL / 6 mice were administered by subcutaneous administration of 150 μg of Qβ VLP chemically similar to LCMV-peptide gp33 (KAVYNFATM) (Qβx33). Immunized. One group of mice receives complete Qβ VLPx33 particles, and the other group has different amounts of poly-L-glutamic acid (0.1 mg / ml, 0.2 mg / ml and 0.4 mg / ml poly-L- Treatment with Qβ VLPx33 reconstituted in the presence of Qβ with glutamic acid / poly L-Glu / x33). The blood of the animals 8 days after immunization was analyzed for expansion of gp33-specific CD8 + T cells. Blood was collected in FACS buffer (PBS, 2% FCS, 5 mM EDTA pH 8.2) and stained with PE-labeled H2-Db-tetramer with gp33-peptide (Proimmune, Oxford, UK) at 37 ° C. for 10 minutes. Thereafter, the cells were stained with APC-labeled rat anti-mouse CD8a-antibody (BD PharMingen, San Jose, USA) at 4 ° C. for 30 minutes. After washing, erythrocytes were lysed with BD-Lyzing solution (BD Biosciences, San Jose, USA) for 10 minutes at room temperature. Finally, the cells were analyzed on a FACS Calibur using CellQuest software. First of all, cells were incorporated into forward scatter (forward catcher) and side scatter (side catcher) to gate lymphocytes. From this lymphocyte population, gp33-PE labeled cells and CD8-APC labeled cells were measured by FL2 and FL4 detectors, respectively. The amount of gp33-specific T cells was calculated as percent CD8 positive cells, gp33 positive cells relative to total CD8 positive lymphocytes.
None of the Qβ / polyL-Glu / x33 samples induced the expansion of gp33-specific CD8 positive T cells by flow cytometry analysis compared to animals administered conventional Qβ VLPx33. (See Table 1).

After measuring gp33-specific T cell responses, mice were challenged with 1.5 × 10 ⁶ pfu of recombinant vaccine virus expressing gp33-peptide. Five days later, the virus titer in the ovaries of mice was measured. Single cell suspensions of ovaries were incubated in serial dilutions of BSC40 cells. After incubating overnight at 37 ° C. with 5% CO ₂ , crystal violet (500 ml 96% ethanol, 5 g crystal violet (Sigma C-3886), 8 g to visualize plaques in the cell layer from virus-induced cell lysate The cells were stained with NaCl, 450 ml H ₂ O, 50 ml formaldehyde). The number of viruses remaining in the ovaries was calculated as plaque forming units (pfu). Plaque-forming units were significantly higher in mice that received Qβ VLPx33 vaccine compared to groups of mice that received conventional Qβ VLPx33 (see Table 1).
Table 1

Example 15 Immune Response Induced with RNase A Digested Qβ VLP Coupled to Mouse TNF Coupling of mouse TNF protein to conventional Qβ VLP A fusion protein consisting of a cysteine-containing linker, a hexahistidine tag and a mature mouse TNF protein (SEQ ID NO: 67) (corresponding to amino acids 78-233 of the immature protein) Recombinant expression in E. coli and purified to homogeneity by affinity chromatography. A solution containing 1.4 mg / ml protein in 20 mM Hepes, 150 mM NaCl pH 7.2 was incubated with an equal volume of TCEP for 60 minutes at room temperature.
A 500 μl solution containing 3.06 mg / ml conventional Qβ VLP in 20 mM Hepes, 150 mM NaCl pH 7.2 was then reacted with 4.2 μl SMPH solution (65 mM in DMSO) for 60 minutes at room temperature. The reaction solution was exchanged twice with 3 L of 20 mM Hepes pH 7.2 and dialyzed at 4 ° C. for 2 hours and 14 hours, respectively. 60 μl of the derivatized and dialyzed Qβ solution was mixed with 30 μl of H ₂ O and 180 μl of purified and pre-diluted mouse TNF protein and incubated at 15 ° C. for 4 hours for chemical crosslinking. The uncoupled protein was dialyzed twice with PBS at 4 ° C. for 2 hours using a cellulose ester membrane having a molecular weight of 300,000 Da.

B. RNase digestion of Qβ-mTNF 300 μl of conventional Qβ coupled to mouse TNFα from A1 (Qβ-mTNF, 0.7 mg / ml) with 3 μl RNase A (100 mg / ml) with gentle agitation Incubated for 3 hours at 37 ° C. The degree of digestion of RNA contained in conventional Qβ was confirmed by agarose gel electrophoresis of a certain amount of the reaction and subsequent ethidium bromide staining. After this treatment, approximately 95% of E. coli host RNA was digested. RNase-treated Qβ-mTNF was dialyzed overnight against excess 20 mM Hepes pH 7.2.

C. Immunization of mice Three female balb / c mice were immunized with conventional Qβ-mTNF and five female balb / c mice were immunized with RNase-treated Qβ-mTNF. 25 μg of total protein was diluted to 200 μl with PBS and injected subcutaneously on day 0 and day 14 (100 μl each on two abdominal sites). On day 0 and day 21, blood was collected from the posterior orbit of the mouse, and the mouse anti-TNF specific antibody titer in the serum was measured by ELISA.

D. ELISA
Microtiter plates (Maxisorp, Nunc) were coated overnight with 1 μg / ml recombinant mouse TNFα. By substantially the same ELISA as described in Example 8, titers of different subclasses (IgG1, IgG2a) specific for conventional QQ VLP or reconstituted Qβ VLP and TNFα in serum and total IgG titers were obtained. It was measured.
Table 2 shows that conventional Qβ-mTNF and RNase-treated Qβ-mTNF induced antibodies specific to mouse TNFα. Conventional Qβ-mTNF induced a Th1-type immune response with high IgG2a titers. The average ratio of IgG2a / IgG1 titers was approximately 0.75. Although RNase treatment of Qβ-mTNF did not affect IgG1 titer, IgG2a titer was greatly reduced to a ratio of 0.135. These data indicate that the immune response induced by RNase-treated Qβ-mTNF has changed from more Th1 immune responses to more Th2 responses compared to the immune response induced by conventional Qβ-mTNF. It was shown that
Table 2

Example 16 Coupling of mouse TNFα (4-23) peptide to conventional Qβ VLP and reconstituted Qβ and AP205 VLP 3 ml containing 3.06 mg / ml complete Qβ VLP in 20 mM Hepes, 150 mM NaCl pH 7.2 Was reacted with 99.2 μl of SMPH solution (DMSO containing 65 mM) at room temperature for 60 minutes. The reaction solution was exchanged twice with 20 mM Hepes and 150 mM NaCl pH 7.2, and dialyzed at 4 ° C. for 4 hours and 14 hours, respectively. 69 μl of derivatized and dialyzed Qβ solution was mixed with 265.5 μl of 20 mM Hepes pH 7.2 and 7.5 μl of N-terminal mTNFα (4-23) peptide (23 in DMSO). .6 mg / ml) and incubated at 15 ° C. for 2 hours for chemical crosslinking. Uncoupled peptides were removed by dialysis twice in PBS for 2 hours at 4 ° C. The coupled product was analyzed on a 12% SDS-polyacrylamide gel under reducing conditions.
The same conditions were applied for coupling of mouse TNFα (4-23) to reconstituted Qβ and AP205 VLP, respectively.

Example 17 Determination of the amount of RNA contained in a VLP VLP samples (see Table 2, 1 mg / ml) were incubated in 10 mM MgCl ₂ and 0.1 M NaHCO ₃ pH 9.7 at 60 ° C. for approximately 16 hours. . This is because the encapsulated RNA, preferably nucleic acid, is hydrolyzed to nucleotides to precipitate the protein. Samples were centrifuged for 5 minutes at 14000 rpm using a rotor F45-30-11 (Eppendorf) to separate precipitated proteins from soluble nucleotides.
The pH of the supernatant was adjusted to pH 7 by adding 0.5 M sodium phosphate buffer pH 7. In addition, the nucleotide concentration in each sample was adjusted in stages and UV data was recorded with absorbance varying below 2 AU (AU refers to “absorption unit”). The same buffer was used as a control except that there was no VLP.
The nucleotide concentration corresponding to the amount of original RNA, preferably nucleic acid, was calculated from the absorbance at 260 nm where 1 AU corresponds to 33 μg RNA / ml. The RNA concentration of the sample is summarized in Table 3.

Table 3

Example 18 Coupling of maleimide-GnRH to conventional Qβ VLP and reconstituted Qβ VLP
(A) Preparation of maleimide-GnRH Peptide GnRH was chemically synthesized using standard methods. MBS (m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester) or SMPH (succinimidyl-6 [β-maleimidopropionamido] hexanoate) was covalently attached to the N-terminal amino group of the peptide.
(B) Amino group transformation of Qβ VLP to sulfhydryl group Conventional Qβ VLP and reconstituted Qβ VLP (PBS stock solution) were prepared from 0.1 M sodium phosphate pH 7 and 2-iminothiolane (containing 200 mM stock solution). DMSO) to a final concentration of 2 mg / ml Qβ VLP, 50 mM sodium phosphate pH 7 and 15 mM 2-iminothiolane. The mixture was incubated at room temperature for 2 hours, after which excess unreacted 2-iminothiolane was removed by gel filtration (Sephadex G25, PBS), resulting in a purified concentration of approximately 1.4 mg / ml. In addition, an SH-denatured Qβ VLP solution was obtained.
(C) Coupling of maleimide-GnRH The conventional Qβ VLP modified with SH and the renatured Qβ VLP (1.4 mg / ml) of SH modified obtained in step (B) were mixed with 1/20 amount of maleimide-GnRH. Mixed with stock solution (DMSO containing 10 mM) and incubated for 1 hour at room temperature. Uncoupled maleimide-GnRH was removed by diafiltration using a 300 kDa membrane. Coupling results were confirmed by SDS-PAGE gel.

Example 19 Pyrogen Test This test was performed according to standard pyrogen test procedures as described in European Pharmacopoeia 4th edition, Chapter 2.6.8., Pages 131-132. This document is incorporated herein by reference.
Test samples were Qβ VLP reconstituted in the presence of polyglutamic acid, Qβ VLP treated with RNase, Qβ VLP treated with ZnSO ₄ , and conventional Qβ comprising E. coli RNA. The concentration of Qβ VLP was 0.3 mg / ml. Prior to injection, the sample was pre-warmed to 38.5 ° C.
The animals used in this study are healthy adult rabbits weighing approximately 3 kg. Three rabbits were tested in separate groups.
Briefly, a sample with a volume of 0.3 ml / kg body weight was slowly injected into the peripheral vein of each rabbit ear within 4 minutes. The starting temperature of each rabbit is the average of the two temperatures of that rabbit, recorded for 30 minutes before 40 minutes of sample injection. The maximum temperature for each rabbit is the highest rabbit temperature recorded 3 hours after injection. The recording of the temperature of each rabbit is at most 30 minutes apart, starting at least 90 minutes before injecting the sample and continuing for 3 hours after injection. The difference between the maximum temperature of each rabbit and the starting temperature is considered to be due to the response. The ability of tested VLPs to induce heat in rabbits was investigated and compared to conventional VLPs.

Example 20 IL-5 Coupling to Reconstructed Qβ VLP and Measurement of Induced Immune Response

Example 21 Measurement of IL-5 coupling and induced immune response to Qβ VLP reconstituted in the presence of conventional Qβ VLP and poly-L-glutamic acid N-terminal γ3 amino acid linker (SEQ ID NO: 48 Mouse IL-5 (SEQ ID NO: 46) or human IL-5 (SEQ ID NO: 47) fused to E. coli was recombinantly produced in E. coli and purified. Conventional Qβ VLP and Qβ VLP reconstituted in the presence of poly-L-glutamic acid were derivatized with 10-fold molar excess of SMPH for 30 minutes at 25 ° C. The derivatization reaction was dialyzed twice for 2 hours in a 1000-fold dialysis buffer consisting of 20 mM Hepes, 150 mM NaCl pH 7.4 in a Slide-A-Lyzer dialysis cassette with 10'000 MWCO. Purified mouse IL-5 was pre-reduced with a 2-fold molar excess of pH neutralized TCEP for 60 minutes. Reduced mouse IL-5 (42 μM) was incubated with 40 μM SMPH derivatized conventional Qβ VLP prepared as in Example 1 and 42 μM mouse IL-5, respectively, at 20 ° C. for 3 hours. Incubated with SMPH derivatized Qβ VLP reconstituted in the presence of 40 μM poly-L-glutamic acid prepared as in 4 for 3 hours at 20 ° C. The coupling reaction was dialyzed using a 300 kDa cut-off dialysis membrane at PBS pH 8.0 for 12 hours and then 6 hours.
Five female BalbC mice per group were given 200 μl of PBS with 50 μg, 25 μg and again 50 μg of vaccine of either “conventional Qβ-mouse IL-5” or “reconstructed Qβ-mouse IL-5”, Subcutaneous injections were made on day 0, day 14 and day 28 (100 μl each in two abdominal regions). As controls, 5 mice were treated on day 0, day 14 and day 28 only with either conventional Qβ VLP or Qβ VLP reconstituted in the presence of poly-L-glutamic acid. Immunized. The vaccine was administered without adjuvant. Mice were bled on days 0, 14, 21 and 28 of the immunization protocol.

Microtiter plates (Maxisorp, Nunc) were overnight with 2 μg / ml recombinant mouse IL-5 or 2 μg / ml conventional Qβ or reconstituted Qβ in the presence of 2 μg / ml poly-L-glutamic acid. I coated it. The titers and total IgG potencies of different subclasses (IgG1, IgG2a) specific for conventional Qβ VLP or reconstituted Qβ VLP and IL-5 in serum by substantially the same ELISA as described in Example 8 The value was measured.
Table 4

Table 4 shows that conventional Qβ-mouse IL-5 and reconstituted Qβ-mouse IL-5 induced antibodies specific to mouse IL-5. Conventional Qβ-IL-5 induced a Th1-type immune response with high IgG2a titers. The average ratio of IgG2a / IgG1 titers was approximately 2.5. However, when mice were immunized with Qβ-mouse IL-5 vaccine made from Qβ VLP reconstructed in the presence of poly-L-glutamic acid, IgG2a titers were dramatically reduced, resulting in mean titers Was 0.0077. These data indicate that the immune response induced by reconstituted Qβ-mIL-5 containing poly-L-glutamic acid is more than the Th1 immune response compared to the immune response induced by conventional Qβ-mIL-5. Was shown to have changed to a Th2 response. Sera from 5 mice (5 per group) were pooled for ELISA analysis.
Similarly, anti-Qβ titer showed the same immune response against VLP. Using conventional Qβ VLPs induced a Th1-type immune response against Qβ with high IgG2a titers. In contrast, the use of Qβ VLP reconstituted in the presence of poly-L-glutamic acid induced more Th2-type immune responses to Qβ (data not shown).

Eosinophilia model An experimental asthma model of allergic airway inflammation was used to evaluate the effect of vaccination against eosinophilia. Balb / c mice (5 per group) are vaccinated with reconstituted Qβ VLP coupled to mouse IL-5 as described above. On day 35 of the vaccination program, alum (Alu-Gel-S) containing 50 μg ovalbumin (OVA) was administered intraperitoneally to mice. A third group of 4 animals that were not immunized was also administered. Ten days later (ie, day 33), mice that received PBS containing 100 μg of OVA were administered intranasally daily for 4 days. Mice were sacrificed 24 hours after the last challenge and the lungs were lavaged with PBS. Cells contained in bronchoalveolar lavage (BAL) were stained with Maigrunwald-Giemsa to count the number of eosinophilic cells (Trifilieff A, et al. Clin Exp Allergy. 2001 Jun, 31 (6): 934-42 ).

Example 21 Coupling of Gastrin Fragments to Reconstructed Qβ VLPs The following gastrin fragments with fused linker sequences were chemically synthesized according to standard methods.
G17 (1-9) C2: pEGPWLEEEESSPPPPC (SEQ ID NO: 72)
c1G17: pEGPWLEEEEEAYGWMDFGGC (SEQ ID NO: 73)
nG17 amide: CGGQGPWLEEEEEAYGWMDFCONH ₂ (SEQ ID NO: 74)
nG17-G: CGGQGPWLEEEEEAYGWMDFG (SEQ ID NO: 54)
nG34 amide: CGGQLGPQGPPHLVADPSKKQGPWLEEEEEAYGWMDFCONH ₂ (SEQ ID NO: 75)
nG34-G: CGGQLGPQGPPHLVADPSKKQGPWLEEEEEAYGWMDFG (SEQ ID NO: 76)

Reaction of 2 ml solution containing 2.0 mg / ml reconstituted Qβ VLP in 20 mM Hepes, 150 mM NaCl pH 7.2 with 114.4 μl SMPH solution (50 mM stock solution in DMSO) for 60 minutes at 25 ° C. I let you. Thereafter, the reaction solution was dialyzed twice with 2 L of 20 mM Hepes, 150 mM NaCl pH 7.2 at 4 ° C. for 2 hours.
Subsequently, dialyzed and derivatized Qβ VLP was used to couple with c1G17. Briefly, 1 ml of derivatized Qβ VLP (2 mg / ml concentration) was reacted with 167 μl DMSO containing 10 mM peptide solution and 100 μl acetonitrile at 15 ° C. for 2 hours. The coupling reaction was then centrifuged at 16100 rcf for 5 minutes, the supernatant was collected and dialyzed once for 2 hours at 4 ° C. with 2 L of 20 mM Hepes, 150 mM NaCl pH 7.2, followed by overnight dialysis. The coupling product was designated Qβ-c1G17. Under substantially the same conditions, nG17 amide, nG17-G, nG34 amide and nG34-G were coupled to the reconstructed Qβ VLP.

Example 22 Mice with Qβ-c1G17, Qβ-nG17 amide, Qβ-nG17-G, Qβ-nG34 amide, Qβ-nG34-G, Qβ-G17 (1-9) C2 and DT-G17 (1-9) C2 Detection of Antibody Titers by ELISA and ELISA Adult female C57BL / 6 mice were isolated from Qβ-c1G17 (5 mice per group), Qβ-nG17 amide, Qβ-nG17-G, Qβ-nG34 amide or Qβ-nG34-G. Vaccinated with either (3 mice per group). 50 μg Qβ-c1G17, or 25 μg Qβ-nG17 amide, Qβ-nG17-G, Qβ-nG34 amide and Qβ-nG34-G (obtained in Example 21) were diluted to 200 μl volume with PBS, Subcutaneous injections were given on day 0 and day 14 (100 μl each in two locations on the abdomen). The vaccine was administered without adjuvant. As a control, one group of mice received 50 μg of reconstituted Qβ. Mice immunized with Qβ-C1G17 were bled from the retro-orbital on days 0, 14, 21, 28, 42, 69 and 101, and Qβ-nG17 amide, Qβ-nG17-G, Qβ-nG34 amide and Qβ Mice immunized with -nG34-G were bled from the retro-orbital at 0, 14, 21, 28, 42, 56 and 77 days.
5 mg / ml RNase and 0.2 mM SPDP (final concentration) were incubated at room temperature for 1 hour. The RNase-SPDP solution was purified on a PD10 column (Amersham). After purification, 10 mM EDTA and 1 mM peptide were added to the RNase-SPDP solution.

An ELISA plate (96-well MAXIsorp, NUNC) was applied to the coating buffer (0.1 M NaHCO ₃ , pH 9. In 6), it was coated at 4 ° C. overnight. After washing the plate with wash buffer (PBS-0.05% Tween), the plate is blocked with blocking buffer (2% BSA-PBS-Tween 20 solution) at 37 ° C. for 2 hours and then washed again. And incubated with serially diluted mouse serum. As a control, preimmune serum from the same mice was also tested. Plates were incubated for 2 hours at room temperature. After further washing, the bound antibody was detected with HRPO labeled, Fc specific, goat anti-mouse IgG antibody (Jackson Immunoresearch) and incubated at room temperature for 1 hour. After further washing, the plate was reacted with OPD solution (1 OPD tablet, 25 ul OPD buffer and 8 μl H ₂ O ₂ ) for 6 minutes, and the reaction was stopped with 5% H ₂ SO ₄ solution. The plate was read at 450 nm with an ELISA reader (Biorad Benchmark). ELISA titers are expressed as serum dilutions that produce half-maximal OD in an ELISA assay.

Example 23 Coupling of CXCR4 Fragment to Conventional Qβ VLP and Reconstructed Qβ VLP N-terminally loaded C and C-terminally loaded G were ligated to link the circularized CXCR4 fragment 176-185 ( SEQ ID NO: 65) and CXCR4 fragment 1-39 (SEQ ID NO: 66) having a CGG or GGC linker sequence fused to the N-terminus or C-terminus of CXCR4 fragment 1-39, using standard methods (Peter Henklein, Charite , Berlin, Germany).
3 ml (1.0 mg / ml) solution containing conventional Qβ VLP or reconstituted Qβ VLP in 20 mM Hepes, pH 7.2 was reacted with 85 μl SMPH (50 mM in DMSO, Pierce) for 30 minutes at 25 ° C. . The reaction solution was then dialyzed twice with 3 L of 20 mM Hepes, pH 7.2 for 2 hours at 4 ° C. The dialyzed and derivatized conventional Qβ or reconstituted VLP is then used to couple the peptide CXCR4-CGG-1-39, CXCR4-1-39-GGC, or CXCR4-C-176-185-G. Used for. Briefly, 1 ml of derivatized conventional Qβ VLP or reconstituted Qβ VLP at a concentration of 1 mg / ml was reacted with 70 μl of 5 mM peptide solution in 20 mM Hepes, pH 7.2 at 25 ° C. for 2 hours. The coupling reaction was then centrifuged at 13000 rpm for 5 minutes and the supernatant was collected and dialyzed once for 2 hours at 4 ° C. with 1 L of 20 mM Hepes, pH 7.2, and then dialyzed overnight.

Example 24 Immunization of mice with Qβ-CXCR4-CGG-1-39, Qβ-CXCR4-1-39-GGC, or Qβ-CXCR4-C-176-185-G and detection of antibody subtype Adult female, C57BL / 6 mice (3 mice per group) were vaccinated with conventional Qβ VLP coupled to CXCR4 fragment (obtained in Example 23) or reconstituted Qβ VLP. Only conventional Qβ VLP or reconstituted Qβ VLP was used as a control. 100 μg of the dialyzed vaccine from each sample was diluted with PBS to a volume of 200 μl and injected subcutaneously on day 0 and day 14 (100 μl at two abdomen locations). The vaccine was administered with or without adjuvant (Allhydrogel, 1 mg / injection). Mice were bled from the retro-orbital at day 14, 21 and 28 and titered with different subclasses (IgG1, IgG2a) specific for the CXCR4 fragment of mice by substantially the same ELISA as described in Example 8. IgE titers and total IgG titers were measured.

Example 25 Coupling of CCR5 fragment to conventional Qβ VLP or reconstructed Qβ VLP and immunization of mice Cyclic peptides ECL2A (SEQ ID NO: 61) or PNt (SEQ ID NO: 63) was chemically synthesized according to standard procedures.
2 g / l of conventional Qβ VLP or reconstituted Qβ VLP was derivatized with 1.43 mM SMPH (Pierce, Perbio Science) for 30 minutes at 25 ° C. and then dialyzed against 20 mM Hepes pH 8, 150 mM NaCl. 1 g / l of derivatized Qβ VLP was dissolved in 20% acetonitrile, 150 mM NaCl, 20 mM phosphoric acid pH 7.5. 0.286 mM CCR5 fragment ECL2A or PNt was added and incubated at 25 ° C. for 2 hours.
Six female black mice were primed with 50 μg of conventional Qβ VLP or reconstituted Qβ VLP coupled to CCR5 fragment on day 0 (0.2 ml PBS, administered subcutaneously) to obtain conventional Qβ VLP. Alternatively, reconstructed Qβ VLP alone was compared to BalbC primed with 50 μg. After additional immunization with the same vaccine on the 14th day, α-Qβ and α-CCR5 antibody titers were confirmed by ELISA on the 14th and 21st days.

Example 26 Purification of CCR5-specific mouse polyclonal antibody and its effect on HIV-neutralization assay Serum from immunized mice (obtained in Example 25) was centrifuged at 14000 rpm for 5 minutes. The supernatant was loaded onto a column of 3.3 ml prewashed protein G sepharose (Amersham Biosciences). The column was then washed with PBS and eluted with 100 mM glycine pH 2.8. 1 ml fractions were collected in tubes previously filled with 100 μl of 1M Tris pH8. Fractions with a peak absorbance at 280 nm were pooled.
JR-FL and SF162, cell lines specific for CCR5 co-receptors have been described (O'Brien et al., Nature 1990, 348, page 69, and Shioda et al., Nature 1991, 349, page 167). The HIV-1 inoculum stock solution, in assay media approximately 1000 to 4000 _TCID 50 / ml of: was adjusted to contain _(TCID 50 a dose of 50% of the tissue culture infectivity). Stimulated primary CD8 depleted PBMC (for HIV neutralization assay).
Briefly, a buffy coat obtained from 3 healthy donors was depleted of CD8 + T cells using Rosette Sep reaction mixture (StemCell Technologies Inc., BIOCOBA AG, Switzerland) and transferred to Ficoll-Hypaque (Amersham-Pharmacia Biotech). To isolate PBMC. Cells were adjusted to 4 × 10 ⁶ / ml culture medium (RPMI 1640, 10% FCS, 100 U / ml IL-2, glutamine and antibiotics), divided into three, 5 μg / ml phytohemagglutinin (PHA), 0 Stimulated with either 5 μg / ml PHA or anti-CD3 MAb OKT3. After 72 hours, cells from the three stimulation products were mixed and used as a source of stimulated CD4 + T cells for infection and virus neutralization reactions.
Briefly, cells were incubated with purified polyclonal mouse IgG or control antibody 2D7 serial dilutions (25 μg / ml-25 ng / ml, Pharmingen) for 1 hour at 37 ° C. in 96 well culture plates. Viral seeds (100 TCID ₅₀ , 50% tissue culture infectious dose, Trkola et al., J. Virol., 1999, page 8966) were then added and the plates were cultured for 4-14 days. The total infection volume was 200 μl. As described previously (Moore et al., 1990. Science 250, page 1139), preferably 6 days after infection, supernatant cultures were assayed for HIV-1 p24 antigen production using an immunoassay.

Example 27 Coupling of bradykinin (BK) and des-Arg-bradykinin (Des-Arg9-BK) to conventional Qβ VLP and reconstructed Qβ VLP Bradykinin (BK) with a cysteine fused to the N-terminus of the sequence ( SEQ ID NO: 55) and des-Arg9-bradykinin (SEQ ID NO: 56) or bradykinin (BK) with a cysteine fused to the C-terminus were chemically synthesized according to standard methods.
A solution of 3 ml (1.0 mg / ml) containing conventional Qβ VLP or reconstituted Qβ VLP in 20 mM Hepes, pH 7.2 was reacted with 84 μl SMPH (DMSO containing 50 mM, Pierce) at 25 ° C. for 30 minutes. . The reaction was then dialyzed twice against 3 L of 20 mM Hepes, pH 7.2 for 2 hours at 4 ° C. Subsequently, dialyzed and derivatized Qβ VLP was used to couple bradykinin or des-Arg9-bradykinin. Briefly, 3 ml of derivatized conventional Qβ VLP or reconstituted Qβ VLP at a concentration of 1 mg / ml was added 2 in 20 mM Hepes, pH 7.2 at 25 ° C. with 42 μl of 50 mM bradykinin or des-Arg-bradykinin. Reacted for hours. The coupling reaction was then centrifuged at 13000 rpm for 5 minutes, the supernatant collected, dialyzed once for 3 hours at 4 ° C. with 3 L of 20 mM Hepes, pH 7.2, and then dialyzed overnight.

Example 28 Immunization of mice with Qβ-BK and Qβ-des-Arg9-BK and detection of antibody subtype Adult female, C57BL / 6 mice (10 per group) were treated with conventional Qβ VLP or reconstituted Qβ VLP. Qβ-BK or Qβ-des-Arg9-BK coupled to (obtained in Example 27) was vaccinated. 50 μg of the dialyzed vaccine from each sample was diluted with PBS to a volume of 200 μl and injected subcutaneously on days 0, 14 and 28 (100 μl each in two locations on the abdomen). The vaccine was administered without an adjuvant. As a control, one group of mice was injected with PBS. Mice were bled from the retro-orbital on days 0, 14, 21, and 33.
By substantially the same ELISA as described in Example 8, titers of different subclasses (IgG1, IgG2a) specific for conventional QQ VLP or reconstituted Qβ VLP and TNFα in serum and total IgG titers were obtained. It was measured. ELISA serum titers (Tables 5 and 6) were expressed as the reciprocal of the dilution required to obtain 50% of the optical density measured at saturation.

表６

このデータにより、免疫応答をＴｈ１からＴｈ２に変化する従来のＱβ-ｍＴＮＦにより誘導される免疫応答と比較して、抗原にカップリングしているかにかかわらず再構築Ｑβ ＶＬＰがより多くのＴｈ１免疫応答からより多くのＴｈ２応答へ変化していたことが示された。 Table 5 Anti-conventional Qβ, anti-reconstructed Qβ in mice immunized with conventional Qβ, reconstituted Qβ, Qβ-BK or Qβ-des-Arg9-BK, respectively, on days 0, 14 and 28 Average of anti-BK and anti-des-Arg9-BK specific tIgG, IgG2a and IgG1 (shown as dilution ratio). This data indicates that the reconstituted Qβ VLP has more Th1 immune response regardless of whether it is coupled to the antigen compared to the immune response induced by the conventional Qβ VLP, regardless of whether it is coupled to the antigen. Was shown to have changed to a more Th2 response.

Table 6

This data indicates that the reconstituted Qβ VLP has more Th1 immune response regardless of whether it is coupled to the antigen compared to the immune response induced by conventional Qβ-mTNF that changes the immune response from Th1 to Th2. Was shown to have changed to a more Th2 response.

Example 29 Cloning, expression and purification of CETP fragment fused to the C-terminus of AP205 VLP Annealing two complementary oligonucleotides encoding the CETP fragment and containing the respective restriction enzyme sites of Kpn2I and Mph1103I As a result, a DNA fragment encoding the CETP fragment (SEQ ID NO: 69) was prepared. The resulting fragment was digested with Kpn2I and Mph1103I and under the control of the E. coli tryptophan operon promoter, the same restriction enzyme in vector pAP405-61 (described in Example 1 of US provisional application 60 / 611,308). Cloned into the site. The resulting plasmid is as follows: AP205 coat protein-GTAGGGSG-FGFPEHLLVDFLQSLS.
AP205-ll-CETP1 protein was expressed and purified substantially as described in WO 04/007538. Further, the step of removing E. coli RNA packaged inside the AP205 VLP was performed in substantially the same manner as described in Example 5 above.

Example 30 Chemical Coupling of CETP Peptide CETP1 to Reconstructed Qβ VLP A CETP fragment with an additional CGG linker fused to the N-terminus (SEQ ID NO: 69) was obtained from solid phase chemistry of EMC microcollections GmbH (Germany). Synthesized by the method. The peptide was amidated at the C-terminus.
2 ml (2.0 mg / ml) solution containing reconstituted Qβ VLP (obtained in Example 4) in 20 mM Hepes, 150 mM NaCl pH 7.4, 57 μl SMPH solution (DMSO with 50 mM stock solution, Pierce) And reacted at 25 ° C. for 30 minutes. The reaction was then dialyzed twice against 2 L of 20 mM Hepes, pH 7.2 for 2 hours at 4 ° C. The dialyzed and derivatized reconstituted Qβ VLP was then used to couple the CETP fragment. Briefly, 1 ml of derivatized reconstituted Qβ VLP at a concentration of 2 mg / ml was reacted with 100 μl of 50 mM peptide solution in 15 mM 20 mM Hepes, 150 mM NaCl pH 7.4 for 2 hours. The coupling reaction was then centrifuged at 16000 g for 5 minutes and the supernatant was collected and dialyzed twice for 2 hours at 4 ° C. against 2 L of 20 mM Hepes pH 7.4.

Example 31 CETP Vaccine Test in Cholesterol-Fed Atherosclerotic Model Rabbits New Zealand White rabbits (n = 12 per group) were treated with VLP-CETP fragment vaccine or VLP 200 μg obtained in Example 30 or Example 29. Vaccinated by subcutaneous administration and boosted at weeks 3, 6, 9, 12, 15, 19, 23 and 27. Rabbits had a high cholesterol diet (0.25%) at week 16 and continued on that diet for 16 weeks. Plasma samples were collected at regular intervals from fasted rabbits for antibody titer, lipoprotein, cholesterol and CETP activity measurements. At week 32, the animals were sacrificed and the aorta removed for atherosclerotic lesion analysis. The aorta was “opened to the front” and then stained with Oil Red O, and the proportion of the artery occupied by the lesion was calculated for each animal.

Example 32 Coupling of mC5acys to Reconstructed Qβ VLP The mouse C5a amino acid sequence (SEQ ID NO: 58, hereinafter referred to as mC5acys) containing an N-terminal CGSGG linker was chemically synthesized by Dictagene SA. The C-terminal 19 amino acids of the mouse C5a sequence were synthesized by adding a CGG linker at the N-terminus (EMC Microcollections GmbH, Germany) (SEQ ID NO: 71, hereinafter referred to as mC5acys ^59-77 ).
A solution containing 143 μM reconstituted Qβ VLP in 4- (2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) buffer (20 mM Hepes, 150 mM NaCl pH 7.2) was added in a 2-fold molar excess (286 μM) of SMPH. (Pierce) and reacted at 25 ° C. for 30 minutes with shaking. The reaction product was dialyzed by exchanging twice with Dulbecco's PBS (Gibco) using a dialysis unit with a 10,000 Da molecular weight cut-off (Slide-A-Lyzer, Pierce).
An equimolar amount of mC5acys was added to a 36 μM solution of SMPH-derivatized Qβ VLP. The reaction volume was 100 μl, and multiple reactions were performed simultaneously. The reaction was incubated at 15 ° C. for 2 hours with shaking. After coupling, an aliquot was centrifuged at 16000 g for 3 minutes at 4 ° C. to pellet insoluble material and transfer the vaccine to the soluble fraction.
A 2-fold molar excess of mC5acys ^59-77 was added to a 107 μM solution of a 5 molar excess of SMPH-derivatized Qβ VLP. The reaction volume was 100 μl, and multiple reactions were performed simultaneously. The reaction was incubated at 15 ° C. for 2 hours with shaking. After coupling, a certain amount was centrifuged at 16000 g for 3 minutes at 4 ° C. to pellet insoluble material. The soluble vaccine was dialyzed twice for 2 hours in PBS using a 10,000 MW cut-off dialysis cassette.

Example 33 Immunization of mice with Qβ-mC5acys VLP in a collagen-induced arthritis model 50 μg of reconstituted Qβ-mC5acys diluted in Dulbecco's PBS on the flank of male 6-week old DBA / 1JCrl mice (Charles River, Deutschland) Immunization was performed by subcutaneous administration (n = 8) or 30 μg of reconstituted Qβ VLP alone (n = 8). On days 15 and 24 after the initial immunization, booster immunizations were performed with two additional subcutaneous doses. Anti-mC5acys antibody titer was measured by ELISA. After initial immunization with 100 μg of bovine type II collagen (MD Biosciences) emulsified with a glass syringe in a 1: 1 ratio with complete Freund's adjuvant (CFA), mice were administered twice on days 35 and 57 Immunized by subcutaneous administration at the base of the tail. The mice were then evaluated for the induction and severity of collagen-induced arthritis by daily measurement of forelimb and hindlimb joint thickness and by daily assessment of the joint clinical score. Joint film thickness was measured using a constant-tension caliper.

Example 34 Immunization of mice with Qβ-CCR5 fragment and detection of antibody subtype Conventional female C57BL / 6 mice (3 per group) were coupled to CCR5 fragment (obtained in Example 25). Either Qβ VLP or reconstructed Qβ VLP were vaccinated. Only conventional Qβ VLP or reconstituted Qβ VLP was used as a control. 100 μg of the dialyzed vaccine from each sample was diluted with PBS to a volume of 200 μl and injected subcutaneously on day 0 and day 14 (100 μl at two abdomen locations). The vaccine was administered with or without adjuvant (Allhydrogel, 1 mg / injection). Mice were bled from the retro-orbital at day 14, 21 and 28, and the titers of different subclasses (IgG1, IgG2a) specific to the mouse CCR5 fragment, IgE titer and total IgG titer were measured by ELISA. .
Microtiter plates (Maxisorp, Nunc) were coated overnight with 10 μg / ml CCR5 fragment coupled to RNase. By substantially the same ELISA as described in Example 8, titers of different subclasses (IgG1, IgG2a) specific for conventional QQ VLP or reconstituted Qβ VLP and TNFα in serum and total IgG titers were obtained. It was measured.

Example 35 Efficacy of Vaccination against Qβ-BK, Qβ-des-Arg9-BK for the Treatment of Collagen-Induced Arthritis The efficacy of reconstituted Qβ-BK or Qβ-des-Arg9-BK was demonstrated using mouse collagen induction. Tested in the osteoarthritis (CIA) model. Ten male DBA / 1 mice per group were immunized by subcutaneous administration of 50 μg Qβ-BK, Qβ-des-Arg9-BK or Qβ alone three times (days 0, 14 and 28). Then, 200 μg of bovine type II collagen mixed with complete Freund's adjuvant was subcutaneously administered to mice twice (days 34 and 55).
After the second collagen / CFA injection, mice were examined regularly and given a clinical score varying from 0 to 3 for each limb depending on the degree of redness and swelling observed. Three weeks after the second collagen / CFA injection, an average clinical score was measured for each limb in the three experimental groups.

Example 36 Efficacy of vaccination against Qβ-BK and Qβ-des-Arg9-BK for the treatment of allergic airway inflammation (AAI) against bradykinin (BK) and des-Arg9-bradykinin (des-Arg9-BK) To evaluate the effect of vaccination on Th2-mediated immune responses, an allergic airway inflammation experimental asthma model was used. This Th2-mediated immune response is characterized by eosinophilic influx into the lung, cytokine (IL-4, IL-5, IL-13) production, IgE antibody and mucosal production, and bronchial hypersensitivity (BHR) . Balb / c mice (5 per group) were immunized with Qβ-BK or Qβ-des-Arg9-BK as described in Example 15 or with Qβ alone. 35 days after the first immunization, mice were administered intraperitoneally with 50 μg of ovalbumin (OVA) with or without adjuvant (Allhydrogel, 1 mg AIOH / injection). Ten days later (ie, day 45), all mice were challenged with intranasal administration of PBS containing 50 μg OVA daily for 4 consecutive days. BHR was measured by whole body phlegtismograph 24 hours after the last antigen administration. The mice were then sacrificed at specific time points and analyzed for pulmonary inflammation and Th2-mediated immune responses. PBS / 1% BSA was used for lung lavage. As already described (Trifilieff A, et al. Clin Exp Allergy. 2001 Jun, 31 (6): 934-42), cells contained in the bronchoalveolar lavage (BAL) were measured with a Coulter counter (Instrumenten Gesellschaft AG). Counted and identified by Maigrunwald-Giemsa staining.

FIG. 5 shows analysis of RNA hydrolysis from Qβ VLP by RNase in a 1% agarose gel stained with ethidium bromide. What was run on the gel is as follows. Sample 1: MBI fermented material 1 kb DNA ladder; Sample 2: Conventional Qβ VLP; Sample 3: Qβ VLP treated with RNase in 0.2 × HBS buffer pH 7.2. 2 shows SDS-PAGE gels of conventional Qβ VLP and Qβ VLP reconstructed according to the present invention. Lane 1: Molecular standards; Lane 2: Conventional Qβ VLP under non-reducing conditions; Lane 3: Molecular standards; Lane 4: Reconstituted Qβ VLP electrophoresis under reducing conditions (25 mM DTT); Lane 5: Reconstituted Qβ VLP electrophoresis material under non-reducing conditions. Antibodies against nicotine after immunization of mice with nicotine inducers coupled to conventional Qβ VLP, RNase A treated Qβ VLP, and reconstituted Qβ VLP in the presence of different polyanionic polymers Indicates the titer. IgG1 against nicotine after immunization of mice with nicotine inducer coupled to conventional Qβ VLP, RNase A treated Qβ VLP, and reconstituted Qβ VLP in the presence of different polyanionic polymers And IgG2a antibody titers. The elution characteristics of the construction (assembly) mixture by size exclusion HPLC are shown. (A) AP205 coat protein constructed without polyanionic polymer; (B) AP205 coat protein constructed in the presence of polyglutamic acid. The formation of AP205VLP is indicated by a peak at 7.83 minutes, the same residence time as conventional AP205VLP. Unassembled AP205 coat protein eluted with a residence time of 11.77 minutes.

Claims (35)

(a) a virus-like particle (VLP) comprising an RNA bacteriophage coat protein, mutein or fragment thereof having at least one first attachment site, wherein the VLP is produced recombinantly in a host A virus-like particle, wherein the amount of the host RNA having a secondary structure contained in the VLP is at most 20% of the amount of the host RNA having a secondary structure originally contained in the VLP;
(b) at least one antigen having at least one second attachment site;
A composition comprising the at least one antigen (b) bound to the VLP (a) via the at least one first attachment site and the at least one second attachment site, Composition. (a) a virus-like particle (VLP) comprising an RNA bacteriophage coat protein, mutein or fragment thereof having at least one first attachment site, wherein the VLP is produced recombinantly in a host A virus-like particle, wherein the VLP is essentially free of host RNA, preferably host nucleic acid;
(b) at least one antigen having at least one second attachment site;
A composition comprising the at least one antigen (b) bound to the VLP (a) via the at least one first attachment site and the at least one second attachment site, Composition.   4. The method according to claim 2 or 3, further comprising at least one polyanionic polymer bound to the VLP, preferably the polyanionic polymer is packaged within the VLP. Composition.   The composition of claim 3, wherein the polyanionic polymer has a molecular weight of 10,000 to 100,000 daltons. The polyanionic polymer is
(a) a polyanionic polypeptide;
(b) polyanionic saccharides;
(c) a polyanionic organic polymer; and
(d) a nucleic acid that is not a toll-like receptor ligand,
The composition according to claim 3 or 4, which is selected from the group consisting of:   6. The composition of claim 5, wherein the nucleic acid is tRNA. The polyanionic polypeptide is
(a) polyglutamic acid;
(b) polyaspartic acid;
(c) poly (GluAsp); and
(d) any chemical modification of (a)-(c),
The composition according to claim 5, wherein the composition is selected from the group consisting of:   The composition according to claim 3 or 4, wherein the polyanionic polymer is polyglutamic acid and / or polyaspartic acid, and preferably the polyanionic polymer is polyglutamic acid. The polyanionic saccharide is
(a) anionic dextran;
(b) phosphocellulose;
(c) polyglucuronic acid;
(d) polygalacturonic acid;
(e) polysialic acid;
(f) hyaluronic acid; and
(g) The composition according to claim 5, wherein the composition is selected from the group consisting of glycosaminoglycans. Said anionic dextran is
(a) dextran sulfate;
(b) Carboxymethyl dextran;
(c) sulfopropyldextran;
(d) dextran methyl sulfonate; and
The composition according to claim 9, which is selected from the group consisting of (e) dextran phosphate. The glycosaminoglycan is
(a) heparin;
(b) heparan sulfate;
(c) dermatan sulfate;
(d) chondroitin sulfate; and
(e) The composition according to claim 9, which is selected from the group consisting of keratan sulfate. The polyanionic organic polymer is
(a) polyvinyl sulfate; and
6. The composition of claim 5, wherein the composition is selected from the group consisting of (b) polyacrylate. The RNA phage is
(a) bacteriophage Qβ;
(b) bacteriophage R17;
(c) bacteriophage fr;
(d) bacteriophage GA;
(e) bacteriophage SP;
(f) bacteriophage MS2;
(g) bacteriophage M11;
(h) bacteriophage MX1;
(i) bacteriophage NL95;
(j) bacteriophage f2;
(k) bacteriophage PP7; and
(l) Bacteriophage AP205
The composition according to any one of claims 1 to 12, which is selected from the group consisting of:   The composition according to any one of claims 1 to 13, wherein the VLP comprises a coat protein of RNA phage Qβ, fr, AP205 or GA, and / or a mutein thereof, and / or a fragment thereof.   15. A composition according to any one of the preceding claims, wherein the first attachment site binds to the second attachment site via at least one covalent bond, preferably at least one non-peptide covalent bond.   16. A composition according to any one of the preceding claims, wherein the first attachment site contains an amino group, preferably an amino group of lysine.   17. A composition according to any one of the preceding claims, wherein the second attachment site contains a sulfhydryl group, preferably a cysteine sulfhydryl group.   18. The composition according to any one of claims 1 to 17, wherein the first attachment site contains a lysine amino group and the second attachment site contains a cysteine sulfhydryl group.   The composition according to any one of claims 1 to 18, further comprising a linker. Said at least one antigen is
(a) a polypeptide;
(b) carbohydrates;
(c) steroid hormones;
(d) an organic molecule; and
(e) The composition according to any one of claims 1 to 19, which is selected from the group consisting of haptens.   21. The composition according to any one of claims 1 to 20, wherein the at least one antigen is a self-antigen or a fragment or variant thereof. The autoantigen is
(a) lymphotoxin (preferably lymphotoxin α (LTα), lymphotoxin β (LTβ));
(b) lymphotoxin receptor;
(c) a nuclear factor kB ligand (RANKL) receptor activator;
(d) vascular endothelial growth factor (VEGF);
(e) Vascular endothelial growth factor receptor (VEGF-R);
(f) Interleukin-5;
(g) interleukin-17;
(h) interleukin-13;
(i) IL-23p19;
(j) Ghrelin;
(k) CCL21;
(l) CXCL12;
(m) SDF-1;
(n) M-CSF;
(o) MCP-1;
(p) endoglin;
(q) GnRH;
(r) TRH;
(s) eotaxin;
(t) bradykinin;
(u) BLC;
(v) tumor necrosis factor α;
(w) amyloid β peptide (Aβ _1-42 );
(x) Aβ _1-6 ;
(y) angiotensin;
(z) gastrin and / or progastrin;
(aa) CETP;
(bb) CCR5;
(cc) C5a;
(dd) CXCR4;
(ee) Des-Arg bradykinin;
(ff) GnRH peptide;
(gg) angiotensin peptide;
(hh) TNF-peptide;
(ii) a fragment of said antigen (a)-(hh); and
(jj) The composition according to any one of claims 1 to 21, which is selected from the group consisting of the aforementioned variants of antigen (a)-(hh).   A vaccine composition comprising the composition according to any one of claims 1 to 22.   24. The vaccine composition of claim 23, wherein the vaccine lacks an adjuvant.   24. The vaccine composition according to claim 23, further comprising at least one adjuvant. A method for preparing a VLP of RNA bacteriophage-antigen conjugate and a composition of the present invention, respectively,
(a) a step of recombinantly producing a virus-like particle (VLP) having at least one first attachment site by a host, wherein the VLP contains an RNA bacteriophage coat protein, a mutein or a fragment thereof. is there;
(b) decomposing virus-like particles into the RNA bacteriophage coat protein, mutein or fragment thereof;
(c) purifying the coat protein, mutein or fragment thereof;
(d) reconstructing the purified RNA bacteriophage coat protein, mutein or fragment thereof into a virus-like particle, wherein the virus-like particle is essentially devoid of host RNA, preferably host nucleic acid. Is; and
(e) binding at least one antigen having at least one second attachment site to the VLP obtained from step (d);
Including a method.   27. The method of claim 26, wherein the purified coat protein is reconstituted in the presence of at least one polyanionic polymer. A method for modulating the VLP of an RNA bacteriophage-antigen conjugate comprising:
(a) a step of recombinantly producing virus-like particles (VLP) by a host, wherein the VLP comprises an RNA bacteriophage coat protein, a mutein or fragment thereof, and a fusion protein containing at least one antigen. Is;
(b) decomposing the VLP into the fusion protein;
(c) purifying the fusion protein;
(d) restructuring the purified fusion protein into a VLP, wherein the virus-like particle is essentially devoid of host RNA, preferably host nucleic acid.   30. The method of claim 28, wherein the purified coat protein is reconstituted in the presence of at least one polyanionic polymer.   30. The method of claim 28 or 29, wherein the VLP is a VLP of RNA bacteriophage AP205.   An immunization method comprising administering the vaccine composition according to any one of claims 23 to 25 to an animal or a human.   Use of a composition according to any one of claims 1 to 22 or a vaccine composition according to any one of claims 23 to 25 in the manufacture of a medicament for the treatment of a disease, preferably Use wherein the disease is an inflammatory disease and / or a chronic autoimmune disease.   A method for treating a disease comprising administering the composition according to any one of claims 1 to 22 or the vaccine composition according to any one of claims 23 to 25 to an animal or a human, preferably Is a method of treatment wherein the disease is an inflammatory disease and / or a chronic autoimmune disease. (a) a composition according to any one of claims 1 to 22; and
(b) A pharmaceutical composition comprising an acceptable pharmaceutical carrier.   31. A composition obtainable by the method according to any one of claims 26-30.

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