MXPA01004372A

MXPA01004372A - Enhanced vaccines

Info

Publication number: MXPA01004372A
Application number: MXPA/A/2001/004372A
Authority: MX
Inventors: Lars T Hellman
Original assignee: Resistentia Pharmaceuticals Ab
Priority date: 1998-11-02
Filing date: 2001-05-02
Publication date: 2002-06-05

Abstract

The invention relates to methods and materials involved in the treatment and prevention of various diseases such as infections and IgE-related diseases. Specifically, the invention relates to methods and materials that can be used to vaccinate a mammal against specific self or non-self antigens. For example, the methods and materials described herein can be used to reduce the effects of IgE antibodies within a mammal by reducing the amount of total and receptor bound IgE antibodies in the mammal. In addition, the invention provides vaccine conjugates, immunogenic polypeptides, nucleicacid molecules that encode immunogenic polypeptides, host cells containing the nucleic acid molecules that encode immunogenic polypeptides, and methods for making vaccine conjugates and immunogenic polypeptides as well as nucleic acid molecules that encode immunogenic polypeptides. Further, the invention provides an IgE vaccine that induces an anti-self IgE response in a mammal.

Description

IMPROVED VACCINES THAT COMPRISE PORTIONS INDEPENDENT AND NON-INDEPENDENT IgE OR DIMERIC ANTIGENS Field of the Invention The invention relates to methods and materials involved in the treatment of various diseases such as infections and diseases related to IgE. Specifically, the invention relates to methods and materials that can be used to vaccinate a mammal against specific independent or non-independent antigens. For example, the methods and materials described herein can be used to reduce the effects of IgE antibodies within a mammal.

BACKGROUND OF THE INVENTION Mammals are susceptible to many diseases and ailments, including bacterial infections, viral infections and IgE-related diseases such as allergies. In general, infections are characterized by the invasion and multiplication of microorganisms (eg, bacteria, fungi and viruses) within body tissues. Many types of infections can be treated or prevented through the use of vaccines. For example, the polio vaccine can prevent poliovirus infections. Typically, a vaccine is a suspension of attenuated or killed microorganisms. The IgE-related diseases are mediated by an immunoglobulin class designated as immunoglobulin E (IgE). In fact, IgE antibodies are a major cause of hypersensitivity reactions found within the human population despite their normally very low concentration in human plasma (10-400 ng / mL). The effects are due to the interaction of IgE antibodies with the high affinity receptor for IgE in mast cells and basophilic leukocytes. Transverse ligation of two IgE receptors on the surface of this type of cells, for example by allergenic ligation, initiates the release of a number of physiologically active substances, such as histamine, PAF (platelet activating factor), heparin, leukotrienes, prostaglandins, thromboxanes and chemotactic factors for eosinophilic and neutrophilic granulocytes. Presumably, these mediators give rise to the direct symptoms of IgE-mediated allergenic reactions (type I hypersensitivity). Disease conditions belonging to this group may include asthma, skin allergies, pollen allergies, food allergies and eczema. The high affinity receptor for IgE has been characterized.

This receptor appears to occur in mast cells, basophilic leukocytes, eosinophils, monocytes and Langerhan cells. In addition, the receptor is a complex of three different subunits (a, ß and? Chains). The α-chain is mainly located extracellularly and appears to interact with the IgE molecule. Previous studies of the epsilon chain of the IgE molecule have suggested that a region of 76 amino acids at the boundary between the CH2 and CH3 domains (CH refers to the constant domains in the heavy chain) are important for the interaction between the IgE molecule and its high affinity receptor. In addition, it was shown that a peptide corresponding to this region inhibits the interaction between the native IgE and its high affinity receptor in vitro at a molar ratio of almost 1: 1 compared to the entire region of CH2-CH3-CH4 (Helm ei). al., Nature 331, 180-183 (1998)). This peptide was also shown to inhibit an eruptive IgE-mediated reaction in allergen stimulation. However, in this case, the concentration was approximately 10 times the concentration necessary to exhibit the same inhibitory effect with native IgE (Helm et al., Proc Nati Acad Sci USA 86, 9465-9469 (1989)).

BRIEF DESCRIPTION OF THE INVENTION The invention relates to methods and materials involved in the treatment and prevention of various diseases, such as infections and diseases related to IgE. Specifically, the invention relates to methods and materials that can be used to vaccinate a mammal against specific independent and non-independent antigens. For exampleS. , the methods and materials described herein can be used to reduce the effects of IgE antibodies within a mammal by reducing the amount of bound, total and receptor IgE antibodies in the mammal. Such methods and materials can be used to treat atopic allergies in mammals such as humans, dogs and pigs. The invention is based on the discovery that a vaccine conjugate can be designed to contain at least two polypeptides, each polypeptide having at least two similar amino acid segments, such that administration of the conjugate to the mammal can induce an immune response against at least a portion of one of the polypeptides. Such immune responses may be more potent than the responses induced by any of the polypeptides in an unconjugated form or any polypeptide conjugate that lacks at least two similar amino acid segments. In this manner, the vaccine conjugates described herein can be used to provide mammals with substantial protection against a wide range of antigens, either independent (eg, IgE molecules) or non-independent (eg, viral polypeptides). The invention is also based on the discovery that a vaccine conjugate containing a polypeptide having a cytosine activity can be designed such that a potent immune response is induced against another polypeptide within the conjugate. Such immune responses may be more potent than the responses induced by a conjugate lacking a polypeptide having a cytosine activity. Although not limited to any particular mode of action, a conjugate containing a polypeptide having a cytosine activity as well as an immunogenic polypeptide, presumably concentrates cytosine activity in the localized area containing the immunogenic polypeptide. In this manner, the polypeptide having the cytosine activity can stimulate the cells to participate in the generation of a specific immune response against the immunogenic polypeptide.

In addition, the invention is based on the discovery that polypeptides containing an independent IgE portion and a non-independent IgE portion are immunogenic and induce effective anti-independent IgE responses in mammals. Such immunogenic polypeptides can be used as a vaccine to induce an anti-independent IgE response that acts against the hypersensitivity induced by the independent IgE antibodies. Although not limited to any particular mode of action, the immunogenic polypeptides described herein induce the production of anti-independent IgE antibodies that presumably have specificity for the portion of the IgE molecule that interacts with the high affinity IgE receptor. After production, the anti-independent IgE antibodies can interact with the independent IgE antibodies, such that the independent IgE antibodies are unable to bind to the high affinity IgE receptor. This inhibition of binding to the receptor presumably reduces the hypersensitivity induced by the independent IgE antibodies. In this way, the degree of IgE-induced effects can be reduced as more anti-independent IgE antibodies are produced. In general, the invention highlights an immunogenic polypeptide having an independent IgE portion and a non-independent IgE portion. The immunogenic polypeptide is effective in inducing an anti-independent IgE response in a mammal (eg, a human). The independent portion may contain at least a portion of a CH3 domain of IgE. The polypeptide may be capable of being dimerized to form a soluble immunogenic dimer effective in inducing the anti-independent IgE response in the mammal. The non-independent IgE portion may contain a first region and a second region, the independent IgE portion being located between the first and second regions of the non-independent IgE portion. The first region may contain at least a portion of a CH2 domain of IgE and the second region may contain at least a portion of a CH4 domain of IgE. The non-independent IgE portion may contain an IgE sequence present in a non-placental mammal (eg, opossum, platypus, Koala, kangaroo, wallabi and Australian bear). The independent IgE portion may lack the CH2 domain of an IgE antibody. The immunogenic polypeptide may contain a post-translational eukaryotic modification. In addition, the immunogenic polypeptide may contain a polyhistidine sequence. The anti-independent IgE response can be a polyclonal response. In another embodiment, the invention highlights a nucleic acid molecule that contains a nucleic acid sequence that encodes an immunogenic polypeptide. The immunogenic polypeptide contains an independent IgE portion as well as a non-independent IgE portion and is effective in inducing an anti-independent IgE response in a mammal. The nucleic acid molecule may contain an additional nucleic acid sequence encoding an amino acid sequence that promotes secretion of the immunogenic polypeptide from a eukaryotic cell.

Another embodiment of the invention highlights a host cell (e.g., eukaryotic cell) that contains a nucleic acid molecule having a nucleic acid sequence that encodes an immunogenic polypeptide. The immunogenic polypeptide contains an independent IgE portion as well as a non-independent IgE portion and is effective in inducing an anti-independent IgE response in a mammal. Another embodiment of the invention highlights a soluble immunogenic dimer containing two immunogenic polypeptides that are capable of dimerizing to form the soluble immunogenic dimer. Each of the two polypeptides Immunogenic contains an independent IgE portion and a non-independent IgE portion and the soluble immunogenic dimer is effective in inducing an anti-independent IgE response in a mammal. Another embodiment of the invention highlights a vaccine containing an immunogenic polypeptide having an independent IgE portion and a non-independent IgE portion. The immunogenic polypeptide is effective in inducing an anti-independent IgE response in a mammal. The vaccine may contain a pharmaceutically acceptable vehicle. Another embodiment of the invention highlights a method for making a nucleic acid molecule that encodes an immunogenic polypeptide effective in inducing an anti-independent IgE response in a mammal. The method includes the combination of nucleic acid sequences, first and second, to form the nucleic acid molecule, wherein the first nucleic acid sequence encodes at least a portion of an IgE molecule present within the mammal and wherein the second nucleic acid sequence encodes at least a portion of a molecule of IgE not present in the mammal. Another embodiment of the invention highlights a method for making a nucleic acid molecule that encodes an immunogenic polypeptide effective in inducing an anti-independent IgE response in a mammal. The method includes (a) the selection of a first nucleic acid sequence, wherein the first nucleic acid sequence encodes at least a portion of an IgE molecule present within the mammal, (b) the selection of a second nucleic acid sequence. , wherein the second nucleic acid sequence encodes at least a portion of an IgE molecule not present in the mammal and (c) the combination of the first and second nucleic acid sequences to form the nucleic acid molecule. In another aspect, the invention highlights a vaccine complex for vaccinating a mammal (e.g., human). The complex contains a first and second polypeptide. Each of the polypeptides, first and second, contains at least two similar amino acid sequences of at least five amino acid residues in length. In addition, the polypeptides, first and second, are connected to form the complex and administration of the complex to the mammal induces an immune response against at least a portion of the polypeptide, first or second. The polypeptide, first and / or second, may contain an amino acid sequence expressed by the mammal. The polypeptides, first and second, can be identical and can form a dimer. The connection of the polypeptides, first and second, can include a disulfide ligation, the connection of the polypeptides, first and second, can include a non-covalent interaction. The polypeptide, first and / or second, may contain a linker site (eg, a polyhistidine sequence). The amino and carboxyl terms of the polypeptide, first and / or second, may contain the linker site. The complex can include a linker molecule (e.g., an antibody such as an anti-polyhistidine antibody). A linker molecule can connect the polypeptide, first and second. The complex may contain a third polypeptide, wherein the third polypeptide has a cytosine activity. The activity of the cytosine may be an activity of a cytosine such as an interferon-α, interferon-β, interferon-β, TNF-α, IL-1, IL-2, IL-4, IL-6, IL-1 2. IL-15, IL-18 and a granulocyte-macrophage colony stimulating factor. A linker molecule can connect the third polypeptide to the polypeptide, first or second. Similar amino acid sequences may be greater than about twenty amino acid residues in length. The complex may contain an Fc-gamma receptor II blocking molecule (eg, an anti-CD32 antibody). In another embodiment, the invention highlights a vaccine complex for vaccinating a mammal (e.g., human). The complex contains a first polypeptide connected to a second polypeptide, wherein the first polypeptide contains at least two similar amino acid sequences of at least five amino acids in length. In addition, the second polypeptide has a cytosine activity and administration of the complex to the mammal induces an immune response against at least a portion of the first polypeptide. The first polypeptide may contain an amino acid sequence expressed by the mammal. The connection of the polypeptides, first and second, can include a non-covalent interaction. The first and / or second polypeptide may contain a binding site (eg, a polyhistidine sequence). For example, the amino and carboxyl terms of the first polypeptide may contain a binding site. The complex may contain a linker molecule (e.g., an antibody such as an anti-polyhistidine antibody). The cytosine activity may be an activity of a cytosine such as an interferon-α, interferon-β, interferon-β, TNF-α, IL-1, IL-2, IL-4, IL-6, IL-12. IL-1 5, IL-18 and a granulocyte-macrophage colony stimulating factor. The complex may contain a third polypeptide. The polypeptides, first and third, can be identical and can form a dimer. The connection of the polypeptides, first and third, may include a disulfide ligation. Similar amino acid sequences may be greater than about twenty amino acid residues in length. The complex may contain an Fc-gamma receptor II blocking molecule (eg, an anti-CD32 antibody). Another embodiment of the invention highlights a vaccine complex for vaccinating a mammal (eg, human). The complex contains a polypeptide, first, second and third, where the polypeptides, first, second and third are connected to form the complex. The first polypeptide has a first cytosine activity. The second polypeptide has a second cytosine activity. Administration of the complex to the mammal induces an immune response against at least a portion of the third polypeptide. The third polypeptide may contain an amino acid sequence expressed by the mammal. The connections of the first, second and third polypeptides may include non-covalent interactions. The polypeptide, first, second and / or third, may contain a binding site. The complex may contain a linker molecule. The third polypeptide may contain at least two similar amino acid sequences of at least five amino acids in length. The complex may contain an Fc-gamma receptor II blocking molecule (eg, an anti-CD32 antibody). Another embodiment of the invention highlights a vaccine complex for vaccinating a mammal (eg, human). The complex contains a first polypeptide connected to a second polypeptide, wherein the first polypeptide is a polypeptide having interferon-a or interferon-β activity, and administration of the complex to the mammal induces an immune response against at least a portion of the second polypeptide. The second polypeptide may contain an amino acid sequence expressed by the mammal. The connection of the polypeptides, first and second, can include a non-covalent interaction. The first and / or second polypeptide may contain a binding site. The complex may contain a linker molecule. The second polypeptide may contain at least two similar amino acid sequences of at least five amino acids in length. The complex may contain an Fc-gamma receptor II blocking molecule (eg, an anti-CD32 antibody).

Another aspect of the invention highlights a vaccine for vaccinating a mammal (eg, human). The vaccine contains an Fc-gamma receptor II blocking molecule (e.g., an anti-CD32 antibody) and a polypeptide, wherein administration of the vaccine to the mammal induces an immune response against at least a portion of the polypeptide. The polypeptide may contain an amino acid sequence expressed by the mammal. The Fc-gamma receptor II blocking molecule and the polypeptide can be connected and the connection includes a non-covalent interaction. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary experience in the subject to which this invention pertains. Although methods and materials similar or equivalent to those described herein may be used in the practice or examination of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated in their entirety for reference. In case of conflict, the present specification, including the definitions, will control. In addition, the materials, methods and examples are illustrative only and are not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description and from the claims.

DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram comparing the amino acid sequences of the CH2-CH3-CH4 domains of human, rat and opossum IgE in the upper, middle and lower rows, respectively. The opossum sequence also contains an N-terminal signal sequence followed by six histidine residues. Figures 2A-2B contain diagrams comparing the amino acid sequences of various polypeptides containing the following components: opossum CH2-rat CH3-opossum CH4 (ORO); opossum CH2-rat terminal-N CH3-opossum terminal C CH3-opossum CH4 (ORO-trunc); opossum CH2-mouse CH3-opossum CH4 (OMO); opossum CH2-CH3-CH4 (OOO); platypus CH2-CH3-CH4 (PPP); opossum CH2-human CH3-opossum CH4 (OHO); opossum CH2- pig CH3-opossum CH4 (OPO); and opossum CH2-dog CH3-opossum CH4 (ODO). The arrows indicate domain limits. Figures 3A-C contain diagrams illustrating analyzes of immune responses against an ORO immunogenic polypeptide in a panel of three different strains of rats. The anti-IgE antibody level of rat IgG directed against the native IgE was measured by an ELISA. The native rat IgE was used at a concentration of 5 μg / mL for coating ELISA plates. The 1/5 dilutions of successive sera from each of the individual rats was examined by color reaction in the ELISA. Six vaccinated rats were analyzed together with four control rats of each strain. Figure 4 is a diagram illustrating an analysis of the immune responses against an ORO immunogenic polypeptide as well as OOO and PPP control polypeptides.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES The invention provides methods and materials for the treatment of various diseases such as infections and diseases related to IgE. Specifically, the invention provides methods and materials that can be used to vaccinate a mammal against specific independent and non-independent antigens. For example, the methods and matepales described herein can be used to reduce the effects of IgE antibodies within a mammal by reducing the amount of bound, total and receptor IgE antibodies in the mammal. 1. Vaccine Conjugates The invention provides vaccine conjugates containing at least two polypeptides, each of said polypeptides having at least two similar amino acid segments. As used in this, the term "conjugate" refers to any composition that contains at least two polypeptides that are connected directly or indirectly through one or more covalent or non-covalent linkages. For example, a conjugate can contain ten polypeptides connected in sequence (for example, number one is connected to number two, number two is connected to number three, number three is connected to number four, etc.). As used herein with respect to the polypeptides, the term "connected" refers to any type of covalent or non-covalent linkage, including, without limitation, single bonds, double bonds, triple bonds, disulfide bonds, hydrogen bonds , hydrophobic interactions, van der Waals interactions and any combination thereof. For example, a disulfide bond can connect polypeptide number one to polypeptide number two. Alternatively, an antibody can connect polypeptides numbers one and two. In this case, polypeptides one and two each contain an epitope recognized by the antibody such that the resulting conjugate contains polypeptide number one non-covalently connected to the antibody that is non-covalently connected to polypeptide number two. It is noted that polypeptides numbers one and two in this example may have an identical amino acid sequence. As used herein, the term "amino acid segment" refers to a contiguous extension of amino acid sequence within a polypeptide. For example, the amino acid sequence of residues 30 to 40 within a 100 amino acid polypeptide would be considered an amino acid segment. For the purpose of this invention, an amino acid segment can be of any length greater than about five amino acid residues (eg, greater than about six, seven, eight, nine, dis, 15, 20, 25, 30, 40, 50, 75, 100, 150 or 200 amino acid residues). In this manner, an amino acid segment can be the entire CH3 domain of an IgE antibody. As used herein, the term "similar" with respect to at least two amino acid segments means that the segments are at least about 50 percent identical in an amino acid sequence. For example, similar amino acid segments can be about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100 percent identical. For the purpose of this invention, the percent identity of the amino acid sequence between one amino acid segment and another is calculated as follows. First, the amino acid sequences of the two amino acid segments are aligned by using the MEGALIGN® sequence alignment software (DNASTAR, Madison, Wl, 1997) after the Jotun Heim algorithm with the default settings. Secondly, the number of positions equated between the two aligned amino acid sequences is determined. A matched position refers to a position in which identical residuals occur in the same position when aligned by the MEGALIGN® sequence alignment software. Third, the number of matched positions is divided by the total number of positions and the resulting value is multiplied by 100 to obtain the percentage identity. Again, a vaccine conjugate of the invention contains at least two polypeptides, each of said polypeptides having at least two similar amino acid segments. In this manner, a vaccine conjugate may contain two, three, four, five, six, seven, eight, nine, ten, 15, 20, 25 or 30 polypeptides, each having at least two similar amino acid segments. It is noted that a polypeptide containing at least two similar amino acid segments can contain two, three, four, five, six, seven, eight, nine, ten, or more similar amino acid segments. In addition to polypeptides that contain at least two similar amino acid segments, a vaccine conjugate of the invention can contain any number of polypeptides that do not have at least two similar amino acid segments. For example, a vaccine conjugate may contain four polypeptides, each containing a segment of 30 amino acid residues repeated three times, as well as two polypeptides each lacking similar amino acid segments. Typically, a vaccine conjugate contains a polypeptide that will act as an antigen against which an immune response is desired. In this manner, a vaccine conjugate within the scope of the invention can contain any type of polypeptide, including, without limitation, bacterial polypeptides, fungal polypeptides, viral polypeptides and mammalian polypeptides. For example, a vaccine conjugate may contain five hepatitis C virus polypeptides. It is noted that each polypeptide of a conjugate can have an identical amino acid sequence. In addition, a polypeptide of a vaccine conjugate typically contains similar amino acid segments, each of which can act as a defined antigenic unit against which an immune response is desired. In this manner, a polypeptide of a vaccine conjugate can contain similar amino acid segments that correspond to any region of a polypeptide, including, without limitation, receptor binding regions, ligand-binding regions, enzyme active sites, dissociation sites. of enzyme from polypeptide substrates, antigen-binding regions of antibodies and epitopes recognized by antibodies. For example, a polypeptide of a vaccine conjugate can contain three similar amino acid segments each corresponding to the active site of the enzyme X enzyme. It is noted that similar amino acid segments can be found one after the other or dispersed through all a polypeptide. Typically, administration of a vaccine conjugate results in the formation of antibodies having specificity for an epitope formed by at least a portion of the similar amino acid segments within one of the polypeptides of the vaccine conjugate. Any method can be used to make the polypeptides of a vaccine conjugate, including, without limitation, prokaryotic expression systems, eukaryotic expression systems, and chemical synthesis techniques. In addition, a polypeptide of a vaccine conjugate can be obtained from natural tissue sources. For example, a cerebral glycopolypeptide can be obtained from brain tissue. Typically, each polypeptide other than a conjugate is made independently or is isolated independently and then used to form a conjugate. It is noted that the polypeptides can be purified before being used to form a conjugate. Any method can be used to purify polypeptides, including, without limitation, fractionation, centrifugation and chromatography. For example, polypeptides containing a polyhistidine sequence can be purified by the use of affinity chromatography. Once obtained, the polypeptides can be connected through the use of any method. For example, a polypeptide sample can be incubated with a linker molecule such that the individual polypeptides form conjugates. A binding molecule is any molecule that connects two polypeptides. Typically, a linker molecule is a molecule with two reactive groups or sites that are capable of interacting with, and thus forming, a link between the amino acid residues of two polypeptides. A binding molecule can be a specific binding molecule such as an antibody or a non-specific binding molecule such as a chemical reagent (eg, glutaraldehyde and formaldehyde). Any antibody can be used as a binding molecule. For example, an anti-polyhistidine antibody or an anti-epitope-identifying antibody such as an anti-FLAG® epitope antibody or anti-het agglutinin (HA) -identifying antibody can be used to connect two polypeptides. FLAG® epitopes are described in U.S. Patent Nos. 4, 703.004 and 4782, 37. It is noted that the polypeptides to be connected to a specific binding molecule need to contain the specific site recognized by the binding molecule. For example, to connect two polypeptides with an anti-polyhistidine antibody, each polypeptide must contain the polyhistidine epitope recognized by that antibody. For the purpose of this invention, the specific site recognized by a specific binding molecule, such as an antibody, is referred to as a binding site. Any method can be used to make a polypeptide that contains a binding site, such that a particular antibody can be used as a binding molecule. For example, common molecular cloning techniques can be used to introduce the nucleic acid encoding a FLAG identifier epitope into the nucleic acid encoding a particular polypeptide. It is noted that a binding site can be located in any position. For example, a histidine sequence can be found in the N-terminus, C-terminus or an internal position of a polypeptide. In addition, a polypeptide may contain more than one binding site. For example, a polypeptide can have a polyhistidine sequence in an internal position as well as in the C term. In addition, a polypeptide can contain different binding sites. For example, a polypeptide may have a polyhistidine sequence in an internal position and an epitope of FLAG identifier in the C-terminus. In some cases, two or more polypeptides may be made in such a way that they are connected through a covalent bond. For example, two polypeptides can be made as a fusion protein in such a way that they are connected through a peptide linkage. Alternatively, a polypeptide can be made in a cell line that promotes the formation of disulfide linkages between, for example, two identical polypeptides. In this case, the conjugate would be a homodimer. It is noted that any polypeptide containing one or more cysteine residues can be designed in such a way that the polypeptides form conjugates through cysteine bridges. For example, a polypeptide containing N and C terminal cysteine residues may be made such that conjugates of variant size are formed intracellularly. In addition, the interaction between biotin and avidin can be used to form conjugates. For example, the polypeptides can be designed or chemically treated to contain biotin molecules at the C and N termini. This biotin-containing polypeptide can be incubated with avidin molecules that are capable of interacting simultaneously with two or more biotin molecules. . In this case, a single avidin molecule can bind two biotin-containing polypeptides to form a conjugate. In addition, chelating molecules that can simultaneously bind two or more ions (eg, Ni ++, Cu ++, Co ++ and Zn ++) can be used to form conjugates. For example, a copper chelating molecule that can interact with two copper ions can be used to link two polypeptides containing a polyhistidine sequence. In this case, a single copper ion can interact with each polyhistidine sequence while a single copper chelating molecule links the two polypeptides to form a conjugate. It is observed that immunostimulatory complexes (iscoms) can be used to form conjugates. For example, an iscom containing copper ions can be designed in such a way that polypeptides containing a polyhistidine sequence can be conjugated. Typically, a nucleic acid molecule is constructed in such a manner that a particular polypeptide is expressed. For example, a nucleic acid molecule can be constructed to encode a polypeptide having three similar amino acid segments, as well as a polyhistidine sequence at its C terminus. Once constructed, the nucleic acid molecule can be introduced into a host cell of such that the polypeptide is produced. Any host cell can be used, including, without limitation, prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., human cells). Once produced, the polypeptide can be purified and used to make the desired vaccine conjugate. As used herein, the term "nucleic acid" encompasses both RNA and DNA, including cDNA, genomic DNA, and synthetic DNA (eg, chemically synthesized). The nucleic acid can be double filament or single filament. When it is a single filament, the nucleic acid may be the detection filament or the anti-sense filament. In addition, the nucleic acid may be circular or linear. The nucleic acid can be obtained by the use of common techniques or techniques of molecular cloning or chemical synthesis of nucleic acid in which the target nucleic acid is amplified in a manner similar to that described in the U.S. Patent. No. 4,683, 1 95, and the subsequent modifications to the procedure described herein. In general, the sequence information from the ends of the region of interest or beyond is used to design oligonucleotide feedstock that is identical or similar in sequence to the opposite strands of a potential model to be amplified. Using PCR, a nucleic acid sequence can be amplified from RNA or DNA. For example, a nucleic acid sequence can be isolated by PCR amplification of total cellular RNA, total genomic DNA, and cDNA as well as bacteriophage sequences, plasmid sequences, viral sequences and the like. When RNA is used as a model source, reverse transcriptase can be used to synthesize complementary DNA strands. Any method for introducing nucleic acid into a cell can be used. In fact, many methods for introducing nucleic acid into the cell, either in vivo or in vitro, are well known to those skilled in the art. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and virus-mediated nucleic acid transfer are common methods in the introduction of nucleic acid into cells. In addition, simple DNA can be delivered directly to the cells in vivo as described elsewhere (U.S. Patent No. 5,580,859 and U.S. Patent No. 5,589,466, including the continuations thereof). In addition, nucleic acid can be introduced into the cells by the generation of transgenic animals. It is noted that transgenic animals such as rabbits, goats, sheep and cows can be designed in such a way that large amounts of a polypeptide are secreted in their milk. Transgenic animals can be aquatic animals (such as fish, sharks, dolphins and the like), farm animals (such as pigs, goats, sheep, cows, horses, rabbits and the like), rodents (such as rats, guinea pigs, Indians and mice), non-human primates (such as mandrill, monkeys and chimpanzees) and domestic animals (such as dogs and cats). Several techniques known in the art can be used to introduce nucleic acid into animals to produce the founder lines of the transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (U.S. Patent No. 4,873, 191); gene transfer mediated by retroviruses in germ lines (Van der Putten et al., Proc. Nati, Acad. Sci., USA, 82: 6148-6152 (1985)); genetic transfection in embryonic stem cells (Gossler A et al., Proc. Nati, Acad. Sci. USA 83: 9065-9069 (1986)); genetic targeting in embryonic stem cells (Thompson et al., Cell, 56: 313-321 (1989)); Nucleic transfer of somatic nuclei (Schnieke AE et al., Science 278: 2130-2133 (1997)); and embryo electroporation. For a review of techniques that can be used to generate and produce transgenic animals, the skilled artisan may consult Gordon (Intl. Rev. Cytol., 1 15: 171-229 (1989)), and may obtain additional guidance from, for example: Hogan et al., "Manipulating the Mouse Embryo" Cold Spring Harbor Press, Cold Spring Harbor, NY (1986); Krímpenfort eí al. , Bio / Technology, 9: 844-847 (1991); Palmíter et al., Cell, 41: 343-345 (1985); Kraemer et al., "Genetic Manipulation of the Early Mammalian Embryo" Cold Spring Harbor Press, Cold Spring Harbor, NY (1985); Hammer et al., Nature, 315: 680-683 (1985); Purscel et al., Science, 244: 1281-1288 (1986); Wagner went to., Patent of E.U. No. 5, 175,385; and Krimpenfort eí al. , Patent of E.U. No. 5, 175,384. In addition, a nucleic acid encoding a polypeptide can be maintained within a cell in any form. For example, nucleic acid can be integrated into the genome of a cell or maintained in an episomal state. In other words, a cell can be a stable or transient transformer. In addition, any method can be used to direct the expression of a particular polypeptide. Such methods include, without limitation, the construction of a nucleic acid such that a regulatory element promotes the expression of a nucleic acid sequence encoding a polypeptide. Typically, regulatory elements are DNA sequences that regulate the expression of other DNA sequences at the level of transcription. In this way, the regulatory elements include, without limitation, promoters, enhancers and the like. In one embodiment, a conjugate for vaccinating rats that contains polypeptides having an N-terminal polyhistidine sequence followed by an opossum IgE CH2 domain, a rat IgE CH3 domain, an opossum IgE CH2 domain, a CH3 domain of rat IgE, a CH4 domain of opossum IgE, and a C-terminal polyhistidine sequence. Alternatively, the first CH2 domain of opossum IgE can be followed by three CH3 domains of rat IgE as opposed to only a CH3 domain of rat IgE. In any case, two polypeptides can be connected through disulfide bonds in such a way that dimers are formed. It is noted that affinity chromatography can be used to purify polypeptides containing a polyhistidine sequence. In addition, an anti-polyhistidine antibody can be used as a linker molecule to connect any number of individual polypeptides or dimers through the N-terminal and the C-terminal polyhistidine sequences. For example, three dimers can be linked sequentially to through two anti-polyhistidine antibodies (ie, dimer one connected to dimer two by antibody one, and dimer two connected to dimer three by antibody two). It is noted that mixing the polypeptides with a linker molecule can result in a vaccine containing vaccine conjugates with various sizes, as well as various combinations of polypeptides. For example, a vaccine may contain a substantial amount of vaccine conjugates having less than four polypeptides, with a few more than four polypeptides. It is also noted that the general configuration of the polypeptides within a vaccine conjugate can be adapted to vaccinate different mammals to rats. For example, domains of rat IgE can be replaced with human IgE domains to vaccinate humans. 2. Vaccine conjugates and cytokines The invention provides conjugates containing a polypeptide having a cytosine activity in such a way that an immune response is induced against another polypeptide within the conjugate. Such immune responses may be more potent than the responses induced by a conjugate lacking a polypeptide having a cytosine activity. Although it is not limited to any particular mode of action, a vaccine conjugate containing polypeptide X and a polypeptide having a cytosine activity presumably concentrate cytosine activity in the localized area containing the X polypeptide. In this manner, a vaccine conjugate containing a polypeptide having an activity of Cytosine can stimulate cells involved in the generation of a specific immune response against other polypeptides within a vaccine conjugate. A polypeptide having cytosine activity can have any type of cytosine activity. For example, a polypeptide may have interferon-α, interferon-β, interferon-α, TNF-α, IL-1, IL-2, IL-4, IL-6, IL-12, IL-15, IL activity. -18 or granulocyte-macrophage colony stimulating factor (GM-CSF). It is important to note that a polypeptide having cytosine activity can be a polypeptide that either occurs naturally or occurs unnaturally. A naturally occurring polypeptide is any polypeptide having an amino acid sequence as found in nature, including polymorphic and wild-type polypeptides. Such naturally occurring polypeptides can be obtained from any species including, without limitation, human, chimpanzee, mandrel, rat or mouse. For example, human interferon-a can be used in a vaccine conjugate. A polypeptide that occurs unnaturally is any polypeptide having an amino acid sequence that is not found in nature. In this manner, a polypeptide that occurs unnaturally may be a mutated version of a naturally occurring polypeptide or a designed polypeptide. For example, a non-naturally occurring polypeptide having interferon-α activity may be a mutated version of a naturally occurring polypeptide having interferon-α activity that retains at least some interferon-α activity. A polypeptide can be mutated, for example, by additions, omissions and / or sequence substitutions by the use of standard methods such as site-directed mutagenesis of the corresponding nucleic acid coding sequence. A conjugate can contain any number of polypeptides having cytosine activity. For example, a conjugate may contain two polypeptides having cytosine activity. In addition, a conjugate may contain polypeptides having different cytosine activities. For example, a conjugate may contain a polypeptide having interferon-a activity and another having GM-CSF activity. It is noted that polypeptides having cytosine activity can be obtained by the use of any method. For example, a polypeptide having cytosine activity can be designed to contain a polyhistidine sequence such that affinity chromatography can be used to purify the polypeptide. In addition, any method for forming a conjugate can be used. For example, a polypeptide having cytosine activity that contains a binding site may be designed such that a binding molecule can bind that polypeptide to another polypeptide such as any of the polypeptides described herein. In one embodiment, a conjugate can be designed to vaccinate rats containing polypeptides having cytosine activity as well as polypeptides having an N-terminal polyhistidine sequence followed by an opossum IgE CH2 domain, a rat IgE CH3 domain, a CH2 domain of opossum IgE, a CH3 domain of rat IgE, a CH4 domain of opossum IgE, and a C-terminal polyhistidine sequence. In this case, polypeptides having cytosine activity may contain a polyhistidine sequence of terminal N in such a way that affinity chromatography can be used for purification. In addition, an anti-polyhistidine antibody can be used as a linker molecule to connect any number of polypeptides through the polyhistidine sequences. For example, a conjugate may contain an interferon-α polypeptide followed by three polypeptides containing IgE domains followed by an interferon-β polypeptide, each connection being through an anti-polyhistidine antibody. It is noted that the mixture of polypeptides with a linker molecule can result in a vaccine containing vaccine conjugates with various sizes and various combinations of polypeptides. For example, a vaccine containing a substantial amount of vaccine conjugates having both polypeptides with interferon-a activity and polypeptides with interferon-β activity. It is also noted that the general configuration of the polypeptides within a vaccine conjugate can be adapted to vaccinate different mammals to rats. For example, rat IgE domains can be replaced with human IgE domains to vaccinate humans. 3. Immunogenic polypeptides and IgE vaccines For a successful IgE vaccination, it is essential to obtain a strong immune response that predominantly reacts with native IgE molecules (eg, IgE surface epitopes). This is required in order to achieve efficient competition with the IgE receptor for free IgE, since the interaction between an IgE antibody and its specific IgE receptor is very strong (2.6x1 0"10; Froese A, CRC Crit.

Rev. Immunol. 1: 79-132 (1980)). As described herein, high levels of antibodies having specificity for independent IgE antibodies, in rat strains, were produced by the administration of an immunogenic polypeptide. Several different rat strains were used, including low, median and elevated IgE responders. As described herein, an "immunogenic polypeptide" is a polypeptide that effectively induces an immune response in a mammal. For example, an immunogenic polypeptide can be a polypeptide that effectively induces an anti-independent IgE response in a mammal. Typically, the immunogenic polypeptides contain at least one amino acid sequence (eg, a single amino acid substitution) that would be considered non-independent for a particular mammal. For example, immunogenic polypeptides that induce anti-independent IgE responses may contain two components: an independent IgE portion and a non-independent IgE portion. The independent IgE portion may be responsible for providing the specificity of the anti-independent IgE response and the non-independent IgE portion may serve to promote and stabilize the immunogenic polypeptide in such a way as to elicit the specific anti-independent IgE response. . Typically, the IgE portion independent of the immunogenic polypeptide is a portion of an IgE antibody that either interacts directly with an IgE receptor or indirectly influences the interaction of an IgE antibody with an IgE receptor.

In summary, the binding site for human IgE with the high affinity IgE receptor in mast cells and basophils is not localized at the junction between the CH2 and CH3 domains of IgE as previously suggested, but rather localized in the region of terminal N of the CH3 domain. Due to the fold, this region is located at the junction between the CH3 and CH4 domains of the native polypeptide. Thus, the use of the entire CH2-CH3 domain as an independent IgE portion can potentially induce an anti-independent IgE response with antibodies that interact with independent IgE antibodies already attached to the surface of mast cells, so that they occur anaphylactic reactions. To reduce the risk of inducing an anaphylactic response, the IgE portion independent of an immunogenic polypeptide can be the entire CH3 domain without the CH2 domain. Alternatively, the independent IgE portion may be the N-terminal region of the CH3 domain. For example, when a rat is vaccinated, the independent IgE portion may be the N-terminal half of the rat CH3 domain in the context of a non-independent IgE portion containing the whole opossum IgE CH2 domain, half terminal C of the Ig3 CH3 domain of opossum and the entire CH4 domain of opossum IgE, such immunogenic polypeptide can be designated ORO-trunc (Figure 2). Typically, the IgE portion not independent of an immunogenic polypeptide stabilizes a functional conformation of the independent IgE portion. For example, if the independent IgE portion is a CH3 domain, then the non-independent IgE portion could be a CH2 domain, a CH4 domain or a CH2 and CH4 domain, the CH3 domain being independent between the CH2 and CH4 domains. Specifically, when a rat is vaccinated, the independent IgE portion can be the rat CH3 domain in the context of an IgE portion not independent of, for example, opossum. In this case, the rat CH3 domain can be located between the opossum CH2 and CH4 domains. Such immunogenic polypeptide can be designated ORO (Figure 2). Similarly, when a mouse is vaccinated, the independent IgE portion can be the mouse CH3 domain in the context of an IgE portion not independent of, for example, opossum. Such immunogenic polypeptide can be designated OMO (Figure 2). The immunogenic polypeptides of the invention can be produced by the use of a eukaryotic expression system, such as a mammalian cell expression system. In such cases, the immunogenic polypeptide is soluble, bends properly, and is suitably modified in such a way that an anti-independent IgE response is induced after its administration to a mammal. For example, immunogenic polypeptides having one or more eukaryotic post-translational modifications can produce an anti-independent IgE response that is significantly greater than similar polypeptides that lack eukaryotic post-translational modification (eg, a bacterially produced polypeptide) . Post-translational eukaryotic modifications include, without limitation, glycosylation, acylation, limited proteolysis, phosphorylation and isoprenylation.

In addition, soluble immunogenic polypeptides, suitably bent and suitably modified, can induce a strong anti-independent IgE response in mammals with high concentrations of plasma IgE, the so-called elevated IgE responders. However, bacterially produced polypeptides are unable to produce such a strong response to anti-independent IgE in elevated IgE responders. Therefore, immunogenic polypeptides having high solubility, adequate folding, and appropriate modification, can be obtained and used as described herein to induce effective anti-independent IgE responses in mammals. In addition, the immunogenic polypeptides described herein can be used to treat mammals, including humans, that have high serum IgE concentrations. It is observed that a high percentage of severely allergic patients in the human population belong to this category of patients. The CH3 domain of IgE, or a portion of a CH3 domain of IgE, derived from an organism to be vaccinated, such as a human, can be inserted into the structural context of a distantly related IgE molecule, such as an IgE molecule of a non-placental mammal (eg, opossum, platypus, Koala, kangaroo, wallabi and Australian bear). Gray short-tailed opossum IgE antibodies, a marsupial, exhibit approximately 25 percent sequence identity with human, rat, pig, and dog IgE antibodies. Therefore, the regions of the opossum IgE antibody can be used as the IgE portion not independent of an immunogenic polypeptide in such a way that potent anti-independent IgE responses are induced in a human, rat, pig or dog. A nucleic acid molecule can be produced for the expression of an immunogenic polypeptide by dividing a first nucleic acid encoding a portion of an IgE antibody from an organism by being vaccinated in a second nucleic acid encoding a portion of an IgE antibody. coming from a mammal distantly related to the organism to be vaccinated. For example, a nucleic acid molecule encoding an immunogenic polypeptide containing the CH3 domain of rat, human, pig or dog IgE can be divided into a nucleic acid containing the opossum IgE CH2 and CH4 domains. Such chimeric nucleic acid molecules can be constructed by the use of common molecular cloning techniques. In general, the construction of nucleic acid in such a way that the CH3 domain of an IgE antibody of an organism is placed between the CH2 and CH4 domains of an IgE antibody of another organism, results in a nucleic acid molecule encoding a chimeric IgE molecule having the CH3 domain in a structural context very similar to its native position within native IgE antibodies. When a rat, human, dog or pig is vaccinated, the opossum CH2 and CH4 domains can serve as the IgE portion not independent of the immunogenic polypeptide, since there is approximately 30 percent amino acid identity between the opossum CH2 and CH4 domains and the corresponding domains of rat, human, dog and pig IgE (Figure 1). Such immunogenic polypeptides can be produced in a mammalian host. In addition, the resulting immunogenic polypeptides can be secreted from producing mammalian cells in a suitably bent and suitably glycosylated form. For example, analysis, in the Biacore system, with monoclonal antibodies directed against the CH3 domain of human IgE, revealed that these monoclonal antibodies can bind strongly to immunogenic polypeptides of the invention, indicating that the entire CH3 domain can be properly doubled. It is important to note that the immunogenic polypeptides described herein can be such that no harmful side effects are exhibited, even in mammals that have highly elevated IgE titers prior to vaccination. In addition, vaccination with an immunogenic polypeptide, as described herein, can induce an anti-independent IgE response that is directed against the entire free deposit of IgE. Such response is not limited to a specific allergen. Therefore, these methods and materials can be used to treat human allergies that have a wide variety of different atopic allergies. 4. Additional components and modes of administration The vaccines, vaccine conjugates and immunogenic polypeptides described herein, may be administered alone or in combination with other components. For example, a vaccine conjugate may contain a blocking molecule that inhibits the interaction between an antibody (e.g., an IgG antibody) and a Fc-gamma II receptor (e.g., CD32). Such blocking molecules (i.e., Fc-gamma receptor II blocking molecules) may include, without limitation, anti-CD32 antibodies. Anti-CD32 antibodies can be obtained by the use of common antibody production and selection techniques. It is noted that the Fc-gamma receptor II blocking molecules can be used in combination with any immunogenic polypeptide in such a way that the immune response against the immunogenic polypeptide is improved. For example, a mixture containing an anti-CD32 antibody and an immunogenic polypeptide, whether conjugated or not, can be administered to a mammal to induce a potent immune response against the immunogenic polypeptide. To vaccinate a mammal, an effective amount of any vaccine, vaccine conjugate or immunogenic polypeptide described herein can be administered to a host. An "effective amount" refers to any amount that induces a desired immune response while not inducing significant toxicity to the host. Such an amount can be determined by determining a host immune response after administration of a fixed amount of a particular material (e.g., immunization polypeptide). In addition, the level of toxicity, if any, can be determined by determining the clinical symptoms of a host before and after administering a fixed amount of a particular material. It is noted that the effective amount of a particular material administered to a host can be adjusted according to the desired results, as well as to the host response and the level of toxicity. Significant toxicity can vary for each particular host and depends on multiple factors including, without limitation, the disease state, age and tolerance to the host's pain. In addition, any of the materials described herein can be administered to any part of the host's body, including, without limitation, the joints, bloodstream, lungs, intestines, muscle tissues, skin and peritoneal cavity. In this way, a vaccine conjugate can be administered by intravenous injection, intraperitoneal, intramuscular, subcutaneous, intrathecal and intradermal, by oral administration, by inhalation or by gradual perfusion with time. For example, an aerosol preparation containing an immunogenic polypeptide can be given to a host by inhalation. It is noted that the duration of vaccination with any of the materials described herein can be of any length of time, from as short as one day as long as for life (eg, many years). For example, an immunogenic polypeptide can be administered once a year for a period of ten years. It is also observed that the frequency of treatment can be variable. For example, an immunogenic polypeptide can be administered once (or twice, three times, etc.) per day, per week, per month or per year. Preparations for administration may include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents include, without limitation, propylene glycol, polyethylene glycol, vegetable oils and injectable organic esters. Aqueous vehicles include, without limitation, water as well as alcohol, saline, and regulated solutions. Condoms, flavorings and other additives may also be presented such as, for example, antimicrobials, anti-oxidants, chelating agents, inert gases, and the like. It will be appreciated that any material described herein, which is to be administered to a mammal, may contain one or more commonly known pharmaceutically acceptable carriers. Any method can be used to determine if a particular immune response is induced. For example, antibody responses against particular antigens can be determined by the use of immunological assays (e.g., ELISA). In addition, clinical methods that can determine the degree of a particular disease state can be used to determine whether a desired immune response is induced. The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES Example 1 - Immunogenic Polypeptides Nucleic acid molecules were constructed to encode immunogenic polypeptides containing both independent and non-independent portions of IgE. These nucleic acid molecules were then used to synthesize immunogenic polypeptides soluble in mammalian cells. Such immunogenic polypeptides effectively induce a polyclonal anti-independent IgE response after administration to a mammal. In addition, the immunogenic polypeptides appear to bend and glycosylate in a manner that allows the immunogenic polypeptides to produce a strong and specific anti-independent IgE response that was more potent than the bacterially produced polypeptides lacking the non-independent IgE portion. Therefore, the immunogenic polypeptides described herein contain a majority of the epitope surface in the same conformation as in native plasma IgE. In addition, immunogenic polypeptides that contain an independent IgE portion limited to either its entire CH3 domain or a fragment of the CH3 domain (e.g., the N-terminal region of CH3) reduced the potential for production of anaphylactic antibodies within a mammal . Example 2 - Production and purification of an immunogenic polypeptide A base pair PCR fragment -330 encoding the CH3 domain of rat IgE (Hellman L et al., Nucleic Acid Res. 10: 6041-6049 (1982)) is merged with two fragments of similar size coding for opossum IgE CH2 and CH4 domains (Aveskogh M and Hellman L, Eur. J. Immunol., 28: 2738-2750 (1998)) by ligation in a modified version of the expression vector pCEP4, pCEP-Pu2 (Margolskee RF et al., Mol Cell Biol. 8: 2837-2847 (1988)). This vector contains the CMV promoter-enhancer, located directly at 5 'of the coding region of interest and allows high levels of expression in mammalian cells. This vector also contains the coding regions for the puromycin resistance and the EBNA1 gene of EBV. The EBNA1 gene provides maintenance of episomal copies of stable vector reproduction in human or canine cell lines. The nucleic acid molecule containing the opossum IgE CH2 nucleic acid sequence, rat IgE CH3 CH4, and opossum IgE CH4 also contained nucleic acid sequences encoding a signal sequence and six histidine residues in the region of terminal N. The region containing the signal sequence and six histidine residues facilitates the secretion of the encoded polypeptide from producer cells and allows purification of the polypeptide with Ni ++ chelating columns. After transfection of the expression vector in human 293 cells, the opossum CH2-IgE opossum polypeptide / rat CH3-IgE / CH4-IgE (ORO) was purified from a conditioned medium of 293 cells in a nickel chelate column up to approximately 100 percent purity. After elution of the ORO immunogenic polypeptide with a solution containing 20 mM Tris (pH 8.0), 0.1 M NaCl, and 100 mM of midazole, the levigated was dialyzed against PBS (pH 7.5) overnight at 4 ° C. ° C. The ORO immunogenic polypeptide was then concentrated to approximately 2 mg / mL by the use of an Amicon concentrate. An aliquot of this preparation containing the ORO immunogenic polypeptide was separated on SDS-PAGE and approximately 100 percent pure was found. This purified ORO immunogenic polypeptide preparation was used as the active component of an anti-independent IgE vaccine for treating rats. Example 3- Sensitization procedure Each rat was sensitized to ovalbumin as follows. 10 Mg of ovalbumin in PBS was administered to each rat, intraperitoneally. Three weeks after this initial intraperitoneal injection of ovalbumin, the rats received weekly intraperitoneal injections of 1 μg of ovalbumin for four weeks. During this four-week period, the rats are sensitized to ovalbumin obtaining a total response of IgE and ovalbumin-specific IgE, which was high and persistent. After this four-week period, the rats began a vaccination program. During the entire vaccination program, intraperitoneal injections of ovalbumin continued as follows. During the first two weeks of vaccination, the rats received intraperitoneal injections of 1 μg of ovalbumin weekly. After the first two weeks of vaccination, the rats received intraperitoneal injections of 1 μg of ovalbumin every other week. Example 4 - ELISA measurement of an anti-independent IgE response Thirty-six rats (twelve Lewis rats, twelve Louvain rats and twelve Norwegian brown rats) were divided into two groups of equal size and injected intraperitoneally with either GOLD immunogenic polypeptide or BSA as a negative control. The negative control of BSA was used in the same concentration of polypeptide as that of the immunogenic polypeptide of ORO. In this study, each rat received intraperitoneal injections of approximately 250 μg of antigen (either the ORO or BSA immunogenic polypeptide) dispersed in 0.2 mL of a 50:50 solution of Freund's complete adjuvant and PBS. Three weeks later, the rats were given a booster injection containing approximately 100 μg of dispersed antigen in 0.1 mL of a 50:50 solution of incomplete Freund's adjuvant and PBS. Six weeks later, the rats were given an additional reinforcement identical to the first reinforcement. One week after this third immunization, blood samples were taken and measured in an ELISA as follows. He Anti-IgE antibody level of IgG directed against independent rat IgE was measured by ELISA. The native rat IgE was used at a concentration of 5 μg / mL for coating ELISA plates. Successive 1/5 dilutions of serum from each of the individual rats was examined by color reaction in the ELISA. The presence of rat IgG antibodies having specificity for rat IgE antibodies was determined by the use of two monoclonal mouse monoclonal antibodies., one with specificity for rat IgG2a / b and one for rat IgG1. Streptavidin coupled to alkaline phosphatase was used to detect these monoclonal mouse monoclonal antibodies. Example 5 - Induction of an anti-independent IgE response in a mammal The in vivo effect of the ORO immunogenic polypeptide as an IgE vaccine was investigated by the use of three different strains of rats (Lewis, Louvain and Norway Brown). Lewis rats are low IgE responders, Louvain rats are average IgE responders and Norwegian Brown rats are high IgE responders. After sensitization to ovalbumin, each rat was either vaccinated with the immunogenic polypeptide of ORO or BSA, as described in Example 3. After collecting blood samples, the serum was diluted in steps of five as indicated (FIG. 3). The purified monoclonal rat IgE (IR 162) was used to coat the ELISA plates (5 μg / mL) and two monoclonal mouse antibodies were used to detect anti-IgE rat IgG antibodies. After the second booster dose, high titers of anti-IgE were detected in the low, medium and high IgE responsive rats that received the vaccine containing the immunogenic polypeptide of ORO. No anti-IgE titers were detected in rats treated with the BSA control. In this manner, the ORO immunogenic polypeptide was able to induce an anti-independent IgE response in rats containing low, medium and high amounts of IgE antibodies. A difference in anti-independent IgE levels between the various strains was observed. The low Lewis strain, showed very high anti-independent IgE titers. The serum could be diluted more than 3000 times before a significant decrease in OD values was detected after the ELISA measurements (Figure 3). For the high-responding strain, Norway Brown, however, OD values began to fall for three out of six animals at a dilution of 25 times or more (Figure 3). In another experiment, Wistar rats were used. The rats Wistar are average IgE responders. The anti-independent IgE response produced by Wistar rats was similar to the response observed in Lewis rats. Example 6 - Analysis of cross-reactivity Cross-reactivity between rat antibodies directed against a CH2 or CH4 domain of opossum IgE with the corresponding domain of rat IgE antibodies was evaluated. This potential cross-reactivity could result from a low primary amino acid sequence homology or a close structural similarity between the opossum IgE CH2 and CH4 domains and rat IgE. The induction of an anti-rat IgE response having specificity for the rat CH2 or CH4 domains could lead to mast cell activation. A recombinant polypeptide (OOO) containing the opossum CH2-CH3-CH4 domains was injected into the Wistar strain. After a second booster injection, the serum was collected from these rats and examined for the presence of antibodies having specificity for rat IgE. No anti-rat IgE antibodies were detected in the rats treated with the OOO polypeptide (Figure 4). In addition, Wistar rats treated with the ORO immunogenic polypeptide exhibited an anti-independent IgE response similar to that observed in the Lewis rats. In addition, Wistar rats treated with a recombinant polypeptide (PPP) containing platypus CH2-CH3-CH4 domains did not produce an anti-rat IgE response. Thus, the CH2, CH3 and CH4 domains of opossum and platypus IgE antibodies do not generate, after their administration to rats, rat antibodies having specificity for rat IgE antibodies. The interaction between the rat antibodies induced by the ORO immunogenic polypeptide (IgE anti-rat IgG independent antibodies) and human IgE antibodies was examined. In a few cases, less cross-reactivity was observed. This lower cross-reactivity detected in a few rats was most likely caused by the interaction between rat IgG antirate IgE CH3 antibodies and the CH3 domain of human IgE. Since the rat CH3 and human IgE domains are much more closely related than the human and opossum or platypus IgE, vaccines containing opossum or platypus components can be considered highly safe, posing a minimal risk for the generation of transverse ligation antibodies. Example 7 - Polypeptides for vaccine conjugates The nucleic acid construct encoding the ORO immunogenic polypeptide described in Example 2 was used to produce two polypeptides each having several identical independent epitopes. One polypeptide contained two identical agglomerations of independent epitopes (the Dominican CH3 of the whole rat), while the other polypeptide contained four such agglomerations. First, the nucleic acid encoding six histidine residues was added to the C-terminus of the opossum CH4 domain by including a nucleotide sequence for six histidine residues in the 3 'PCR priming charge. In this manner, each polypeptide contained a polyhistidine sequence at both the N and C terminal ends so that the conjugates can be formed. Second, a fragment of nucleic acid encoding the CH2 domains of opossum and rat CH3 of the original construct was obtained by PCR amplification. This fragment was subsequently ligated into the construct encoding the ORO immunogenic polypeptide. The resulting construct encoded a polypeptide designated ORORO. That ORORO polypeptide contains two rat CH3 domains, two opossum CH2 domains and one opossum CH4 domain in the following order CH2 from opossum, rat CH3, opossum CH2, rat CH3, and opossum CH4. In this way, this polypeptide has two identical CH3 domains, each with multiple independent epitopes. The nucleic acid construct encoding ORORO was used as a raw material to produce the second immunogenic polypeptide. This polypeptide contains additional rat CH3 domains that were added to a 3 'position of the first rat CH3 domain in the ORORO polypeptide. The resulting polypeptide has a polyhistidine tag followed by a possum CH2 domain, three identical rat CH3 domains, a possum CH2 domain, a rat CH3 domain, a possum CH4 domain and a C terminal polyhistidine tag (6his-ORRRORO-6his). Each recombinant polypeptide was produced in the vector system based on pCEP4. In addition, the polypeptides were purified using Ni ++ chelation columns according to the method described in Example 2. Similar vaccine constructions are produced using CH3 domain of human or dog IgE in place of the rat IgE CH3 domain. Example 8-Vaccine Conjugates To determine a favorable combination of polypeptide to monoclonal antibody, the purified polypeptides of Example 7 are mixed with a monoclonal anti-polyhistidine antibody in various combinations ranging from a ratio of 1/1 to 10/1 (ratio from polypeptide to monoclonal antibody). This mixture results in the generation of long multimeric conjugates with a large number of identical independent epitopes one after the other. The biological activity of the various combinations is assessed in rats as described herein. The non-conjugated ORO immunogenic polypeptide is used as a reference to assess immune responses. Example 9 - Polypeptides having cytokine activity The PCR initiator charge are designed so that the cDNAs encoding rat, dog and human cytokines (e.g., interferon-a, interferon-α, and GM-CSF) can be isolated from mRNA of total spleen. The nucleotide sequence encoding six histidine residues is introduced into each 5'PCR primer charge so that the cytokines can be purified through affinity chromatography and can be conjugated non-covalently to the polypeptides described herein through an anti-DNA antibody. -polyistidine. Recombinant cytokines are produced using any of the following three expression systems: bacteria, yeast (e.g., Pichia pastoris), mammalian cells (e.g., 293-EBNA cells using an expression system based on pCEP-4). Example 10 - Vaccine conjugates having a polypeptide with cytokine activity The cytokines produced according to Example 9 are used to make vaccine conjugates. A mixture of three different cytokines (e.g., mouse interferon-a, rat interferon-γ and rat GM-CSF) is produced and tested in combination with the ORORO polypeptide and an anti-polyhistidine antibody. Initially, a mixture of 1/10 ratio (ratio of cytokine to immunogenic polypeptide) is tested. With three cytokines, this results in a mixture of 30% cytokine per molar basis and 70% immunogenic polypeptide. In addition, this mixture contains an anti-polyhistidine antibody at a 1/10 ratio (ratio of immunogenic polypeptide to monoclonal antibody). A large number of proportion combinations is evaluated such that a ratio of cytokine to immunogenic polypeptide is determined as well as a ratio of monoclonal antibody to optimal immunogenic polypeptide. In addition, polypeptides having activity of several species are evaluated to determine the optimal combination for a particular species. OTHER MODALITIES It should be understood that although the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims

CLAIMS 1. An immunogenic polypeptide, characterized in that it comprises an independent IgE portion and a non-independent IgE portion, wherein said immunogenic polypeptide is effective in inducing an anti-independent IgE response in a mammal.
2. The immunogenic polypeptide according to claim 1, characterized in that said mammal is a human.
3. The immunogenic polypeptide according to claim 1, characterized in that said independent portion comprises at least a portion of a CH3 domain of IgE.
4. The immunogenic polypeptide according to claim 1, characterized in that said polypeptide is capable of being dimerized to form a soluble immunogenic dimer, effective in the induction of said anti-independent IgE response in said mammal.
The immunogenic polypeptide according to claim 1, characterized in that said non-independent IgE portion comprises a first region and a second region, said independent IgE portion being located between said first and second regions of said non-independent IgE portion.
6. The immunogenic polypeptide according to claim 5, characterized in that said first region comprises at least a portion of a CH2 domain of IgE.
The immunogenic polypeptide according to claim 5, characterized in that said second region comprises at least a portion of a CH4 domain of IgE.
8. The immunogenic polypeptide according to claim 1, characterized in that said non-independent IgE portion comprises an IgE sequence present in a non-placental mammal.
The immunogenic polypeptide according to claim 8, characterized in that said non-placental mammal is selected from the group consisting of opossum, platypus, koala, kangaroo, wallaby and Australian bear.
10. The immunogenic polypeptide according to claim 1, characterized in that said independent IgE portion lacks the CH2 domain of an IgE antibody. eleven .
The immunogenic polypeptide according to claim 1, characterized in that said immunogenic polypeptide contains a post-translational eukaryotic modification.
The immunogenic polypeptide according to claim 1, characterized in that said immunogenic polypeptide comprises a polyhistidine sequence. 3.
The immunogenic polypeptide according to claim 1, characterized in that said anti-independent IgE response is a polyclonal response.
14. A nucleic acid molecule comprising a nucleic acid sequence encoding an immunogenic polypeptide, said immunogenic polypeptide comprising an independent IgE portion and a non-independent IgE portion, wherein said immunogenic polypeptide is effective in inducing a response of anti-independent IgE in a mammal.
15. The nucleic acid molecule according to claim 14, characterized in that said nucleic acid molecule comprises an additional nucleic acid sequence encoding an amino acid sequence that promotes the secretion of said immunogenic polypeptide from a eukaryotic cell.
16. A host cell, characterized in that it comprises a nucleic acid molecule, wherein said nucleic acid molecule comprises a nucleic acid sequence encoding an immunogenic polypeptide, said immunogenic polypeptide comprising an independent IgE portion and a non-independent IgE portion. , wherein said immunogenic polypeptide is effective in inducing an anti-independent IgE response in a mammal.
17. The host cell according to claim 16, characterized in that said host cell is a eukaryotic cell.
18. A soluble immunogenic dimer, characterized in that it comprises two immunogenic polypeptides that are capable of being dimerized to form said soluble immunogenic dimer, wherein each of said two immunogenic polypeptides comprises an independent IgE portion and a non-independent IgE portion, said soluble immunogenic dimer b effective in inducing an anti-independent IgE response in a mammal.
19. A vaccine, characterized in that it comprises an immunogenic polypeptide comprising an independent IgE portion and a non-independent IgE portion, whersaid immunogenic polypeptide is effective in inducing an anti-independent IgE response in a mammal.
The vaccine according to claim 19, characterized in that said vaccine further comprises a pharmaceutically acceptable carrier. twenty-one .
A method for making a nucleic acid molecule, which encodes an immunogenic polypeptide effective in inducing an anti-independent IgE response in a mammal, said method comprising the combination of the first and second nucleic acid sequences for forming said nucleic acid molecule, whersaid first nucleic acid sequence encodes at least a portion of an IgE molecule present within said mammal and whersaid second nucleic acid sequence encodes at least a portion of a non-IgE molecule. present in said mammal.
22. A method for making a nucleic acid molecule encoding an immunogenic polypeptide effective in inducing an anti-independent IgE response in a mammal, said method comprising: a) selecting a first nucleic acid sequence, whersaid first nucleic acid sequence encodes at least a portion of an IgE molecule present within said mammal, b) selects a second nucleic acid sequence, whersaid second nucleic acid sequence encodes at least a portion of an IgE molecule not present in said mammal, and c) combining said nucleic acid sequences, first and second, to form said nucleic acid molecule.
23. A vaccine complex for vaccinating a mammal, characterized in that said complex comprises a first and second polypeptides, whereach of said polypeptides, first and second, contains at least two similar amino acid sequences of at least five amino acid residues of length, whersaid first and second polypeptides are connected to form said complex, and wherthe administration of said complex to said mammal induces an immune response against at least a portion of said first or second polypeptide.
24. The complex according to claim 23, characterized in that said mammal is a human.
25. The complex according to claim 23, characterized in that said first or second polypeptide comprises an amino acid sequence expressed by said mammal.
26. The complex according to claim 23, characterized in that said polypeptides, first and second, are identical.
27. The complex according to claim 23, characterized in that said polypeptides, first and second, form a dimer.
28. The complex according to claim 23, characterized in that the connection of said polypeptides, first and second, comprises a disulfide bond.
29. The complex according to claim 23, characterized in that the connection of said polypeptides, first and second, comprises a non-covalent interaction.
30. The complex according to claim 23, characterized in that said first or second polypeptide comprises a binding site.
31 The complex according to claim 30, characterized in that said binding site is a polyhistidine sequence.
32. The complex according to claim 23, characterized in that the amino and carboxyl terms of said first or second polypeptide contain a binding site.
33. The complex according to claim 23, characterized in that said complex comprises a binding molecule.
34. The complex according to claim 33, characterized in that said binding molecule connects said first and second polypeptides.
35. The complex according to claim 33, characterized in that said binding molecule comprises an antibody.
36. The complex according to claim 35, characterized in that said antibody is an anti-polyhistidine antibody.
37. The complex according to claim 23, characterized in that said complex comprises a third polypeptide, said third polypeptide having a cytosine activity.
38. The complex according to claim 37, characterized in that said cytosine activity is an activity of a cytosine selected from the group consisting of interferon-a, interferon-β, interferon-α, TNF-α, IL-1, IL -2, IL-4, IL-6, IL-12, IL-15, IL-18 and granulocyte-macrophage colony stimulating factor.
39. The complex according to claim 37, characterized in that a linker molecule connects said third polypeptide to said first or second polypeptide.
40. The complex according to claim 23, characterized in that said first and second polypeptides comprise a binding site.
41 The complex according to claim 23, characterized in that the amino and carboxyl terms of said first and second polypeptides contain a binding site.
42. The complex according to claim 23, characterized in that said similar amino acid sequences are greater than about twenty amino acid residues in length.
43. The complex according to claim 23, characterized in that said complex comprises a blocking molecule of receptor II Fc-gamma.
44. A vaccine complex for vaccinating a mammal, said complex comprising a first polypeptide connected to a second polypeptide, wherein said first polypeptide contains at least two similar amino acid sequences of at least five amino acids in length, wherein said second polypeptide has a cytosine activity and wherein the administration of said complex to said mammal induces an immune response against at least a portion of said first polypeptide.
45. The complex according to claim 44, characterized in that said mammal is human.
46. The complex according to claim 44, characterized in that said first polypeptide comprises an amino acid sequence expressed by said mammal.
47. The complex according to claim 44, characterized in that the connection of said polypeptides, first and second, comprises a non-covalent interaction.
48. The complex according to claim 44, characterized in that said first or second polypeptide comprises a binding site.
49. The complex according to claim 44, characterized in that said complex comprises a binding molecule.
50. The complex according to claim 44, characterized in that said cytosine activity is an activity of a cytosine selected from the group consisting of interferon-a, interferon-β, interferon- ?, TNF-α, IL-1, IL-2 , IL-4, IL-6, IL-12. IL-1 5, IL-18 and granulocyte-macrophage colony stimulating factor.
51 The complex according to claim 44, characterized in that said complex comprises a third polypeptide.
52. The complex according to claim 51, characterized in that said polypeptides, first and third, are identical.
53. The complex according to claim 51, characterized in that said polypeptides, first and third, form a dimer.
54. The complex according to claim 44, characterized in that said similar amino acid sequences are greater than about twenty amino acid residues in length.
55. The complex according to claim 44, characterized in that said complex comprises a blocking molecule of receptor II Fc-gamma.
56. A vaccine complex for vaccinating a mammal, said complex comprising a polypeptide, first, second and third, wherein said polypeptides, first, second and third, are connected to form said complex, wherein said first polypeptide has a first cytosine activity, wherein said second polypeptide has a second cytosine activity, and wherein administration of said complex to said mammal induces an immune response against at least a portion of said third polypeptide.
57. The complex according to claim 56, characterized in that said mammal is human.
58. The complex according to claim 56, characterized in that said third polypeptide comprises an amino acid sequence expressed by said mammal.
59. The complex according to claim 56, characterized in that the connections of said first, second and third polypeptides comprise non-covalent interactions.
60. The complex according to claim 56, characterized in that said first, second or third polypeptide comprises a binding site.
61. The complex according to claim 56, characterized in that said complex comprises a binding molecule.
62. The complex according to claim 56, characterized in that said third polypeptide comprises at least two similar amino acid sequences of at least five amino acids in length.
63. The complex according to claim 56, characterized in that said complex comprises a blocking molecule of receptor II Fc-gamma.
64. A vaccine complex for vaccinating a mammal, said complex comprising a first polypeptide connected to a second polypeptide, wherein said first polypeptide is a polypeptide having an interferon-a or interferon-β activity, and wherein the of said complex to said mammal induces an immune response against at least a portion of said second polypeptide.
65. The complex according to claim 64, characterized in that said mammal is a human.
66. The complex according to claim 64, characterized in that said second polypeptide comprises an amino acid sequence expressed by said mammal.
67. The complex according to claim 64, characterized in that the connection of said polypeptides, first and second, comprises a non-covalent interaction.
68. The complex according to claim 64, characterized in that said first or second polypeptide comprises a binding site.
69. The complex according to claim 64, characterized in that said complex comprises a binding molecule.
70. The complex according to claim 64, characterized in that said second polypeptide comprises at least two similar amino acid sequences of at least five amino acids in length.
71 The complex according to claim 64, characterized in that said complex comprises a blocking molecule of receptor II Fc-gamma.