KR100780158B1 - Immunotherapy and Improved Vaccines - Google Patents

Abstract

In the present invention, IL-12, GM-CSF, IL-1, TNF-α, TNF-β, IL-2, IL-4, IL-5, IL-10, IL-15, operably linked with regulatory factors Improved vaccines comprising nucleotide sequences encoding IL-18 and / or BL-1 are disclosed. Improved vaccines include DNA vaccines, recombinant vaccines carrying foreign antigens, and attenuated live vaccines. Methods of immunization of individuals are also disclosed. Consisting of IL-12, GM-CSF, IL-1, TNF-α, TNF-β, IL-2, IL-4, IL-5, IL-10, IL-15, IL-18, and BL-1 Pharmaceutical compositions comprising nucleic acid molecules encoding one or more immunomodulatory proteins selected from the group are disclosed. Immunregulatory proteins, BL1, and nucleic acid molecules encoding BL1 are also disclosed. Methods of making and using BL1 are also disclosed.

Antigen, vaccine, immunomodulatory protein, BL1, regulatory factor, target protein, IL-12

Description

Immunotherapy and Improved Vaccines

The present invention relates to immunotherapeutic compositions and immunotherapeutics, and improved protective and therapeutic vaccines, and improved prophylaxis and / or therapies for inducing an immune response to an antigen.

Vaccines are useful for immunizing an individual against target antigens, such as pathogen antigens or antigens associated with cells involved in human disease. Cell-associated antigens involved in human disease include cancer-associated tumor antigens and antigens associated with autoimmune disease-related cells.

The overall goal of any immunization is to induce a specific immune response that defends the immunized individual from any pathogen throughout its life. One major problem in meeting this objective is that the correlating factor of defense against each pathogen is different for each infectious agent. Thus, clinically more effective vaccines must elicit a more specific immune response to the target pathogen. Immunization should be designed to be "focused" depending on the correlating factors of known defense against specific pathogens.

In such vaccine designs, it is known that vaccines that produce target antigens in the cells of vaccinated individuals are effective in inducing cellular arms of the immune system. Specifically, attenuated live vaccines, recombinant vaccines using non-toxic vectors, and DNA vaccines both induce antigen production in cells of vaccinated individuals, leading to cellular mineralization of the immune system. On the other hand, subunit vaccines containing only proteins and dead or inactivated vaccines, which induce humoral responses, do not induce a good cellular immune response.

Cellular immune responses are often essential for providing effective immune mediated therapies for the defense against pathogen infections and for the treatment of pathogen infections. Thus, vaccines that produce target antigens in cells of the vaccinated individual are preferred, eg, attenuated live vaccines, recombinant vaccines using nontoxic vectors and DNA vaccines.

Nucleic acid immunization is a new vaccination technique that transports a DNA construct encoding a specific immunogen to a host. In addition to the ability of DNA vaccines to elicit immune responses of both antigen-specific cells and body fluids, this technique has the potential to modulate the immune response caused by the co-transportation of immunologically important molecules.

While such vaccines are often effective in prophylactically or therapeutically immunizing an individual against a pathogen infection or human disease, there is a need for improved vaccines and there is a need for compositions and methods that produce an enhanced immune response. .

Summary of the Invention

The present invention relates to genetic constructs comprising nucleotide sequences encoding immunoregulatory proteins that can be administered to a subject under prophylactic or therapeutic vaccination, or therapeutic immunomodulation. Immunomodulatory proteins include human proteins IL-12, GM-CSF, IL-1, TNF-α, TNF-β, IL-2, IL-4, IL-5, IL-10, IL-15, IL-18 , And novel molecules designated BL-1.

The present invention relates to IL-12, GM-CSF, IL-1, TNF-α, TNF-β, IL-2, IL-4, IL-5, IL-10, IL-15, IL-18, or BL- Nucleotide sequence encoding 1 or IL-12, GM-CSF, IL-1, TNF-α, TNF-β, IL-2, IL-4, IL-5, IL-10, IL-15, IL- A genetic construct comprising a nucleotide sequence encoding two or more of 18, or BL-1. Gene constructs may comprise multiple copies of the same nucleotide sequence.

The present invention provides a subject by administering a vaccine composition, wherein the subject is administered by injecting an immunogen with the gene construct comprising a nucleotide sequence encoding one or more immunoregulatory proteins to enhance and / or elicit a more desirable immune response. It relates to a method of vaccination. Moreover, the present invention relates to a method of modulating an individual's immune response by administering a genetic construct comprising a nucleotide sequence encoding one or more immunoregulatory proteins. Modulation of the immune response may be achieved by first administering the vaccine composition with an immunomodulatory protein that favors one immune response form, and then boosting the vaccine composition with the immunomodulatory protein that favors another immune response form. This may be one step of vaccination that is primarily switched from Th1 to Th2 responses or vice versa.

The vaccine composition is preferably a plasmid injected directly into the subject. Similarly, genetic constructs comprising nucleotide sequences encoding one or more immunoregulatory proteins are preferably plasmids.

The present invention provides a nucleotide sequence encoding a human IL-12 protein operably linked with a regulatory factor required for expression in eukaryotic cells, and a nucleotide sequence encoding an immunogenic target antigen operably linked with a regulatory factor required for expression in eukaryotic cells. It relates to a plasmid containing. In certain preferred embodiments, the immune target antigen is a pathogen antigen, a cancer associated antigen or an antigen associated with an autoimmune disease associated cell. In certain embodiments, the plasmid comprises a nucleotide sequence encoding a human single chain IL-12 protein operably linked with a regulatory factor required for expression in eukaryotic cells. Single chain IL-12 protein is a single protein that is encoded by a single coding sequence and comprises a linker that connects two subunits. The linker is large and flexible enough to allow a single protein to be folded into a biologically active form (estimated by a functional, natural two subunit IL-12 protein).

The present invention provides a nucleotide sequence encoding human IL-12 operably linked to a regulatory factor necessary for expression in a cell of an individual, and a nucleotide sequence encoding a target antigen operably linked with a regulatory factor required for expression in a cell of an individual. It relates to a method of inducing an immune response to an antigen in a subject, comprising administering to the subject a plasmid comprising a. In certain preferred embodiments, the target antigen is a pathogen antigen and an antigen associated with cancer associated antigens or autoimmune related cells. In a preferred embodiment, the immune response directed against the target antigen is directed to a cell having a protein in which an antigen-antibody immune response is involved and / or a protective immune response cross reacts with a pathogen or an immune response generated against the antigen. Provides therapeutic advantages in the infections, diseases, disorders and diseases associated with the protein that are induced. In certain embodiments, the human IL-12 protein is a single chain IL-12 protein.

The present invention collectively comprises two subunits of human IL-12 and a nucleotide sequence encoding a target antigen, each coding sequence comprising a plurality of plasmids operably linked with regulatory factors required for gene expression. To a composition. In certain embodiments, the composition comprises two plasmids, a first plasmid comprising a nucleotide sequence encoding an IL-12 protein operably linked with a regulatory factor for expression in eukaryotic cells, and a regulatory factor for expression in eukaryotic cells. And a second plasmid comprising a nucleotide sequence encoding an immunogenic target antigen operably linked with. In certain embodiments, the first plasmid comprises a predetermined nucleotide sequence encoding an immunogenic target protein and one subunit of human IL-12, and the second plasmid is predetermined to encode another subunit of human IL-12. Nucleotide sequences. In certain embodiments, three different plasmids, a plasmid comprising a nucleotide sequence encoding an immunogenic target protein, a plasmid encoding a p35 subunit of human IL-12, and a p40 subunit of human IL-12 Provide a plasmid encoding. In certain embodiments, the composition comprises two plasmids, a first plasmid comprising a nucleotide sequence encoding a single chain of IL-12 protein operably linked with a regulatory factor required for expression in eukaryotic cells, and for expression in eukaryotic cells. A second plasmid comprising a nucleotide sequence encoding an immunogenic target antigen operably linked with a required regulatory factor.

The invention comprises a plurality of plasmids collectively comprising two subunits of human IL-12 and a nucleotide sequence encoding a target antigen, wherein each coding sequence is operably linked with a regulatory factor required for gene expression. A method of inducing an immune response in an individual, comprising administering to the individual. In certain embodiments, the two or three plasmids described above comprise two subunits of a human IL-12 protein and a nucleotide sequence encoding an immunogenic target protein, wherein the nucleotide sequence encoding the protein is a cell of the individual Is operably linked to regulatory factors required for expression. In a preferred embodiment, the immune response induced by the target antigen cross reacts with an antigen associated with a pathogen antigen, a cancer associated antigen or an autoimmune disease associated cell. The present invention relates to a method of immunizing an individual against a pathogen, cancer or autoimmune disease. In a preferred embodiment, the target antigen is a pathogen antigen, a cancer associated antigen or an antigen associated with an autoimmune disease related cell. In certain embodiments, human IL-12 is a single chain of IL-12 protein.

The present invention relates to GM-CSF, IL-1α, TNF-α, TNF-β, IL-2, IL-15, IL-18, IL-4, human operably linked to regulators required for expression in eukaryotic cells. A nucleotide sequence encoding at least one of IL-5 and IL-10, and a nucleotide sequence encoding an immunogenic target antigen operably linked with regulatory factors required for expression in eukaryotic cells. In certain preferred embodiments, the immunogenic target antigen is a pathogen antigen, a cancer associated antigen or an antigen associated with an autoimmune disease associated cell.

The present invention relates to GM-CSF, IL-1α, TNF-α, TNF-β, IL-2, IL-15, IL-18, IL-4 in humans operably linked to regulators required for expression in cells of an individual. Administering to the subject a plasmid comprising a nucleotide sequence encoding at least one of an IL-5 or IL-10 protein, and a nucleotide sequence encoding a target antigen operably linked to a regulatory factor required for expression in a cell of the subject. It relates to a method of inducing an immune response against an antigen in a subject, comprising the step. In certain preferred embodiments, the target antigen is a pathogen antigen, a cancer associated antigen or an antigen associated with an autoimmune disease related cell. In a preferred embodiment, the immune response directed against the target antigen is directed to a cell having a protein in which an antigen-antibody immune response is involved and / or a protective immune response cross reacts with a pathogen, or an immune response generated against the antigen. Provides therapeutic advantages in the infections, diseases, disorders and diseases associated with the protein that are induced.

The present invention relates to human GM-CSF, IL-1α, TNF-α, TNF-β, IL-2, IL-15, IL- operably linked to two plasmids, that is, regulatory factors required for expression in eukaryotic cells. A first plasmid comprising a nucleotide sequence encoding one or more of 18, IL-4, IL-5 or IL-10 proteins, and an immunogenic target antigen operably linked with regulatory factors required for expression in eukaryotic cells It relates to a composition comprising a plurality of plasmids, including a second plasmid comprising a nucleotide sequence. In certain embodiments, the composition is human GM-CSF, IL-1α, TNF-α, TNF-β, IL-2, IL-15, IL-18 operably linked with regulatory factors required for expression in eukaryotic cells. Three plasmids, including a third plasmid comprising a nucleotide sequence encoding one or more of the IL-4, IL-5 or IL-10 proteins. In certain embodiments, the composition is human GM-CSF, IL-1α, TNF-α, TNF-β, IL-2, IL-15, IL-18 operably linked with regulatory factors required for expression in eukaryotic cells. A third plasmid comprising a nucleotide sequence encoding at least one of an IL-4, IL-5 or IL-10 protein, and a human GM-CSF, operably linked with regulatory factors required for expression in eukaryotic cells, A fourth comprising a nucleotide sequence encoding one or more of the IL-1α, TNF-α, TNF-β, IL-2, IL-15, IL-18, IL-4, IL-5 or IL-10 proteins Four plasmids, including plasmids.

The present invention relates to two plasmids, ie, human GM-CSF, IL-1α, TNF-α, TNF-β, IL-2, IL-15, IL-18, which are operably linked to regulatory factors required for expression in genes. , A first plasmid comprising a nucleotide sequence encoding at least one of an IL-4, IL-5 or IL-10 protein, and a nucleotide sequence encoding an immunogenic target antigen operably linked with a regulatory factor required for expression. A method for inducing an immune response against an antigen comprising administering to a subject a composition comprising a plurality of plasmids, including a second plasmid comprising. In a preferred embodiment, the immune response induced by the target antigen cross reacts with an antigen associated with a pathogen antigen, a cancer associated antigen or an autoimmune disease associated cell. The present invention relates to a method of immunizing an individual against a pathogen, cancer or autoimmune disease. In a preferred embodiment, the target antigen is a pathogen antigen, a cancer associated antigen or an antigen associated with an autoimmune disease related cell. In certain embodiments, the method comprises human GM-CSF, IL-1α, TNF-α, TNF-β, IL-2, IL-15, IL-18 operably linked with regulatory factors required for expression in eukaryotic cells. And administering a composition comprising three plasmids, including a third plasmid comprising a nucleotide sequence encoding one or more of the IL-4, IL-5 or IL-10 proteins. In certain embodiments, the method comprises human GM-CSF, IL-1α, TNF-α, TNF-β, IL-2, IL-15, IL-18 operably linked with regulatory factors required for expression in eukaryotic cells. A third plasmid comprising a nucleotide sequence encoding at least one of an IL-4, IL-5 or IL-10 protein, and a human GM-CSF, operably linked with regulatory factors required for expression in eukaryotic cells, A fourth comprising a nucleotide sequence encoding one or more of the IL-1α, TNF-α, TNF-β, IL-2, IL-15, IL-18, IL-4, IL-5 or IL-10 proteins Administering a composition comprising four plasmids, including plasmids.

The present invention relates to IL-12, GM-CSF, IL-1α, TNF-α, TNF-β, IL-2, IL-15, IL-18, in humans operably linked to regulatory factors required for expression in eukaryotic cells. An improved nucleotide sequence encoding a target antigen operably linked with a nucleotide sequence encoding one or more of the IL-4, IL-5, IL-10, or BL1 proteins and regulatory factors required for expression in eukaryotic cells It relates to a recombinant vaccine vector. In certain embodiments, the gene encoding human IL-12 protein encodes IL-12 as a single chain protein. In a preferred embodiment, the target antigen is a pathogen antigen, a cancer associated antigen or an antigen associated with an autoimmune disease related cell.

The present invention relates to human IL-12, GM-CSF, IL-1α, TNF-α, TNF-β, IL-2, IL-15, IL- operably linked to regulatory factors required for expression in cells of an individual. A nucleotide sequence encoding at least one of 18, IL-4, IL-5, IL-10, or BL1 protein, and a nucleotide sequence encoding a target antigen operably linked with a regulatory factor required for expression in a cell of the individual. Immunizing the subject against a pathogen, cancer or autoimmune disease, comprising administering to the subject a recombinant vaccine vector comprising a target antigen, wherein the target antigen is a pathogen antigen, a cancer associated antigen or an antigen associated with an autoimmune disease related cell. It is about how to make anger.

The present invention provides human IL-12, GM-CSF, IL-1α, TNF-α, TNF-β, IL-2, IL-15, IL-18 operably linked to regulatory factors required for expression in eukaryotic cells. , And an improved attenuated live vaccine comprising a nucleotide sequence encoding at least one of IL-4, IL-5, IL-10, or BL1 protein.

The present invention provides human IL-12, GM-CSF, IL-1α, TNF-α, TNF-β, IL-2, IL-15, IL-18 operably linked to regulatory factors required for expression in eukaryotic cells. Pathogen, cancer or autoimmune disease, comprising administering to the individual an attenuated vaccine comprising a nucleotide sequence encoding at least one of IL-4, IL-5, IL-10, or BL1 protein. To a method for immunizing an individual against.

The present invention provides human IL-12, GM-CSF, IL-1α, TNF-α, TNF-β, IL-2, IL-15, IL-18 operably linked to regulatory factors required for expression in eukaryotic cells. , And a plasmid comprising a nucleotide sequence encoding at least one of IL-4, IL-5, IL-10, or BL1 protein.

The present invention includes a nucleotide sequence encoding a p35 subunit of IL-12 of a human operably linked to a regulatory factor necessary for expression in eukaryotic cells, and the other is operable with a regulatory factor required for expression in eukaryotic cells. It relates to a pair of plasmids, comprising a nucleotide sequence encoding a p40 subunit of IL-12 of a human being linked.

The present invention relates to a plasmid comprising a single nucleotide sequence encoding a single chain of human IL-12 protein operably linked to a regulatory factor required for expression in eukaryotic cells, wherein the single chain of human IL-12 protein comprises p35 and p40 sub The unit is a single protein linked to each other by a linker sequence, wherein the expressed single chain protein can form a biologically active IL-12 molecule.

The present invention relates to IL-12, GM-CSF, IL-1α, TNF-α, TNF-β, IL-2, IL-15, IL-18, IL, which are operably linked to regulatory factors required for expression in eukaryotic cells. A pharmaceutical composition comprising a plasmid comprising a nucleotide sequence encoding at least one of -4, IL-5, IL-10, or BL1 protein, and a pharmaceutically acceptable carrier or diluent.

The present invention includes a nucleotide sequence encoding a human IL-12 protein p35 subunit operably linked to a regulatory factor for expression in eukaryotic cells, and the other is operable with a regulatory factor required for expression in eukaryotic cells. A pharmaceutical composition, comprising a pair of plasmids, and a pharmaceutically acceptable carrier or diluent, comprising a nucleotide sequence encoding a humanly linked human IL-12 protein p40 subunit.

The present invention relates to a plasmid comprising a single nucleotide sequence encoding a human single stranded IL-12 protein operably linked to a regulatory factor required for expression in eukaryotic cells, wherein the single stranded human IL-12 protein comprises p35 and p40 sub Wherein the unit is a single protein linked to each other by a linker sequence, wherein the expressed single-chain protein may form a biologically active IL-12 molecule), and a pharmaceutically acceptable carrier or diluent It is about.

The invention includes administering to a subject a plasmid comprising a nucleotide sequence encoding a single chain of human IL-12 protein operably linked to a regulatory factor necessary for expression in a cell of the subject, allergic reactions, pathogen infection, A method of treating a patient with cancer or autoimmune disease.

The present invention includes one nucleotide sequence encoding a p35 subunit of human IL-12 protein operably linked to a regulatory factor necessary for expression in eukaryotic cells, and the other acts with a regulatory factor required for expression in eukaryotic cells. Treating a patient with an allergic reaction, a pathogen infection, cancer or an autoimmune disease, comprising administering to the subject a pair of plasmids comprising a nucleotide sequence encoding a p40 subunit of an IL-12 protein of a possibly linked human It is about how to.

The present invention relates to a plasmid comprising a single nucleotide sequence encoding a single chain of human IL-12 protein operably linked to a regulatory factor required for expression in eukaryotic cells, wherein the single chain of human IL-12 protein comprises p35 and p40 sub The unit is a single protein linked to each other by a linker sequence, wherein the expressed single-chain protein may form a biologically active IL-12 molecule), the method comprising administering to the subject an allergic reaction, pathogen infection, cancer Or to a method for treating an autoimmune disease patient.

The present invention relates to a Th1 type immune response comprising administering to a subject a plasmid comprising a nucleotide sequence encoding a single chain of human IL-12 protein operably linked to a regulatory factor required for expression in a cell of the subject. To a method of enhancing or driving an immune response in a subject.

The present invention includes a nucleotide sequence encoding a p35 subunit of IL-12 of a human operably linked to a regulatory factor necessary for expression in eukaryotic cells, and the other is operable with a regulatory factor required for expression in eukaryotic cells. Enhancing a Th1 type immune response in a subject, comprising administering to the subject a pair of plasmids comprising a nucleotide sequence encoding a p40 subunit of IL-12 To a method for driving an immune response in a subject to a response.

The present invention relates to a plasmid comprising a single nucleotide sequence encoding a single chain of human IL-12 protein operably linked to a regulatory factor required for expression in eukaryotic cells, wherein the single chain of human IL-12 protein comprises p35 and p40 sub Unit is a single protein linked to each other by a linker sequence, wherein the expressed single-chain protein may form a biologically active IL-12 molecule), the method comprising administering to the subject an Th1 type immune response Or to drive an immune response in an individual against a Th1 type of immune response.

The present invention relates to a recombinant vector comprising a nucleotide sequence encoding a human IL-12 protein operably linked with regulatory factors required for expression in eukaryotic cells.

The present invention provides a single nucleotide sequence encoding a single chain human IL-12 protein operably linked to a regulatory factor required for expression in eukaryotic cells, wherein the single chain human IL-12 protein is a linker sequence in which the p35 and p40 subunits are Is a single protein linked to each other, wherein the expressed single-chain protein is capable of forming a biologically active IL-12 molecule).

The present invention relates to human IL-12, GM-CSF, IL-1α, TNF-α, TNF-β, IL-2, IL-15, IL-18, which are operably linked to regulatory factors required for expression in eukaryotic cells. A pharmaceutical composition comprising a recombinant vector comprising a nucleotide sequence encoding one or more of the IL-4, IL-5, IL-10 or BL-1 proteins, and a pharmaceutically acceptable carrier or diluent.

The present invention provides a recombinant vector comprising a nucleotide sequence encoding a human single-chain IL-12 protein operably linked to a regulatory factor required for expression in eukaryotic cells, wherein the single-chain human IL-12 protein is p35 and p40. The subunit is a single protein linked to each other by a linker sequence, wherein the expressed single-chain protein may form a biologically active IL-12 molecule), and a pharmaceutically acceptable carrier or diluent It is about.

The present invention comprises administering to a subject a recombinant vector comprising a nucleotide sequence encoding a human IL-12 protein operably linked to a regulatory factor required for expression in eukaryotic cells, allergic reactions, pathogen infection, cancer or A method of treating an autoimmune disease patient.

The present invention provides a recombinant vector comprising a nucleotide sequence encoding a single strand of human IL-12 protein operably linked to a regulatory factor required for expression in eukaryotic cells, wherein the single strand of human IL-12 protein comprises p35 and p40 subs. The unit is a single protein linked to each other by a linker sequence, wherein the expressed single-chain protein may form a biologically active IL-12 molecule), the method comprising administering to the subject an allergic reaction, pathogen infection, cancer Or to a method for treating an autoimmune disease patient.

The present invention includes administering to a subject a recombinant vector comprising a nucleotide sequence encoding a single chain of human IL-12 protein operably linked to a regulatory factor required for expression in eukaryotic cells, the Th1 type of immunity in the subject. To enhance a response or to drive an immune response in an individual against a Th1 type of immune response.

The present invention provides a recombinant vector comprising a single nucleotide sequence encoding a single chain of human IL-12 protein operably linked to a regulatory factor required for expression in eukaryotic cells, wherein the single chain of human IL-12 protein is p35 and p40. The subunit is a single protein linked to each other by a linker sequence, wherein the expressed single-chain protein can form a biologically active IL-12 molecule), the method comprising administering to the subject a Th1 type of immunity A method of enhancing a response or enhancing or driving an immune response in a subject against a Th1 type immune response.

The present invention relates to pro-inflammatory cytokines (IL-1α, TNF-α and TNF-β), Th1 cytokines (IL-2, IL-15 and IL-18), and Th2 cytokines (IL) in one or more humans. -4, IL-5 and IL-10) composition comprising a nucleic acid molecule encoding a protein as a primary agent and a method of using said composition for driving and inducing an immune response by administering said nucleic acid molecule to an individual will be. The present invention relates to a method for enhancing antigen specific humoral response by co-transporting IL-5 or IL-18 with a vaccine that introduces a target immunogen, such as a DNA vaccine construct. The present invention provides antigen-specific T helper cells by co-transporting IL-2, IL-5, IL-10, IL-18, TNF-α or TNF-β with a vaccine that introduces a target immunogen, such as a DNA vaccine construct. It relates to a method of increasing the proliferation of. The present invention relates to a method of enhancing a cytotoxic response due to the coadministration of TNF-α or IL-15 with a vaccine into which a target immunogen is introduced, such as a DNA vaccine construct.

The present invention provides compositions comprising nucleic acid molecules encoding GM-CSF of humans, and methods of inducing and regulating immune responses by transporting or co-transporting gene constructs encoding GM-CSF. The present invention relates to a method for enhancing antigen specific antibody and T helper cell proliferation by co-administering the GM-CSF gene with a DNA vaccine construct.

The present invention relates to substantially pure BL1 and immunoregulatory fragments thereof.

The present invention relates to an isolated nucleic acid molecule encoding BL1 and immunoregulatory fragments thereof.

The present invention relates to nucleic acid probes and primers specifically directed to nucleic acid molecules encoding BL1 or immunoregulatory fragments thereof.

The present invention relates to oligonucleotide molecules consisting of nucleotide sequences complementary to specific regions of the nucleotide sequence encoding BL1.

The present invention relates to a vector comprising a nucleic acid molecule encoding BL1 or an immunoregulatory fragment thereof.

The present invention relates to a recombinant expression vector comprising a nucleic acid sequence encoding BL1 or an immunoregulatory fragment thereof.

The present invention relates to a host cell comprising a recombinant expression vector comprising a nucleic acid sequence encoding BL1 or an immunoregulatory fragment thereof. The present invention relates to a genetic therapeutic vector comprising a nucleic acid molecule encoding BL1 or an immunoregulatory fragment thereof.

The present invention relates to an isolated antibody that binds to a specific epitope on BL1.

The present invention relates to a method of making BL1 and immunoregulatory fragments thereof.

The present invention relates to a method of modulating an immune response in an individual, comprising administering a vector comprising a nucleotide sequence encoding each BL1 or immunoregulatory fragment thereof or BL1 protein or immunoregulatory fragment thereof. According to the method of the invention, the vector comprising the BL1 coding sequence is sufficient to modulate the immune response.

The present invention provides a method for enhancing an immune response in an individual, comprising administering a vaccine composition for the transport of an immunogen and BL1 protein, or an immunoregulatory fragment thereof, or a vector comprising a nucleotide sequence encoding a BL1 protein or an immunoregulatory fragment thereof. It is about how to adjust. According to the method of the invention, the vector comprising the BL1 coding sequence is sufficient to modulate the immune response.

1A and 1B show the drawings from Example 4. FIG. In FIG. 1A, 50 μg of each cDNA expression cassette was administered intramuscularly prior to measurement. After 14 days of immunization, spleens harvested from all immunized animals were weighed. Negative control animals were immunized. Spleen mass from rats injected with Gag / Pol only and IL-12 only was similar to that of non-immunized control rats (about 100 mg). However, the spleen mass from mice injected with the Gag / Pol + IL-12 gene was nearly three times that of the control spleen. In contrast, the spleen of Gag / Pol + GM-CSF immunized mice did not grow. In FIG. 1B, leukocytes were prepared and purified from each spleen. Depending on the mass difference of each spleen, the number of cells from the Gag / Pol + IL-12 immunized spleen exceeded three times that of the control spleen. The spleen of Gag / Pol + GM-CSF immunized mice did not significantly increase the number of lymphocytes in the cell count of the control spleen.

In FIG. 2, 50 μg of each cDNA expression cassette was administered intramuscularly before measurement. After 14 days of immunization, the spleen was harvested and photographed. The visual size of the spleen corresponded directly to the mass, where the immunogen + IL-2 vaccinated spleen was much larger than the unimmunized control spleen. Community: (-) not immunized; IL-12 immunized; Envelope + IL-12 immunized; Gag / Pol + IL-12 immunized.

In FIG. 3, each chain of IL-12 was coadministered. 50 μg of each plasmid was used. In addition to Gag / Pol, both p35 and p40 chains were required for splenic expansion.

In FIG. 4, 50 μg of each cDNA expression cassette was administered intramuscularly before measurement. Antisera from immunized mice were collected and analyzed for specific antibody responses to HIV-1 by ELISA. The results of ELISA of samples collected on day 28 are shown. At a 1: 100 dilution ratio, serum from the group immunized with Invelop + GM-CSF showed an antibody response to HIV-1 gp120 greater than the group immunized with Invelop alone. On the other hand, the group immunized with Invelop + IL-12 showed less humoral response during the same period.

 In Figure 5, activation and proliferation of T helper lymphocytes play an important role in inducing both the immune response of humor through the expansion of antigen activated B cells and the cellular immune response through the expansion of CD8 + cytotoxic T cells. 50 μg of each cDNA expression cassette was administered intramuscularly before measurement. Harvested spleen cells were tested for T cell proliferation. 20 μg / ml of recombinant p55 protein was plated in each well to promote proliferation of T cells. 10 μg / ml of lectin PHA was used as a polyclonal promoter positive control. Promoting index is the degree of radioactivity detected from cells promoted with a particular protein divided by the level measured from the cells in the medium. The PHA promotion index was 58.8.

In FIG. 6, 50 μg of each cDNA expression cassette was administered intramuscularly before measurement. CTL analysis was performed using cells prepared from harvested spleen without in vitro palpation. Controls immunized with the IL-12 gene cassette alone did not result in specific lysis of target cells above the background level. In addition, a low level (3%) of non-solubility was observed when the effector: target ratio was immunized with Gag / Pol only at 50: 1. In contrast, 62% of the nasal involvement was observed with Gag / Pol + IL-2 at 50: 1 effector: target ratio and was determined to be 9% at 12.5: 1 effector: target ratio. . Immunization with Gag / Pol + GM-CSF plasmid showed no detectable CTL activity. The same CTL analysis performed on targets made with inappropriate antigen-expressing vaccinia did not result in a meaningful CTL response.

In FIG. 7, 50 μg of each cDNA expression cassette was administered intramuscularly before measurement. CTL analysis performed without in vitro palpation used cells prepared from harvested spleen. Colonies immunized with envelope alone and with envelope + GM-CSF at a 50: 1 effector: target ratio resulted in low levels of specific CTLs of 4% and 1%, respectively. On the other hand, with Envelop + IL-2, a dramatic increase in CTL activity of 59% was observed. The same CTL analysis performed on targets made with inappropriate antigen-expressing vaccinia did not result in a meaningful CTL response.

8A, 8B and 8C show plasmids useful in the present invention. 8A shows a plasmid containing the coding sequence of IL-12 as a single chain protein. 8B and 8C show plasmids comprising two coding sequences for each of two subunits.

9 shows Table 3.

As shown in FIG. 10, each cytokine gene is cloned into an expression plasmid under the control of the CMV promoter and transfected into RD cells in vitro. Expression of each cytokine was confirmed using immunoprecipitation or cytokine ELISA.

11A-11O show the results from experiments to determine MHC class I-limiting CTL. As shown in FIGS. 11A-11E, two weeks after co-administration (50 μg each) of the first DNA with pCEnv, mice (4 per colony) were re-inoculated at the same dose. After one week, the spleens were collected from immunized mice, their lymphocytes isolated, and using target cells prepared using envelope specific peptides (RIHIGPGRAFYTTKN) reported as MHC class I-limiting in balb / c. Tested for CTL reaction. As shown in FIGS. 11F-11O, two weeks after co-administration (50 μg each) of the first DNA with pCGag / POL, mice (4 per colony) were re-inoculated at the same dose. After one week, the spleens were collected from immunized mice, their lymphocytes were isolated and tested for CTL response. CTL analysis was performed by removing CD8 + T cells by complement lysis. Effector cells were prepared in the presence of CD8 + T cells (top) and in the absence of CD8 + T cells (bottom). This experiment was repeated twice with similar results.

12 shows the results from experiments that directly assess antigen specific CTL (without in vitro promotion of effectors). Two weeks after the first DNA co-administered with pCEnv (50 μg each), mice (4 per colony) were re-inoculated at the same dose. After one week, the spleens were collected from immunized mice, their lymphocytes were isolated and tested for CTL responses using target cells infected with specific (vMN462) and nonspecific vaccinia (vSC8). This experiment was repeated twice with similar results.

FIG. 13 summarizes the effect of the accompanying administration of each cytokine on antibodies (Y axis), T helpers (X axis), and cytotoxic T lymphocyte response (Z axis). Each cytokine was plotted on three dimensions according to their effect on three modes of immune response.

14 depicts the nucleotide and predicted amino acid sequence of BL1.

Figure 15 depicts ligation of BL1 with a PCR3 eukaryotic expression vector and vector pBBKan.

FIG. 16 shows the results of an ELISA assay comparing the anti-HIV antigen response regulated in HIV antigen Nef, with and without the accompanying administration of BL1.

17A, 17B, 17C and 17D show assay results comparing the anti-HIV antigen immune response regulated in HIV antigen Gag / Pol, with and without the accompanying administration of BL1.

As used herein, the term “immunoregulatory protein” refers to human IL-12, GM-CSF, IL-1, TNF-α, TNF-β, IL-2, IL-15, IL-18, IL-4, One of the novel molecules named IL-5 and IL-10, and BL-1.

 As used herein, the term “IL-12 gene construct” encodes one or both of human IL-12 protein subunits and / or immunogenic target proteins, and includes regulatory factors required for expression of the coding sequence in eukaryotic cells. Refers to a plasmid comprising a coding sequence operably linked. Regulatory factors for DNA expression include promoters and polyadenylation signals. In addition, other factors, such as Kozak regions, may also be included in the gene construct. Initiation and termination signals are necessary regulatory factors that are often considered part of the coding sequence. Coding sequences of the genetic constructs of the invention include functional initiation and termination signals.

As used herein, the term “target IL-12 protein” refers to a single chain IL-12 protein, wherein two subunits are encoded by a single coding sequence and expressed as a single protein with a linker sequence connecting the two subunits. One or all of the IL-12 subunits of the person comprising a.

As used herein, the term “protein of interest” refers to an immunogenic target protein encoded by the coding sequence of a vaccine comprising a nucleic acid molecule encoding an immunogenic target protein.

As used herein, the terms “single chain protein” and “single chain IL-12 protein” refer to two subunit regions of a single protein joined together by sufficiently long and flexible amino acid linkers of the IL-12 p35 and p40 subunits. Acts to form a biologically active complex form, i.e. a single chain protein presumed IL-12. Single chain IL-12 functions essentially the same as IL-12 consisting of p35 and p40. The present invention includes the use of single chain IL-12 in all cases where IL-12 is used. A single protein is encoded by a single nucleotide sequence.

Interleukin-12 (IL-12), a heterodimeric cytokine, is produced primarily by macrophages and B cells. IL-12 consists of two different subunits, designated p35 and p40 (Podlaski, FJ et al. (1991) Arch. Biochem. Biophysics. 294 (1): 230-237, incorporated herein by reference) ).

Different immune responses are involved in T cell populations. Specifically, there are two different types of T cells, type 1 T-helper cells (Th1) and type 2 T-helper cells (Th2), which differ from each other in cytokine production. IL-12 has been shown to play an important role in the Th1 immune response by primarily inducing the production of Th1-associated cytokine interferon-gamma. Natural killer (NK) and T cells are activated through the induction and release of various cytokines including IFN-gamma.

A feature of the present invention relates to the use of a nucleic acid molecule encoding human IL-12 protein as an immunomodulator. Nucleic acid molecules encoding human IL-12 protein may be transported as a primary active agent, ie a gene therapy vaccine, such as a vaccine comprising a nucleic acid molecule encoding an immunogenic target protein, or a portion or a mixture of vaccine compositions. .

In the use of a nucleic acid molecule encoding a human IL-12 protein as a primary agent, the present invention administers an immune response or enhances a Th1 immune response by administering to a subject a nucleic acid molecule encoding a human IL-12 protein. Provided are compositions and methods. According to certain aspects of the invention, a patient with an allergic disease, pathogen infection, cancer or autoimmune disease is a nucleic acid comprising a nucleotide sequence encoding IL-12 of a person operably linked with a regulatory factor such that the nucleic acid is expressed in a cell of the individual. The molecule can be treated by administering to the patient. The nucleic acid molecule is taken up by the cell and the nucleotide sequence encoding human IL-12 is expressed. IL-12 in humans so produced by the cell is biologically active, and with its activity the immune response elicited by the individual is induced and / or enhanced. In certain preferred embodiments, the nucleic acid molecule encoding human IL-12 protein is a plasmid.

Another aspect of the invention involves the use of nucleic acid molecules encoding granulocyte-macrophage colony enhancing factor (GM-CSF), wherein GM-CSF is a neutrophil monocytic / macrophage, and hematopoietic proliferation factor that promotes eosinophil colony formation. to be. It also induces proliferation and differentiation of red blood cells and megakaryocyte proliferating cells. In addition, GM-CSF increases the antibody dependent cell mediated cytotoxicity of neutrophils, eosinophils, and macrophages, but has not been reported to play a direct role in inducing CTL responses in vivo. The present invention provides compositions and methods for driving an immune response by administering to a subject a gene construct comprising a nucleotide sequence encoding GM-CSF. The nucleic acid molecule is taken up by the cell so that the nucleotide sequence encoding GM-CSF is expressed and produced by the cell. GM-CSF is biologically active, and with its activity the immune response elicited by the subject is induced and / or enhanced. In certain preferred embodiments, the nucleic acid molecule encoding GM-CSF is a plasmid.

Other aspects of the invention include human pro-inflammatory cytokines (IL-1α, TNF-α and TNF-β), Th1 cytokines (IL-2, IL-15 and IL-18), and Th2 cytokines (IL -4, IL-5 and IL-10), including the use of nucleic acid molecules encoding proteins as primary agents. The present invention relates to human pro-inflammatory cytokines (IL-1α, TNF-α and TNF-β), Th1 cytokines (IL-2, IL-15 and IL-18), and Th2 cytokines (IL-4, Provided are compositions and methods for driving an immune response or enhancing an immune response by administering a nucleic acid molecule encoding IL-5 and IL-10) to the subject. According to some aspects of the invention, human pro-inflammatory cytokines (IL-1α, TNF-α and TNF-β), Th1 cytokines (operably linked to regulatory factors such that nucleic acid molecules are expressed in cells of an individual) The subject is treated by administering a nucleic acid molecule comprising nucleotide sequences encoding IL-2, IL-15 and IL-18), and Th2 cytokines (IL-4, IL-5 and IL-10). The nucleic acid molecule is taken up by the cell and the nucleotide sequence that encodes the protein is expressed. The human protein thus produced by the cell is biologically active and its activity induces and / or enhances the immune response elicited by the individual. In a preferred embodiment, the nucleic acid molecule is a plasmid.

Human IL, in the use of a nucleic acid molecule encoding human IL-12 protein as part of or together with a nucleic acid molecule encoding an immunogenic target protein, ie as part of a vaccine for eliciting an immune response against an immunogenic protein. The nucleic acid molecule encoding the -12 protein is a component of a vaccine comprising a nucleic acid molecule encoding an immunogenic target protein, a component of a vaccine comprising an immunogenic target, or a vaccine comprising a nucleic acid molecule encoding an immunogenic standard protein. Or in separate compositions co-administered with the vaccine comprising the immunogenic target. In a preferred embodiment, the nucleic acid molecule encoding human IL-12 protein is a plasmid and the vaccine is a DNA vaccine comprising a plasmid encoding an immunogenic target protein. In a preferred embodiment, the DNA vaccine comprises a plasmid encoding an immunogenic target protein and human IL-12 protein.

IL-12 is disclosed in PCT Application WO90 / 05147, published May 17, 1990, incorporated herein by reference. Wolf, S.F. et al., 1991, J. Immunol. 146 (9); 3074-3081 (incorporated herein by reference) discloses the nucleotide sequence of cDNA encoding IL-12 and the predicted amino acid sequence of IL-12 protein. Human native IL-12 protein consists of two subunits, p35 and p40. Two subunits form a biologically active heterodimer complex.

According to an embodiment of the invention, the nucleotide sequence encoding each subunit of IL-12 is on a single plasmid, or on a nucleic acid molecule other than the plasmid, or on a viral or bacterial genome, wherein the nucleotides encoding each subunit The sequence is controlled by its regulatory factor. In a preferred embodiment, the coding sequences for all of the subunits of IL-12 are on a single plasmid, and each coding sequence is operably linked with its own regulatory factor. In certain embodiments, the coding sequence of the target immunogenic protein operably linked with a regulatory factor is in the same plasmid as the coding sequence for the two subunits. In certain embodiments, the coding sequence of the target immunogenic protein operably linked with a regulatory factor is on a plasmid separate from the plasmid containing the coding sequence for the two subunits, and the two plasmids are transported to the individual.

According to an embodiment of the invention, the nucleotide sequence encoding the p35 subunit is present on the first plasmid, the nucleotide sequence encoding the p40 subunit on the second plasmid, and the two plasmids accompany the same site in the individual. Administered. In certain embodiments, the coding sequence for the target immunogenic protein operably linked with a regulatory factor is on the same plasmid as the coding sequence for the p35 subunit. In certain embodiments, the coding sequence for the target immunogenic protein, operably linked with a regulatory factor, is on the same plasmid as the coding sequence for the p40 subunit. In certain embodiments, the coding sequence for the target immunogenic protein, operably linked with a regulatory factor, is present on a plasmid separated from the plasmid containing the coding sequence for each subunit, and the three plasmids are transported to the individual .

The IL-12 protein, and the nucleotide sequence encoding it, can be modified so that two subunits are encoded by a single nucleotide sequence and expressed as a single chain (fusion) protein molecule. According to the invention, the linker amino acid sequence is essentially flexible so that two subunits are combined, but different regions of the single chain protein can be complexed to form a biologically active protein. 8A shows an example of a single chain protein in which the coding sequence for the single chain protein is regulated by the human cytomegalovirus promoter. The coding sequence of the single-chain protein includes, as the single coding sequence, the coding sequence of the p35 subunit, the coding sequence of the linker and the coding sequence of the p40 subunit in the 5 'to 3' direction. In another arrangement, the coding sequence of a single chain protein includes the coding sequence of the p40 subunit, the coding sequence of the linker and the coding sequence of the p35 subunit as a single coding sequence. The linker must be long and flexible enough so that the two parts of a single protein are placed in close proximity to each other to form a biologically active complex.

According to certain embodiments of the invention, the nucleotide sequence encoding a single-chain IL-12 protein, wherein two subunits are joined by a linker to form a single protein, is operably linked to regulatory factors required for expression in eukaryotic cells, It is incorporated into a plasmid or a nucleic acid molecule that is not a plasmid, or a viral or bacterial genome. In a preferred embodiment, the nucleotide sequence encoding the single chain IL-12 protein, which encodes a single chain protein wherein two subunits are joined by a linker to form a single protein, is incorporated into the plasmid. In certain embodiments, the coding sequence of the target immunogenic protein, operably linked with a regulatory factor, is on the same plasmid as the coding sequence of single chain IL-12 protein. In certain embodiments, the coding sequence of the target immunogenic protein, operably linked with a regulatory factor, is present on a plasmid separated from the plasmid containing the coding sequence of the single chain protein, and the two plasmids are transported to the individual.

In accordance with aspects of the present invention regarding improved methods and improved compositions for vaccination, the DNA encoding the target immunogen in a particular DNA vaccination is administered to a subject so that the DNA is absorbed and expressed to induce an immune response against the immunogen. According to aspects of the invention. The DNA encoding the immunoregulatory protein is transported to the individual and the DNA is expressed to produce an immunoregulatory protein, which induces a specific immune response so that it can be defended against various diseases through closer immunization. To regulate the degree and direction of the immune response.

It has been found that co-production of IL-12 protein in cells of vaccinated individuals expressing the target antigen surprisingly enhances the immune response to the target antigen. By providing an expression form of the nucleotide sequence encoding the IL-12 protein, it improves vaccines, such as DNA vaccines, recombinant vector vaccines and attenuated vaccines, which function by expressing the target antigen in the cells of the vaccinated individual. .

Co-production of IL-12 in cells producing antigens enhances cellular immunogens for antigens. Accordingly, the present invention is improved by providing nucleotide sequences encoding IL-12 operably linked to regulatory factors required for expression in a vaccine as part of a vaccine such as a DNA vaccine, a nontoxic recombinant vector vaccine and an attenuated live vaccine. Provide a vaccine.

The present invention provides for the induction and regulation of immune responses from concomitant transport of gene constructs encoding GM-CSF. Concomitant administration of the GM-CSF gene and DNA vaccine constructs enhances antigen specific antibodies and T helper cell proliferative responses.

The present invention relates to pro-inflammatory cytokines (IL-1α, TNF-α and TNF-β), Th1 cytokines (IL-2, IL-15 and IL-18), and Th2 cytokines (IL-4, IL- 5 and the induction and regulation of immune responses from concomitant transport of the gene construct encoding IL-10).

Certain aspects of the present invention provide meaningful enhancement of antigen specific humoral responses by co-transportation of IL-5 or IL-18.

Certain aspects of the present invention are to increase antigen specific T helper cell proliferation by co-transportation of IL-2, IL-5, IL-10, IL-18, TNF-α or TNF-β.

Certain aspects of the present invention provide for the enhancement of cytotoxic responses to co-administration of TNF-α or IL-15 genes.

Thus, when the T cell mediated response is excellent, the response of the humor is not necessary or even harmful, and the IL-12 gene is preferred as an immunomodulator to be transported with a specific DNA immunogen. On the other hand, to prepare a vaccine against target extracellular bacteria, for example, the IL-4, IL-5 or IL-10 gene may be coadministered. Moreover, where CD4 + T helper cells and antibodies play a more important function in defense, GM-CSF and IL-2 can be transported in tandem. Recently, where all three immune response weapons are important, TNF-α can be co-injected to enhance the antibody, T-helper cells and CTL responses together.

Nucleotide and amino acid sequences of human IL-1α are known and are incorporated herein by reference in Telford, et al. (1986) Nucl. Acids Res. 14: 9955-9963, Furutani, et al. (1985) Nucl. Acids Res. 14: 3167-3179, March, et al. (1985) Nature 315: 641-647, and deposited code swissprot PO1583.

Nucleotide and amino acid sequences of human IL-2 are known and are incorporated herein by reference in Holbrook, et al. (1984) Proc. Natl. Acad. Sci. USA 81: 1634-1638, Fujita, et al. (1983) Proc. Natl. Acad. Sci. USA 80: 7437-7441, Fuse, et al. (1984) Nucl. Acids Res. 12: 9323-9331, Taniguchi, et al. (1983) Nature 302: 305-310, Maeda, et al. (1983) Biochem. Biophys. Res. Comm. 115: 100-1047, Devos, et al. (1983) Nucl. Acids Res. 111: 4307-4323, and deposited code Swissprot PO1585.

Nucleotide and amino acid sequences of human IL-4 are known and are incorporated herein by reference in Arai, et al. (1989) J. Immunol. 142: 274-282, Otsuka et al. (1987) Nucl. Acids Res. 15: 333-344, Yokota, et al. (1986) Proc. Natl. Acad. Sci. USA 83: 5894-5898, Noma, et al. (1984) Nature 319: 640-646, Lee, et al. (1986) Proc. Natl. Acad. Sci. USA 83: 2061-2063, and the deposit code Swissprot 05112 (the deposited code of rat IL-4 is Swissprot 07750).

Nucleotide and amino acid sequences of human IL-5 are known and are incorporated herein by reference in Campbell, et al. (1987) Proc. Natl. Acad. Sci. USA 84: 6629-6633, Tanabe et al. (1987) J. Biol. Chem. 262: 16580-16584, Campbell, et al. (1988) Eur. J. Biol. Chem. 174: 345-352, Azuma et al. (1986) Nucl. Acids Res. 14: 9149-9158, Yokota, et al. (1986) Proc. Natl. Acad. Sci. USA 84: 7388-7392, and Deposit Code Swissprot P05113.

Nucleotide and amino acid sequences of human IL-10 are known and described in Viera, et al. (1991) Proc. Natl. Acad. Sci. USA 88: 1172-1176, and the deposit code Swissprot P22301.

Nucleotide and amino acid sequences of human IL-15 are known and are incorporated herein by reference Grabstein, et al. (1994) Science 264: 965-968, and Deposit Code Swissprot U03099.

Nucleotide and amino acid sequences of human IL-18 are known and are incorporated herein by reference Ushio, et al. (1996) J. Immunol. 156: 4274-4279, and deposited code Swissprot D49950.

The nucleotide and amino acid sequences of human TNF-α are known and are presented in Pennica, (1984) Nature 312: 724-729, and the deposit code Swissprot PO1375, which is hereby incorporated by reference.

The nucleotide and amino acid sequences of human TNF-β are known and are presented in Gray, (1984) Nature 312: 721-724, and the deposit code Swissprot PO1374, which is incorporated herein by reference.

DNA vaccines are described in US Pat. Nos. 5,593,972, 5,589,466, PCT Application No. PCT / US90 / 01515, PCT / US93 / 02338, PCT / US93 / 048131, and PCT / US94 / 00899 The priority applications described in the patent application and cited in the above patents and publications are incorporated herein by reference. In addition to the transport methods described in this application, alternatives to DNA transport are described in US Pat. Nos. 4,945,050 and 5,036,006, which are incorporated herein by reference.

An improvement of the present invention is that in addition to the production of antigen targets encoded by the nucleic acid sequences of DNA vaccines, it contains genetic material for the co-production of immunoregulatory proteins.

The present invention relates to a method of injecting a genetic material into a cell of an individual to induce an immune response against proteins and peptides encoded by the genetic material. The method comprises a single nucleic acid molecule comprising a nucleotide sequence encoding a target protein and a nucleotide sequence encoding an immunoregulatory protein, or one nucleotide sequence encoding a target protein, in a tissue of an individual and the other Administering a composition comprising two nucleic acid molecules comprising a nucleotide sequence encoding a regulatory protein. The nucleic acid molecule may be part of a plasmid DNA, a nucleic acid molecule of a recombinant vector or a genetic material provided in an attenuated vaccine.

In accordance with the present invention, compositions and methods are provided for prophylactically and / or therapeutically immunizing an individual against pathogens or abnormal, disease related cells. The genetic material encodes a target protein, ie a peptide or protein that shares at least an epitope with an immunogenic protein found on a pathogen or cell to be targeted, and the genetic material also encodes an immunoregulatory protein. The genetic material is expressed by the individual's cells and acts as an immunogenic target to produce an immune response. The resulting immune response is based on reacting with the pathogen or cell to be targeted and, in addition to the immune response of the body fluids, eliciting two cellular immune responses. The methods of the invention are useful for conferring prophylactic and / or therapeutic immunization. Thus, immunization methods include methods of protecting an individual from pathogen infiltration, or the development or proliferation of certain cells, and methods of treating patients with pathogen infection, hyperproliferative diseases or autoimmune diseases.

As used herein, “target protein” refers to peptides and proteins encoded by the gene constructs of the invention that act as target proteins in an immune response. The term "target protein" refers to a protein capable of eliciting an immune response. Target proteins are immunogenic proteins that share at least an epitope with proteins from pathogens or undesirable cell types, such as cancer cells or cells involved in autoimmune diseases requiring immunization. The immune response to the target protein will defend and treat the individual against the specific infection or disease to which the target protein is associated. The target protein need not be identical to the protein for which an immune response is required. Rather, the target protein should be capable of inducing an immune response that cross reacts with the protein for which the immune response is desired.

The present invention is useful for eliciting a broad immune response to a target protein, ie, a protein specifically associated with a "abnormal" cell of a pathogen or individual. The invention is useful for immunizing an individual against pathogens and microorganisms, where the immune response to the pathogen protein provides protective immunization against the pathogen. The present invention is useful in combating hyperproliferative diseases and disorders, such as cancer, by eliciting an immune response against hyperproliferative cells and certain related target proteins. The present invention is useful in combating autoimmune diseases and disorders by eliciting an immune response against target proteins specifically associated with autoimmune disease related cells.

According to the invention, the DNA or RNA encoding the target protein and immunoregulatory protein is injected into a tissue cell of an individual, where it is expressed to produce the target protein. DNA or RNA encoding the target protein and immunoregulatory protein is linked to regulatory factors required for expression in the cells of the individual. Regulatory factors required for DNA expression include promoters and polyadenylation signals. In addition, other factors, such as Kozak regions, may also be included in the gene construct.

As used herein, the term “expressable form” refers to a gene in which the coding sequence is expressed when the necessary regulatory factor is operably linked to a coding sequence encoding a target protein or immunoregulatory protein in a gene construct so that it is present in the cell of an individual. Means a construct.

As used herein, the term "sharing an epitope" means a protein comprising one or more epitopes that are identical or substantially similar to epitopes of other proteins.

As used herein, the term “substantially similar epitope” refers to an epitope whose structure does not coincide with the epitope of a given protein, but has a structure that elicits a cellular or humoral immune response that cross reacts with that protein.

Gene constructs comprise nucleotide sequences encoding target proteins and / or immunoregulatory proteins, operably linked with regulatory factors necessary for gene expression. According to the present invention, a combination of gene constructs is provided, one comprising an expressible form of a nucleotide sequence encoding a target protein and one comprising an expressible form of a nucleotide sequence encoding an immunoregulatory protein. DNA or RNA molecules, including combinations of gene constructs, can be incorporated into living cells to allow expression of DNA or RNA and the production of target and immunoregulatory proteins. As a result, the immune response to the target protein is enhanced.

When the gene construct (s) are taken up into the cell, it may remain in the cell as a functional extrachromosomal molecule or be incorporated into the cell's chromosomal DNA. DNA can be injected into cells in the form of plasmid (s) to remain as separate genetic material. In addition, linear DNA that can be integrated into the chromosome can also be introduced into the cell. When introducing DNA into cells, reagents may be added that promote the integration of DNA into the chromosome. DNA sequences useful for facilitating integration may also be included in the DNA molecule. RNA can also be administered to cells. It is also contemplated to provide the gene construct as a linear mini chromosome with isotopes, telomeres, and origins of replication. The genetic construct may be a recombinant microbial vector that survives in cells or as part of a genetic material in an attenuated live vaccine microorganism. The genetic construct may be part of the genome of a recombinant viral vaccine, where the genetic material is integrated into the chromosome of the cell or remains extrachromosomal.

Gene constructs contain regulatory factors required for gene expression of nucleic acid molecules. Such factors include promoters, start codons, stop codons, polyadenylation signals, and the like. In addition, enhancers are often required for gene expression of sequences encoding immunogenic target proteins or immunoregulatory proteins. The factors must be operably linked to the sequence encoding the desired protein and the regulatory factor must be operable in the subject to which it is to be administered.

In general, the start codon and the stop codon are considered part of the nucleotide sequence encoding the protein of interest. However, these factors need to be functional in the subject to which the gene construct will be administered. The start and end codons must be within the frame of the coding sequence.

The promoter and polyadenylation signal used must be functional in the cells of the individual.

Examples of promoters useful in the practice of the present invention, particularly in the manufacture of genetic vaccines for humans, include monkey virus 40 (SV40), mouse breast tumor virus (MMTV) promoter, human immunodeficiency virus (HIV), for example HIV Long terminal repeat (LTR) promoter, moronivirus, ALV, cytomegalovirus (CMV), eg CMV early early promoter, Epstein Barr virus (EBV), Loews sacoma virus (RSV) In addition, there are, but are not limited to, promoters derived from human actin, human myosin, human hemoglobin, human muscle creatine, human metallothionein.

Examples of polyadenylation signals useful in the practice of the present invention, particularly in the manufacture of human genetic vaccines, include, but are not limited to, SV40 polyadenylation signal and LTR polyadenylation signal. Among others, the SV40 polyadenylation signal in pCEP4 plasmid (Invitrogen, San Diego, Calif.) Called SV40 polyadenylation signal is used.

In addition to the regulatory factors required for DNA expression, other factors may be included in the DNA molecule. Such an additional argument is an enhancer. Enhancers can be selected from the group including, but not limited to, human actin, human myosin, human hemoglobin, human muscle creatine, and viral enhancers such as CMV, RSV, and EBV.

In order to generate multiple copies in cells while maintaining the gene construct as extrachromosomal, the origin of replication of a mammal can be provided. Plasmids pCEP4 and pREP4 (Invitrogen, San Diego, Calif.) Include the replication origin of Epstein Barr virus and the nuclear antigen EBNA-1 coding region, which causes multiple copies of episome replication without integration.

In some preferred embodiments related to the field of immunity, nucleic acid molecule (s) comprising a target protein, a nucleotide sequence encoding an IL-12 protein, and additionally a gene for a protein that further enhances an immune response to such target protein, Are transported. Examples of such genes include cytokines and lymphokines such as α-interferon, gamma-interferon, platelet derived growth factors (PDGF), GC-SF, GM-SCF, TNF, epidermal growth factor (EGF), IL And those that encode -1, IL-2, IL-4, IL-6, IL-8, IL-10, and B7.2. In some embodiments, it is preferred that the gene of GM-CSF is included in the gene construct used in the immune composition.

In any case, if it is necessary to remove the cells containing the gene construct, additional factors can be added that serve as targets for cell destruction. The herpes thymidine kinase (tK) gene may be included in the gene construct in an expressible form. The drug, gangcyclovir, can be administered to a subject, which selectively kills all cells that produce tK, thus providing a means of selectively destroying cells containing the gene construct.

To maximize protein production, the regulatory sequences can be chosen to be well suited for gene expression in the cells to which the construct is administered. Codons can also be chosen to be most effectively transcribed in that cell. Those skilled in the art will be able to produce functional DNA constructs in cells.

Examples of two types include types used in two plasmids, and types used in a single plasmid. In two plasmid systems, one plasmid has a target coding sequence in an expressible form and the other has an IL-12 coding sequence in an expressible form. In a single plasmid system, a single plasmid comprises a target coding sequence and an IL-12 coding sequence in an expressible form.

The method of the present invention comprises administering a nucleic acid molecule to a tissue of an individual. In some embodiments, the nucleic acid molecule is a mucosal membrane selected from the group consisting of intramuscular, intranasal, intraperitoneal, subcutaneous, intradermal, intravenous, aerosol administration or topical administration to lung tissue, or the vagina, rectum, urethra, oral cavity, and sublingual Administered by lavage into tissue.

In some embodiments, the nucleic acid molecules are transported together by administering a promoter. Promoters are also referred to as polynucleotide function enhancers or gene vaccine promoters. Promoters include U.S. Patent Application 08 / 008,342, filed Jan. 26, 1993, U.S. Patent Application 08 / 029,336, filed March 11, 1993, and U.S. Patent No. 5,593,972, incorporated herein by reference. (Patented January 14, 1997), PCT Application No. PCT / US94 / 00899 (January 26, 1994). Accelerators are also described in PCT Application Nos. PCT / US95 / 1502 (September 28, 1995) and PCT / US95 / 04071 (March 30, 1995), incorporated herein by reference. Promoters administered with a nucleic acid molecule may be administered as a mixture with the nucleic acid molecule, or may be administered simultaneously or before or after the nucleic acid molecule separately. In addition, transfectants and / or replication agents and / or other agents that function as inflammatory agents may be co-administered with or without promoters, such as growth from α-interferon, gamma-interferon, platelets. Factor (PDGF), GC-SF, GM-CSF, TNF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-8, IL-10 and B7.2 etc. LPS analogues, including Murine's growth factor, cytokines and lymphokines, as well as surfactants such as fibroblast growth factor, immune-stimulating complex (ISCOMS), Freund's incomplete adjuvant, monophosphoryl lipid A (MPL) Vesicles and squalenes such as peptides, quinone analogs, squalene and the like, and hyaluronic acid.

In some preferred embodiments the gene constructs of the invention are prepared with or administered with an promoter selected from the group consisting of benzoic acid esters, anilides, amidines, urethanes, hydrochloride salts thereof, such as those belonging to the group of local anesthetics. In some preferred embodiments the promoter can be a compound having one of the following formulas.

Ar-R ¹ -OR ² -R ³

or

Ar-NR ¹ -R ² -R ³

or

R ⁴ -NR ⁵ -R ⁶

or

R ⁴ -OR ¹ -NR ⁷ .

Wherein Ar is benzene, p-aminobenzene, m-aminobenzene, o-aminobenzene, substituted benzene, substituted p-aminobenzene, substituted m-aminobenzene, substituted o-aminobenzene (aminobenzene compound The amino group of may be amino, C ₁ -C ₅ alkylamine, C ₁ -C ₅ , C ₁ -C ₅ dialkylamine, and in the substituted compound the substituents are halogen, C ₁ -C ₅ alkyl, and C _1- C ₅ alkoxy),

R ¹ is C═O,

R ² is C ₁ -C ₁₀ alkyl including branched alkyls,

R ³ is hydrogen, amine, C ₁ -C ₅ alkylamine, C ₁ -C ₅ , C ₁ -C ₅ dialkylamine,

R ² + R ³ is cyclic alkyl, C ₁ -C ₁₀ alkyl substituted cyclicalkyl, cyclic aliphatic amine, C ₁ -C ₁₀ alkyl substituted cyclic aliphatic amine, heterocycle, C ₁ -C ₁₀ alkyl N Form a C ₁ -C ₁₀ alkyl substituted heterocycle including a-substituted heterocycle,

R ⁴ is Ar, R ² or C ₁ -C ₅ alkoxy, cyclic alkyl, C ₁ -C ₁₀ alkyl substituted cyclic alkyl, cyclic aliphatic amine, C ₁ -C ₁₀ alkyl substituted cyclic aliphatic amine, hetero A C ₁ -C ₁₀ alkoxy substituted heterocycle comprising a cycle, a C ₁ -C ₁₀ alkyl substituted heterocycle, a C ₁ -C ₁₀ alkyl N-substituted heterocycle,

R ⁵ is C = NH,

R ⁶ is Ar, R ² or C ₁ -C ₅ alkoxy, cyclic alkyl, C ₁ -C ₁₀ alkyl substituted cyclic alkyl, cyclic aliphatic amine, C ₁ -C ₁₀ alkyl substituted cyclic aliphatic amine, hetero A C ₁ -C ₁₀ alkoxy substituted heterocycle, including a cycle, a C ₁ -C ₁₀ alkyl substituted heterocycle, and a C ₁ -C ₁₀ alkyl N-substituted heterocycle,

R ⁷ is Ar, R ² or C ₁ -C ₅ alkoxy, cyclic alkyl, C ₁ -C ₁₀ alkyl substituted cyclicalkyl, cyclic aliphatic amine, C ₁ -C ₁₀ alkyl substituted cyclic aliphatic amine, hetero a cycle, C ₁ -C ₁₀ alkyl substituted heterocycle and a C ₁ -C ₁₀ alkyl N- substituted C ₁ -C ₁₀ alkoxy substituted heterocycle including a heterocycle.

Examples of esters include benzoic acid esters (piperocaine, meprylcaine, isobucaine, etc.), paraaminobenzoic acid esters (procaine, tetracaine, butetamine, propoxycaine, chloroprocaine, etc.), metaaminobenzoic acid esters (meta Butamine, primacaine and the like), paraethoxybenzoic acid ester (such as parethoxycaine) and the like. Examples of anilide include lidocaine, ethidocaine, mepivacaine, bupivacaine, pyrocaine, prilocaine and the like. Other examples of such compounds include, but are not limited to, dibucaine, benzocaine, diclonin, pramoxine, propparacaine, butacaine, benoxynate, carbocaine, methyl bupivacaine, butacin picrate, phenacaine, diotan, Leucaine, intracaine, nupercaine, metabutoxycaine, pyridocaine, bifenamine, vegetable bicycles (e.g. cocaine, cinnamoylcocaine, truccillin, cocaethylene) and complexes of all these compounds with hydrochloride, etc. There is this.

In a preferred embodiment, the promoter is bupivacaine. The difference between bupivacaine and mepivacaine is that bupivacaine has an N-butyl group instead of the N-methyl group of mepivacaine. The compound has C ₁ -C ₁₀ in N. Compounds can be substituted with halogens, such as procaine and chloroprocaine. Anilide is preferred.

Promoter is administered prior to or concurrently with the gene construct or after the gene construct. Promoters and gene constructs may be formulated in the same composition.

The chemical name of bupivacaine-HCl is 2-piperidinecarboxamide, 1-butyl-N- (2,6-dimethylphenyl) -monohydrochloride, monohydrate, and several sources for pharmaceutical use (Astra Pharmaceutical Products Inc. (Westboro, Mass.), Sanofi Winthrop Pharmaceuticals (New York, NY), Eastman Kodak (Rochester, NY), and the like. Bupivacaine is commercially formulated with or without methylparaben, with or without epinephrine. All such agents can be used. 0.25%, 0.5%, 0.75% concentrations are commercially available for pharmaceutical use and may be used in the present invention. If desired, other ranges of concentrations, among others, may be prepared in concentrations of 0.05 to 1.0% which lead to the desired effect. According to the present invention, about 250 μg to about 10 mg of bupivacaine is administered. In some embodiments, about 250 μg to about 7.5 mg is administered. In some embodiments, about 0.05 mg to about 5.0 mg is administered. In some embodiments, about 0.5 mg to about 3.0 mg is administered. In some embodiments, about 5-50 μg is administered. For example, in some embodiments about 50 μl to about 2 ml, preferably about 50 μl to about 1500 μl, more preferably about 50 μl of 0.5-0.50% bupivacaine-HCl and 0.1% methylparaben in an isotonic pharmaceutical carrier. 1 ml is administered at the same location prior to or at the same time as the vaccine or after the vaccine. Similarly, in some embodiments from about 50 μl to about 2 ml, preferably from about 50 μl to about 1500 μl, more preferably about 1 ml of 0.25-0.50% bupivacaine-HCl in an isotonic pharmaceutical carrier. Before or at the same time as the vaccine or after the vaccine. Bupivacaine and any other similar active compound, particularly those belonging to the kin of local anesthetics, may be administered at a concentration that provides the necessary rate of acceleration of obtaining the gene construct of the cell.

In some embodiments of the invention, the promoter is first injected prior to administering the gene construct to the subject. In other words, the promoter is first injected into the subject, for example about 1 week to about 10 days before the gene construct is administered. In some embodiments, the promoter is infused about 1 to 5 days before and after administering the gene construct, and in some embodiments 24 hours. In addition, if it is used, the promoter is administered simultaneously with the gene construct or within minutes before and after the gene construct is administered. Thus, promoters and gene constructs can be combined to produce a single pharmaceutical composition.

In some embodiments, there is no promoter in the formulation because the gene construct is administered without a promoter, that is to say that the administration of the gene construct uses an administration protocol that is not accompanied by the administration of the promoter.

Nucleic acid molecules delivered to cells according to the present invention may function as gene templates for proteins that function as prophylactic and / or therapeutic immunological agents. In a preferred embodiment, the nucleic acid molecule comprises regulatory sequences necessary for transcription and translation of the coding region in the animal cell.

The invention can be used to immunize an individual against all pathogens such as viruses, prokaryotes, unicellular pathogenic organisms and pathogenic eukaryotes such as multicellular parasites. The present invention is particularly useful for immunizing individuals against viruses and pathogens that infect cells against unencapsulated pathogens such as prokaryotes such as gonococci, Listeria, Shigella and the like. The invention is also useful for immunizing an individual against protozoan pathogens that have one stage of their life cycle where they are present as intracellular pathogens. The term "intracellular pathogen" as used herein means that at least part of the breeding or life cycle of a virus or pathogenic organism is in the host cell, in which the pathogenic protein is produced or produced. Table 1 lists the viruses and parts of the genus in which the vaccine according to the invention may be made. DNA constructs comprising a DNA sequence encoding a peptide comprising at least one epitope that is identical or substantially similar to an epitope that appears on a pathogen antigen, such as the antigens listed in this table, are useful in vaccines. Moreover, the present invention is useful for immunizing individuals against prokaryotic and eukaryotic protozoan pathogens as well as other pathogens, including multicellular parasites such as those listed in Table 2.

To prepare genetic vaccines to protect against pathogen infection, genetic material encoding immunogenic proteins that elicit a protective immune response must be included in the genetic construct as the coding sequence of the target. Whether pathogens infect intracellularly (in which case the invention is particularly useful) or extracellularly, not all pathogen antigens will induce a protective response. Since both DNA and RNA can be made relatively small and relatively easy, the present invention has the additional advantage of being able to inoculate with multiple pathogen antigens. Gene constructs used in genetic vaccines may include genetic material encoding a number of pathogen antigens. For example, multiple targets can be provided by including several viral genes in one construct.

Tables 1 and 2 include a list of some of the pathogenic mediators and organisms that can make genetic vaccines against them to protect individuals from infection by them. In some preferred embodiments, the method of immunizing an individual against a pathogen is against HIV or HTLV or HBV.

Another aspect of the invention relates to a method of eliciting a broad based protective immune response on a broad basis for hyperproliferative cells characteristic of hyperproliferative diseases and to treating individuals suffering from hyperproliferative diseases. As used herein, the term "hyperproliferative disease" means diseases and disorders characterized by hyperproliferation of cells. Examples of hyperproliferative diseases are all forms of cancer and psoriasis.

It has been found that injecting a genetic construct comprising a nucleotide sequence encoding an immunogenic “hyperproliferative cell” related protein into a subject results in the production of a protein in the subject's vaccinated cells. As used herein, the term "hyperproliferative associated protein" refers to a protein associated with a hyperproliferative disease. To immunize against a hyperproliferative disease, the subject is administered a genetic construct comprising a nucleotide sequence encoding a protein associated with the hyperproliferative disease.

In order for a hyperproliferative related protein to be an effective immunogenic target, the protein must be produced exclusively or in high concentration only in hyperproliferative cells as compared to normal cells. Target antigens include peptides including at least such proteins, fragments thereof, and epitopes found on such proteins. In certain cases, hyperproliferative related proteins are the product of mutation of the genes encoding the proteins. The mutated gene encodes a protein that is nearly identical to the normal protein, except that it has slightly different amino acid sequences that result in epitopes not found on the normal protein. Such target proteins include tumor genes such as myb, myc, fyn, and proteins encoded by the translocation genes bcr / abl, ras, src, P53, neu, trk, and EGRF. In addition to tumor gene products, target proteins for anticancer treatment and protective curing as target antigens include variable regions of antibodies produced by T cell receptors of various regions of B cell lymphocytes and T cell lymphocytes, which in certain embodiments It is also used as a target antigen for autoimmune diseases. Other tumor related proteins can be used as target proteins, such as proteins found by monoclonal antibody 17-1A and folate binding proteins, as well as proteins found in high concentrations in tumor cells.

Although the present invention can be used to immunize an individual against one or more cancer types, in particular the present invention is useful for prophylactically immunizing an individual who has a predisposition to develop a particular cancer or that has developed a cancer and so is likely to relapse. . Advances in genetic engineering and dermatology make it possible to determine probability and risk judgments for the development of cancer in a subject. Genetic screening and / or family history can be used to predict the likelihood of developing any of several types of cancer in a particular individual.

Similarly, individuals who have already developed cancer, have been treated to fight cancer, or whose illness has become particularly prone to relapse. As part of the treatment regimen, these individuals may be immunized against cancer that has already been diagnosed to prevent recurrence. Thus, individuals whose cancer has developed and are at risk of relapse can be immunized to prepare their immune system to prevent any cancer from appearing in the future, if known.

The present invention provides a method for treating a subject from a hyperproliferative disease. In this method, the injection of the gene construct acts to direct and enhance the individual's immune system to combat hyperproliferative cells that are immunotherapeutic and produce the target protein.

The present invention provides a method of treating patients with autoimmune diseases and disorders by conferring a broad defensive immune response against targets associated with autoimmunity, including cell receptors and cells that produce cell receptors and "self-directed" antibodies. .

T cell mediated autoimmune diseases include rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome, sarcoidosis, insulin dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, Dermatitis, psoriasis, vasculitis, Wegner's granulomatosis, Crohn's disease and ulcerative colitis. Each disease is characterized by T cell receptors that bind to endogenous antigens and initiate inflammatory cascades associated with autoimmune diseases. Vaccination against the variable region of T cells elicits an immune response including CTLs that eliminate T cells.

In rheumatoid arthritis, several specific variable regions (TCRs) of T cells involved in the disease have been characterized. This TCR includes Vβ-3, Vβ-14, Vβ-17 and Vα-17. Thus, vaccination with a DNA construct encoding at least one of these proteins will elicit an immune response that targets T cells involved in rheumatoid arthritis. Howell, M.D., et al., 1991 Proc. Natl. Acad. Sci. USA 88: 10921-10925; Paliard, X., et al., 1991 Science 253: 325-329; Williams, W. V., et al., 1992 J. Clin. Invest. 90: 326-333, which are incorporated herein by reference.

In multiple sclerosis, several specific variable regions of TCR involved in the disease have been characterized. This TCR includes Vβ-7 and Vα-10. Thus, vaccination with a DNA construct encoding at least one of these proteins will elicit an immune response that targets T cells involved in multiple sclerosis. Wucherpfenning, K.W. et al., 1990 Science 248: 1016-1019; Oksenberg, J. R., et al., 1990 Nature: 345: 344-346; These documents are incorporated herein by reference.

In sclerosis, several specific variable regions of TCR involved in the disease have been characterized. These TCRs include Vβ-6, Vβ-8, Vβ-14 and Vα-16, Vα-3C, Vα-7, Vα-14, Vα-15, Vα-16, Vα-2β and Vα-12. Thus, vaccination with a DNA construct encoding at least one of these proteins will elicit an immune response that targets T cells involved in rheumatoid sclerosis.

To treat T cell mediated autoimmune diseases, particularly disease patients for which the variable regions of TCR have not yet been characterized, biopsies of synovial fluid can be performed. Samples of T cells present were taken and the variable region of TCR was identified using standard techniques. Genetic vaccines can be prepared using this information.

B cell mediated autoimmune diseases include lupus erythematosus (SLE), Grav's disease, dystonia, autoimmune hemolytic anemia, autoimmune thrombocytopenia, asthma, Hans globulinemia, primary gallbladder sclerosis and pernicious anemia. Each disease is characterized by antibodies that bind to endogenous antigens and initiate inflammatory cascades associated with autoimmune diseases. Vaccination against the variable region of an antibody will trigger an immune response, including CTLs, to eliminate B cells that produce the antibody.

In order to treat patients with B cell mediated autoimmune disease, variable regions of antibodies involved in autoimmune activity must be identified. A biopsy can be performed to take antibody samples present at the site of inflammation. Variable regions of such antibodies can be identified using standard techniques. Genetic vaccines can be prepared using this information.

In the case of lupus, one house is believed to be DNA. Thus, in patients immunized against lupus, their serum can be screened for anti-DNA antibodies and a vaccine can be prepared comprising a DNA construct encoding the variable region of the anti-DNA antibody found in such serum. have.

Common structural features among the variable regions of both TCRs and antibodies are known. DNA sequences encoding specific TCRs or antibodies are described in Kanat, et al., 1987 Sequence of Proteins of Immunological Interest U.S. And the commonly known methods described in the Department of Health and Human Services, Bethesda MD, incorporated herein by reference. In addition, general methods for cloning functional variable regions from antibodies are described by Chaudhary, V.K., et al., 1990 Proc. Natl. Acad. Sci. USA 87: 1066, incorporated herein by reference.

In addition to using expressible forms of immunoregulatory protein coding sequences to improve genetic vaccines, the present invention relates to improved vaccines using improved attenuated live vaccines and recombinant vectors for transporting foreign genes encoding antigens. will be. Vaccines using recombinant vectors for transporting attenuated live and foreign antigens are described in US Pat. Nos. 4,722,848, 5,017,487, 5,077,044, 5,110,587, 5,112,749, 5,174,993, 5 5,223,424, 5,225,336, 5,240,703, 5,242,829, 5,294,441, 5,294,548, 5,310,668, 5,387,744, 5,389,368, 5,424,065 5,451,499, 5,453,364, 5,462,734, 5,470,734, and 5,482,713, which are incorporated herein by reference. Provided are gene constructs comprising nucleotide sequences encoding immunoregulatory proteins that are operably linked to regulatory factors and can be expressed in a vaccine. Gene constructs are injected into attenuated live and recombinant vaccines to produce the improved vaccines according to the present invention.

The present invention provides an improved method for immunizing an individual comprising transporting the gene construct to a cell of the individual as part of a vaccine composition, including DNA vaccines, attenuated live vaccines and recombinant vaccines. Gene constructs comprise nucleotide sequences encoding immunoregulatory proteins that can be operably linked to regulators and expressed in a vaccine. Improved vaccines produce an enhanced cellular immune response.

In certain aspects of the invention, nucleic acid molecules encoding human immunoregulatory proteins are transported as gene therapeutics without co-administration of immunogenic targets or nucleic acid molecules encoding immunogenic target proteins. Gene therapy results from the ability of immunomodulatory proteins to drive immune responses.

Gene constructs that are transported as genetic therapeutics are nucleic acid molecules encoding human IL-12. Such gene constructs are preferably plasmids. Other nucleic acid based vectors are also contemplated, such as recombinant viruses, recombinant microorganisms and linear nucleic acid molecules, all of which are known to those skilled in the art. It is known that plasmids useful for administration to a subject are administered, absorbed and expressed in tissue. Recombinant viruses contemplated include recombinant vaccinia virus vectors, recombinant adenovirus virus vectors, and recombinant retroviral vectors. Recombinant microorganisms contemplated include recombinant BCG vectors, and recombinant Salmonella vectors. Linear nucleic acid molecules and plasmid DNA molecules can be encapsulated in microspheres and liposomes.

In certain preferred embodiments, the plasmid encoding the human immunoregulatory protein is a genetic construct as described above. In essence, for gene therapeutics encoding human immunoregulatory proteins, the gene therapy compositions do not contain coding sequences for immune target proteins, except that the same composition as described in the genetic immunization compositions and methods and The method can be used.

This disclosure describes gene therapies encoding human immunoregulatory proteins by referring to these gene constructs. The genetic construct comprising a nucleotide sequence encoding a human immunoregulatory protein, operably linked to a regulatory factor necessary for expression, is intended to describe a gene therapy agent. Such constructs can be administered to a subject to ameliorate and / or drive an immune response.

For example, IL-12 can be transported to treat an allergy, cancer, autoimmune disease or infected patient. Gene constructs encoding IL-12 are administered in the presence or absence of a promoter. In certain embodiments, the gene construct is administered with one or more promoters described above. In certain embodiments, the gene construct is transported without any promoter. In certain preferred embodiments, the gene encoding IL-12 is free of any infectious agent. In certain embodiments, the gene constructs are administered using a non-injectable infusion device. In certain embodiments, the gene encoding IL-12 is transported using microinjection. In certain embodiments, the gene encoding IL-12 transports without any solid particles.

The pharmaceutical composition according to the invention, which is a gene vaccine or gene therapy composition, comprises about 1 ng to about 1000 μg of DNA. In certain preferred embodiments, the composition comprises about 10 ng to about 800 μg DNA. In certain preferred embodiments, the composition comprises about 0.1 to about 500 μg DNA. In certain preferred embodiments, the composition comprises about 1 to about 350 μg DNA. In certain preferred embodiments, the composition comprises about 25 to about 250 μg DNA. In certain preferred embodiments, the composition comprises about 100 μg DNA.

Pharmaceutical compositions of the invention are prepared according to the mode of administration used. One skilled in the art can readily prepare pharmaceutical compositions comprising gene constructs. When intravenous infusion is used as the infusion route, isotonic formulation is preferably used. Additives to isotonic solutions generally include sodium chloride, dextrose, mannitol, sorbitol and lactose. In certain cases, isotonic solution, such as phosphate buffered saline, is preferred. Stabilizers include gelatin and albumin. In certain embodiments, vasoconstrictors are added to the formulation. Pharmaceutical formulations according to the invention are provided that are sterile and pyrogen free.

The method of the present invention is useful in both medicine and veterinary medicine. Thus, the present invention relates to the genetic immunization of stray animals, poultry and fish. The methods of the present invention may be particularly useful for mammalian species, including human, cow, sheep, pig, horse, dog, and cat species.

BL1 in humans, a novel immunomodulator. DNA and predicted amino acid sequences are shown in FIG. 14. Such proteins and fragments thereof enhance the immune response when administered in combination with vaccine compositions capable of injecting an immunogen into a subject. The term “immunoregulatory fragment” as used herein refers to a fragment of BL1 that is shorter than the full length sequence shown in FIG. 14 but retains the immunomodulatory activity of the full length compound. According to the present invention, the transport of the BL1 gene sequence results in immune regulation. In certain experiments, protein expression is not detected but the immune response is nevertheless affected by cotransportation, indicating that the transport of BL1 DNA is an important step in regulating and directing the immune response. The disclosures set forth herein disclose the use of BL1 DNA in immunomodulatory compositions and methods regardless of protein production. Thus, the disclosure uses BL1 DNA and a vector comprising the same as an immunomodulatory agent in an immunomodulatory composition useful for modulating and directing an immune response as a primary active agent or as an operating agent administered with an immunogen or DNA encoding an immunogen. It is about a method.

Isolating and purifying the protein encoded by the BL1 DNA, generating a hybridoma that produces an antibody bound to the protein, isolating, sequencing, injecting into the vector, including the expression vector, the cDNA encoding the protein, The protein is recombinantly expressed by injection into a host cell. Coadministration of the vector with the nucleotide sequence encoding the immunogen enhances the immune response to the immunogen.

Discovery of BL1 provides a means of designing and using vaccine protocols that enhance, drive and direct immune responses.

Isolated cDNA encoding BL1 is useful as a starting material in the construction of recombinant expression vectors capable of producing BL1 or immunoregulatory fragments thereof. cDNA is incorporated into vectors, including expression vectors, which are introduced into host cells to recombinantly express the protein. Nucleic acid molecules and fragments thereof can be used as probes to detect the presence of a BL1 coding sequence. Such probes hybridize specifically with the BL1 coding sequence. As used herein, the term “specific BL1 sequence” refers to a sequence unique to BL1. Nucleic acid molecules comprising nucleotide sequences complementary to specific fragments of cDNA encoding BL1 can be used as antisense molecules and primers for inhibiting translation of mRNA and amplifying gene sequences, respectively.

The BL1 and predicted amino acid sequences encoded by cDNA are shown in FIG. 14. The BL1 coding sequence can be conventionally synthesized and the BL1 protein can be produced by recombinant DNA methods or synthesized by standard protein synthesis.

Nucleic acid molecules encoding BL1 can be prepared using standard methods and readily available starting materials. The present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding BL1. The present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of FIG. 14, or an immunoregulatory fragment thereof. In certain embodiments, the nucleic acid molecule consists of a nucleotide sequence encoding BL1. In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence consisting of the coding sequence of FIG. 14. In certain embodiments, the nucleic acid molecule consists of the nucleotide sequence in FIG. 14. Isolated nucleic acid molecules of the invention are useful for preparing constructs and recombinant expression vectors.

Probes or primers specific for BL1 have 16 or more nucleotides, preferably 24 or more nucleotides. Probes or primers are used to screen cDNA libraries using standard hybridization methods.

The cDNA encoding BL1 can be used to design PCR primers or to amplify nucleic acid sequences. PCR methods are commonly performed by those skilled in the art, and their use in diagnostics is known and accepted. Methods of performing PCR methods are described in "PCR Protocols: A Guide to Methods and Applications", Innis, M.A., et al. Eds. Academic Press, Inc. San Diego, Calif. (1990), incorporated herein by reference. Application of the PCR method is described in "Polymerase Chain Reaction", Erlich, H.A., et al., Eds. Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), incorporated herein by reference. Some simple rules design efficient primers. Typical primers are 18 to 28 nucleotides with a 50% to 60% G + C composition. The entire primer is preferably complementary to the sequence to be hybridized. Preferably, the primer produces a PCR product of 100 bp to 2000 bp. However, it is possible to produce PCR products of 50 bp to 10 kb or more.

The PCR method provides 5 'and 3' primers that hybridize the sequences present in the nucleic acid molecule and further provides free nucleotides and enzymes to complementary DNA strands by filling the complementary bases of the nucleotide sequences with free nucleotides between the primers. It is possible to quickly generate multiple copy numbers of nucleotide sequences by generating. The enzyme will fill the complementary sequence adjacent to the primer. When both the 5 'primer and the 3' primer hybridize to nucleotide sequences on the complementary strand of the same fragment of nucleic acid, exponential amplification of certain double stranded products occurs. If only a single primer hybridizes to the nucleic acid molecule, linear amplification produces a single stranded product of variable length.

The present invention relates to a vector or recombinant expression vector comprising a nucleotide sequence encoding BL1 comprising the amino acid sequence of FIG. 14. As used herein, the term “recombinant expression vector” refers to a plasmid, phage, viral particle or other vector, which, when introduced into a suitable host, contains the genetic factors necessary for the direct expression of the coding sequence encoding BL1. do. Those skilled in the art can insert nucleic acid molecules encoding BL1 into expression vectors using standard techniques and readily available starting materials. The coding sequence is operably linked to the necessary regulatory sequence. Expression vectors are known and are readily available. Examples of expression vectors include plasmids, phages, viral vectors, and other nucleic acid molecules that transform host cells and facilitate the expression of coding sequences. In certain embodiments, the recombinant expression vector comprises the nucleotide sequence shown in FIG. 14. The recombinant expression vector of the present invention is preferably a plasmid.

The present invention relates to a host cell comprising a recombinant expression vector comprising a nucleotide sequence encoding BL1. In certain embodiments, the host cell comprises a recombinant expression vector comprising the nucleotide sequence of FIG. 14. Host cells used in known recombinant expression systems for protein production are known and readily available. An example of a homework cell is this. Bacterial cells, such as E. Coli, S. Yeast cells, such as S. cerevisias, S. Insect cells such as S. frugiperda, tissue culture cells of non-human mammals, human tissue culture cells such as Chinese hamster ovary cells and HeLa cells.

In certain embodiments, for example, those skilled in the art can insert DNA molecules into commercial expression vectors for use in known expression systems using known techniques. For example, commercially available plasmid pSE420 (Invitrogen, San Diego, Calif.) It can be used to produce BL1 in Collie. Commercially available plasmid pYES2 (Invitrogen, San Diego, CA) is a yeast strain S. It can be used to produce BL1 in cerevisiae. A commercially available MAXBAC® complete baculovirus expression system (Invitrogen, San Diego, CA) can be used to produce in insect cells. Commercially available plasmid pcDNA I or pcDNA 3 (Invitrogen, San Diego, Calif.) Can be used for production in mammalian cells, such as, for example, Chinese hamster ovary cells. Those skilled in the art can use these commercially available expression vectors and expression systems, or conventional methods for producing hVIP, and readily available starting materials. See Sambrook et al., Molecular Cloning a Laboratory Manual, 2nd Ed. Cold spring Harbor Press (1989), incorporated herein by reference. Thus, the protein of interest can be prepared in both prokaryotic and eukaryotic systems to obtain the protein in a broadly processed form.

Those skilled in the art use other commercially available expression vectors and expression crabs or known methods and generate the vectors using readily purchased starting materials. Expression systems containing the desired regulatory sequences, such as promoters and polyadenylation signals, and preferably enhancers, are readily available and are known in the art for various hosts. See, eg, Shambrook et al., Molecular Cloning a Laboratory Manual, 2nd Ed. Cold Spring Harbor Press (1989).

The expression host containing the DNA encoding the BL1 is used to transform the compatible host, followed by incubation and maintenance under the conditions under which expression of the foreign DNA occurs. The protein of the present invention thus produced is recovered from the medium by lysing the cells or, as the case is known to those skilled in the art. One skilled in the art can isolate the BL1 produced using such expression systems in a known manner. As described above, the method of purifying BL1 from a natural source using an antibody that specifically binds to BL1 can be equally used to purify BL1 produced by recombinant DNA methods.

Examples of genetic constructs include BL1 coding sequences operably linked to a human or a promoter acting on the cell line to which the construct is transfected, as set forth above. Examples of constitutive promoters include promoters from cytomegavirus or SV40. Examples of inducible promoters include mammalian leukemia virus or metallothionein promoters in mice.

In addition to producing BL1 by recombination, an automated peptide synthesizer can also be used to generate BL1. Such methods are known to those skilled in the art and are useful in the case of derivatives with substituents not provided in the production of DNA encoded proteins.

Nucleic acid molecules encoding BL1 can be transported using any one of a variety of transport components, such as direct plasmid administration, recombinant viral expression vectors, or other suitable means of transport, for introduction and expression in an individual or compatible host cell. have. In general, the viral vector may be a DNA virus such as recombinant adenovirus and recombinant vaccinia virus, or an RNA virus such as recombinant retrovirus. Other recombinant vectors include recombinant prokaryotes that can infect cells and express recombinant genes. In addition to recombinant vectors, other transport components are also contemplated, such as encapsulation in liposomes, transferrin mediated transfection and other receptor mediated means. The present invention includes other forms of expression vectors and other suitable means of transport that function equally and are known to those skilled in the art.

The invention also relates to a transgenic and non-human mammal comprising a recombinant expression vector comprising a nucleic acid sequence encoding BL1. Mammalian transgenic and non-human mammals useful for producing recombinant proteins are known, and expression vectors and methods necessary for producing transgenic animals are also known. Generally, transgenic animals include recombinant expression vectors in which the nucleotide sequence encoding BL1 is operably linked to a flow cell specific promoter, so that the coding sequence is expressed only in the flow cell, and the expressed recombinant protein is Collected from milk. Those skilled in the art will be familiar with US Patent No. 4873191 issued to Wagner on October 10, 1989 and US Pat. No. 4736866, issued April 12, 1988 to Reader, all of which are incorporated herein by reference. Standard techniques as taught in can be used to generate transgenic animals that produce BL1. Preferred animals are goats, sheep or rodents, especially rats and mice.

The BL1 protein or expression vector for producing it may be formulated in a pharmaceutical composition.

Pharmaceutical compositions according to the invention comprise a transport component in admixture with a nucleic acid molecule and further comprise a pharmaceutically acceptable carrier or vehicle, for example saline. Any medium can be used to successfully transport the nucleic acid. Those skilled in the art will understand that many pharmaceutically acceptable media that can be used in the present invention can be used. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, A. Osol, incorporated herein by reference in the art. The pharmaceutical composition of the present invention may be administered by any device in which the active substance may migrate to the target cell. In oral administration, since the peptides are digested, parenteral administration, ie intravenous, subcutaneous, transdermal, intramuscular, will typically optimize absorption. Intravenous administration can be performed using an infusion pump. Pharmaceutical compositions of the invention may be formulated in emulsions. Alternatively, they may be formulated as aerosol pharmaceuticals for intranasal or inhaled administration. In some cases, topical administration may be desirable.

Dosage will vary depending on pharmacokinetic properties, dosage form and route, age, health and weight of the recipient, the nature and extent of symptoms, the type of current treatment, and the number of treatments. Typically, the dosage of peptide can be 1 to 3000 mg per 50 kg body weight, preferably 10 to 1000 mg per 50 kg body weight, more preferably 25 to 800 mg per 50 kg body weight. Typically 8 to 800 mg per day is administered to a subject once or six times or sustained release is effective to achieve the desired result. Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous or powdery or oily bases, thickeners and the like may be necessary or desirable. Compositions for oral administration include powders or granules, suppositories or aqueous solutions or non-aqueous media, capsules, sachets or tablets. Thickeners, fragrances, diluents, emulsifiers, dispersants or binders may be preferred. Compositions for parenteral, intravenous, intraretinal or intraventricular administration may include sterile aqueous solutions comprising buffers, diluents and other suitable additives, preferably sterile and pyrogen free. Pharmaceutical compositions suitable for intravenous administration according to the invention are sterile and pyrogen free.

For parenteral administration, the peptides of the invention may be formulated in solution, suspensions, emulsions or lyophilized powders with pharmaceutically acceptable parenteral vehicles. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and fixative oils may also be used. Vehicle or lyophilized powder may contain isotonic agents (eg sodium chloride, mannitol) and chemical stabilizers (eg buffers and preservatives). The formulation is sterilized by conventional methods. For example, parenteral compositions suitable for injection administration are prepared by dissolving 1.5% by weight of active substance in 0.9% sodium chloride solution.

The pharmaceutical composition of the present invention may be administered by any means by which the active substance can reach the site of action in the mammal's body. The pharmaceutical compositions of the present invention can be administered in several ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration can be performed topically (including ocular, vaginal, rectal, nasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drips, subcutaneous, intraperitoneal or intramuscular injection, administration through the lungs, such as inhalation or insufflation, or intravesical or intraventricular administration.

Hybridomas that produce antibodies that bind BL1, and their antibodies, are useful for the isolation and purification of BL1 and protein complexes comprising BL1. In addition, the antibody is a specific inhibitor of BL1 activity. Antibodies that specifically bind to hVIP can be used to purify proteins from natural sources using known techniques and readily available starting materials. Such antibodies can also be used to purify proteins from substances present when the proteins are produced by recombinant DNA techniques.

As used herein, the term “antibody” refers to a complete and intact antibody, Fab fragment thereof and F (ab) ₂ fragment. Complete and intact antibodies include monoclonal antibodies such as murine monoclonal antibodies, chimeric antibodies and humanized antibodies. Antibodies that bind to epitopes are useful for isolating and purifying proteins from both natural sources or presynthetic expression systems using known techniques, such as affinity chromatography (ie, antibodies do not cross react with other proteins). . Such antibodies are useful for detecting the presence of such proteins in a sample and for detecting whether the cell expresses the protein.

The production of antibodies and the organization of protein sequences of complete and intact antibodies, their Fab fragments and F (ab) ₂ fragments, and the genetic sequences encoding such molecules are known and described, for example, in Harlow, E. and D. Lane (19880 ANTIBODIES; A Laboratory Manual, Col Spring Harbor Laboratory, Cold Spring Harbor, NY, hereby incorporated by reference). Remove the spleen of the mouse, isolate the spleen cells and fuse them to the cells of an infinite growth mouse, cultivate hybrid cells, ie hybridomas, and select cells releasing antibodies.Analyze the antibodies, BL1 If specific binding is found, the hybridomas that produce them are cultured to continuously supply antibodies.

The following examples illustrate the invention. This example is intended to illustrate, but not to limit the scope of the invention. In addition, various aspects of the invention may be summarized by the following description. However, this description is not intended to limit the scope of the invention but to various aspects of the invention. Those skilled in the art can readily understand further aspects and embodiments of the present invention.

<Example 1>

The following is a list of constructs used in the present invention. The vector pBabe · puro, which is used as a starting material to generate the various constructs listed below, is constructed in aid and described in Morganstern, J. P., and H. Land, 1990 Nucl. Acids Res. 18912): 3587-3596, incorporated herein by reference. The pBabepuro plasmid is particularly useful for the expression of exogenous genes in mammalian cells. The DNA sequence to be expressed is inserted into the cloning site under the control of the long terminal repeat (LTR) promoter of the Molecular Rat Leukemia Virus (MoMuVL). Plasmids include selective markers for furomycin resistance.

Plasmid pBaVa3-IL-12 is operably linked with a CMV promoter and a 2.7 kb EcoRI genomic fragment encoding a T cell receptor Vα3 region comprising L, V and J fragments cloned into the EcoRI site of pBabepuro Plasmid comprising a -12 coding sequence and an SV40 polyadenylation sequence. Target proteins derived from T cell receptors are useful for immunization and treatment for T cell mediated autoimmune diseases and clonotype T cell lymphoma and leukemia.                 

Plasmid pBa.gagpol-vpr-IL-12 is a plasmid containing gag / pol and vif genes from HIV / MN cloned into pBabepuro. The vpr gene is missing. HIV plasmids encoding HIV target proteins, and plasmids comprising an SV-12 polyadenylation sequence and an IL-12 coding sequence operably linked with a CMV promoter, are useful for immunization and treatment for HIV infection and AIDS. HIV DNA sequences are described in Reiz, M.S., 1992, AIDS Res. human Retro. 8: 1549. The sequence is deposited in GenBank M17449, which is incorporated herein by reference.

Plasmid pM160 is a plasmid containing a 2.3 kb PCR fragment encoding HIV-I / 3B envelope protein and a rev / tat gene cloned into pMAMneoBlue. The nef region is missing. HIV plasmids encoding HIV target proteins, plasmids comprising an IL-12 coding sequence operably linked with a CMV promoter, and an SV 40 polyadenylation sequence are useful for immunization and treatment for HIV infection and AIDS. DNA sequences of HIV-1 / 3B are disclosed in Fisher, A. 1985, Nature 316: 262, which is incorporated herein by reference. The sequence is deposited in GenBank K03455, which is incorporated herein by reference.

Plasmid pBaVL-IL-12 is a PCR fragment encoding a VL region of an anti-DNA antibody cloned into pBabepuro at the XbaI EcoRI site, an IL-12 coding sequence operably linked with a CMV promoter, and an SV 40 polya Plasmid comprising a denylation sequence. Target proteins derived from antibodies are useful for immunization and treatment for B cell mediated autoimmune diseases and chlorotype B cell lymphoma and leukemia. General methods for cloning functional variable regions from antibodies are described in Chaudhary, V.K., et al., 1990 Proc. Natl. Acad. Sci. USA 87: 1066.

Plasmid pOspA-B-IL-12 is a coding region encoding the OspA and OspB antigens of the spiroheta Borrelia burgdorferi, the cause of Lyme disease, cloned at the BamHI and SalI sites in pBabepuro, and the CMV promoter And an IL-12 coding sequence operably linked with the SV40 polyadenylation sequence. Williams, W. V., et al. 1992, DNA and Cell. Biol. 11 (3); 207. Plasmids containing pathogen genes encoding target proteins are useful for immunization against Lyme disease.

Plasmid pBa.Rb-G-IL-12 is a PCR-producing fragment encoding a rabies G protein cloned to the BamHI site in pBabepuro, and an IL-12 coding sequence operably linked to the CMV promoter and SV40 polyadenylation sequence. It is a plasmid containing. Plasmids containing the above pathogen gene encoding rabies G protein are useful for immunization of rabies. DNA sequences are disclosed in GenBank M32571, incorporated herein by reference. See Anilionis, A. et al., 1981, Nature, 294: 275, which is incorporated herein by reference.

Plasmid pBaHPV-L1 is a PCR-producing fragment encoding the L1 capsid protein of human papillomavirus (HPV), including HPV strains 16, 18, 31 and 33 cloned at the BamHI and EcoRI sites in pBabepuro, and CMV Plasmid comprising an IL-12 coding sequence operably linked with a promoter and an SV40 polyadenylation sequence. Such plasmids are useful for immunization against HPV infection and resulting cancer. DNA sequences are disclosed in GenBank M15781, which is incorporated herein by reference. Howley, P., 1990 Fields Virulogy, Volume 2, Eds., Incorporated herein by reference; Channock, R.M. et al. Chapter 58: 1625; and Shah, K. and P. Howley, 1990 Fiels Virology, Volume 2, Eds .; Channock, R.M. et al. Chapter 59].

Plasmid pBaHPV-L2-IL-12 produced a PCR encoding the L2 capsid protein of human papillomavirus (HPV), including HPV strains 16, 18, 31 and 33 cloned at the BamHI and EcoRI sites in pBabepuro Fragment, and a plasmid comprising an IL-12 coding sequence operably linked with a CMV promoter and an SV40 polyadenylation sequence. Such plasmids are useful for immunization against HPV infection and resulting cancer. DNA sequences are disclosed in GenBank M15781, which is incorporated herein by reference. Howley, P., 1990 Fields Virulogy, Volume 2, Eds., Incorporated herein by reference; Channock, R.M. et al. Chapter 58: 1625; and Shah, K. and P. Howley, 1990 Fiels Virology, Volume 2, Eds .; Channock, R.M. et al. Chapter 59].

Plasmid pBaMNp7-IL-12 is a PCR-induced fragment encoding a p7 coding region, including HIV MN gag (core protein) cloned into pBabepuro at the BamHI site, and an IL-12 coding sequence operably linked with a CMV promoter , And a plasmid comprising the SV 40 polyadenylation sequence. Such plasmids containing HIV viral genes encoding HIV target proteins are useful for immunization and treatment for HIV infection and AIDS. Reiz, M.S., 1992 AIDS Res., Incorporated herein by reference. Human Retro. 8: 1549. Such sequences are disclosed in GenBank M17449, which is incorporated herein by reference.

Plasmid pGA733-2-IL-12 is a pCDM8 vector from colorectal cancer cell line SW948 (Seed, B. and A. Aruffo, 1987 Proc. Natl. Acad. Sci. USA 84: 3365). And a plasmid comprising a GA733-2 tumor surface antigen cloned at the BstXI site within, and an IL-12 coding sequence operably linked with a CMV promoter and an SV40 polyadenylation sequence. Tumor related target proteins are examples of target proteins useful for immunization and treatment for hyperproliferative diseases such as cancer. GA733-2 antigen is a useful target antigen for colon cancer. GA733 antigens are described in Szala, S. et al., 1990 Proc. Natl. Acad. Sci. USA 87: 3542-3546.

Plasmid pT4-pMV7-IL-12 comprises a cDNA encoding a human CD4 receptor cloned into a pMv7 vector at an EcoRI site, an IL-12 coding sequence operably linked with a CMV promoter, and an SV 40 polyadenylation sequence Plasmid. CD4 target proteins are useful for immunization and treatment for T cell lymphoma. Plasmid pT4-pMV7 is available from AIDS Inventory No. 158.

Plasmid pBa.RAS-IL-12 was first subcloned from pZIPneoRAS and was cloned into pBabepuro at the BamHI site, the IL-12 coding sequence operably linked with the CMV promoter, and the SV 40 polyadenylation sequence. It is a plasmid containing. The ras target protein is an example of a molecule for cytoplasm signal. Cloning methods of ras are described in Weinberg, 1984, Mol. Cell. Biol. 4: 1577. Ras coding plasmids are useful for the immunization and treatment of cancer, in particular ras-related cancers such as bladder cancer, muscle cancer, lung cancer, brain tumors and bone cancer.

Plasmid pDJGA733-IL-12 is a plasmid comprising a GA733 tumor surface antigen cloned into pBabepuro at the BamHI site, an IL-12 coding sequence operably linked with a CMV promoter, and an SV 40 polyadenylation sequence. Tumor related target proteins are examples of target proteins useful for immunization and treatment for hyperproliferative diseases such as cancer. GA733 antigen is a useful target antigen for colon cancer.

Plasmid pBa.MNp55-IL-12 is a PCR-induced fragment encoding a p55 coding region, including HIV MN gag (core protein) cloned into pBabepuro at the BamHI site, and an IL-12 coding sequence operably linked with a CMV promoter. , And a plasmid comprising the SV 40 polyadenylation sequence. Such plasmids comprising HIV viral genes encoding HIV target proteins are useful for immunization and treatment for HIV infection and AIDS. Reiz, M.S., 1992 AIDS Res., Incorporated herein by reference. Human Retro. 8: 1549. Such sequences are disclosed in GenBank M17449, which is incorporated herein by reference.

Plasmid pBa.MNp24-IL-12 is operable with PCR-induced fragments from the pMN-SF1 template encoding the p24 coding region, including the entire HIV MN gag coding region cloned into pBabepuro at the BamHI and EcoRI sites, and the CMV promoter Plasmid comprising the linked IL-12 coding sequence, and the SV 40 polyadenylation sequence. Such plasmids comprising HIV viral genes encoding HIV target proteins are useful for immunization and treatment for HIV infection and AIDS. Reiz, M.S., 1992 AIDS Res., Incorporated herein by reference. Human Retro. 8: 1549. Such sequences are disclosed in GenBank M17449, which is incorporated herein by reference.

Plasmid pBa.MNp17-IL-12 is a PCR-induced fragment encoding a p17 coding region including HIV MN gag (core protein) cloned into pBabepuro at the BamHI and EcoRI sites, and IL-12 operably linked to the CMV promoter. Plasmid comprising the coding sequence, and the SV 40 polyadenylation sequence. Such plasmids comprising HIV viral genes encoding HIV target proteins are useful for immunization and treatment for HIV infection and AIDS. Reiz, M.S., 1992 AIDS Res., Incorporated herein by reference. Human Retro. 8: 1549. Such sequences are disclosed in GenBank M17449, which is incorporated herein by reference.

Plasmid pBa.SIVenv-IL-12 worked with 2.7 kb PCR generated fragments amplified from constructs containing SIV 239 in pBR322 and cloned into the BamHI and EcoRI sites in pBabepuro, and the CMV promoter and SV40 polyadenylation sequence. Plasmid comprising an IL-12 coding sequence possibly linked. The plasmids are available from AIDS Research and Reference Regents Program (List No. 210).

Plasmid pcTSP / ATK.env-IL-12 is a PCR generated fragment that encodes a complete HTLV envelope coding region from HTLV-1 / TSP and / ATK isolates subcloned into pcDNA1 / neo vectors, and CMV promoter and SV40 polyade Plasmid comprising an IL-12 coding sequence operably linked with a nilation sequence. Plasmid pcTSP / ATK.env is described in the literature [1988, Proc. Natl. Acad. Sci. USA 85: 3599. HTLV envelope target proteins are useful for immunization and treatment for infection by T HTLV and T cell lymphoma.

Plasmid pBa.MNgp160-IL-12 was operable with 2.8 kb PCR generated fragments amplified from constructs containing MNenv in pSP72 and cloned into BamHI and EcoRI sites in pBabepuro, and with CMV promoter and SV40 polyadenylation sequence. Plasmid comprising an IL-12 coding sequence. Reiz, M.S., 1992 AIDS Res. Human Retro. 8: 1549. Such sequences are disclosed in GenBank M17449, which is incorporated herein by reference. Such plasmids comprising HIV viral genes encoding HIV target proteins are useful for immunization and treatment for HIV infection and AIDS.

Plasmid pC.MNp55-IL-12 is a plasmid containing a 1.4 kb PCR generated fragment amplified from the gag region of the MN isolate and cloned into the pCEP4 vector. This plasmid comprising an HIV viral gene encoding an HIV target protein, and an IL-12 coding sequence operably linked with a CMV promoter and an SV40 polyadenylation sequence, is useful for immunization and treatment for HIV infection and AIDS. Reiz, M.S., 1992 AIDS Res. Human Retro. 8: 1549. Such sequences are disclosed in GenBank M17449, which is incorporated herein by reference.

Plasmid pC.Neu-IL-12 was operably linked to a 3.8 kb DNA fragment containing a human neu oncogene coding region cut from the LTR-2 / erbB-2 construct and subcloned into the pCEP4 vector, and the CMV promoter. Plasmid comprising an IL-12 coding sequence, and an SV 40 polyadenylation sequence. pC.Neu plasmids are reported in DiFiore, 1987, Science 237: 178, which is incorporated herein by reference. neu oncogenic target proteins are examples of growth factor receptors useful as target proteins in the immunization and treatment of cancer, particularly hyperproliferative diseases such as colon cancer, breast cancer, lung cancer and brain cancer.

Plasmid pC.RAS-IL-12 is first operably cloned from pZIPneoRAS and contains 1.4 kb of DNA fragment containing the ras oncogene coding region cloned to the BamHI site in pCEP4, and the CMV promoter and SV40 polyadenylation sequence. Plasmid comprising the linked IL-12 coding sequence. pC.RAS plasmids are described in Weinberg, 1984, Mol. Cell. Biol. 4: 1577. The ras target protein is an example of a molecule for cytoplasm signal. Ras coding plasmids are useful for the immunization and treatment of cancer, in particular ras-related cancers such as bladder cancer, muscle cancer, lung cancer, brain tumors and bone cancer.

Plasmid pNLpuro-IL-12 is a plasmid containing HIV gag / pol and SV40-puro inserts. HIV plasmids encoding HIV target proteins, plasmids comprising an IL-12 coding sequence operably linked with a CMV promoter, and an SV 40 polyadenylation sequence are useful for immunization and treatment for HIV infection and AIDS.

<Example 2>

Plasmid pCSIL-12 comprises an IL-12 coding sequence operably linked with a CMV promoter, and an SV 40 polyadenylation sequence.

<Example 3>

Compositions according to embodiments of the invention could be prepared by combining one of the following plasmids and plasmid pCSIL-12 together.

Plasmid pBa.Vα3 is a 7.8 kb plasmid containing a 2.7 kb EcoRI genomic fragment encoding a T cell receptor Vα3 region comprising L, V and J fragments, cloned into the EcoRI site of pBabepuro. Target proteins derived from T cell receptors are useful for immunization and treatment for T cell mediated autoimmune diseases and clonotype T cell lymphomas and leukemias.

Plasmid pBa.gagpol-vpr is a 9.88 kb plasmid containing gag / pol and vif genes from HIV / MN cloned into pBabe.puro. The vpr gene is missing. Plasmids containing HIV viral genes encoding HIV target proteins are useful for immunization and treatment for HIV infection and AIDS. HIV DNA sequences are described in Reiz, M.S., 1992, AIDS Res. human Retro. 8: 1549. The sequence is deposited in GenBank M17449, which is incorporated herein by reference.

Plasmid pM160 is a 11.0 kb plasmid containing a 2.3 kb PCR fragment encoding HIV-I / 3B envelope protein and a rev / tat gene cloned in pMAMneoBlue. The nef region is missing. Plasmids containing HIV viral genes encoding HIV target proteins are useful for immunization and treatment for HIV infection and AIDS. DNA sequences of HIV-1 / 3B are disclosed in Fisher, A. 1985, Nature 316: 262, which is incorporated herein by reference. The sequence is deposited in GenBank K03455, which is incorporated herein by reference.

Plasmid pBaVL is a 5.4 kb plasmid containing a PCR fragment encoding the VL region of an anti-DNA antibody cloned at the XbaI and EcoRI sites in pBabepuro. Target proteins derived from antibodies are examples of target proteins useful for immunization and treatment for B cell mediated autoimmune diseases and clonotype B cell lymphoma and leukemia. General methods for cloning functional variable regions from antibodies are described in Chaudhary, V.K., et al., 1990 Proc. Natl. Acad. Sci. USA 87: 1066.

Plasmid pOspA.B is a 6.84 kb plasmid containing coding regions encoding the OspA and OspB antigens of the spirochete Borrelia burgdorferi, the cause of Lyme disease cloned at the BamHI and SalI sites in pBabepuro. . Williams, W. V., et al. 1992, DNA and Cell. Biol. 11 (3); 207. Such plasmids containing these pathogen genes encoding target proteins are useful for immunization against Lyme disease.

Plasmid pBa.Rb-G is a 7.10 kb plasmid containing a PCR generated fragment encoding a rabies G protein cloned at the BamHI site in pBabepuro. Plasmids containing the above pathogen gene encoding rabies G protein are useful for immunization of rabies. DNA sequences are disclosed in GenBank M32751, which is incorporated herein by reference. See Anilionis, A. et al., 1981, Nature, 294: 275, which is incorporated herein by reference.

Plasmid pBaHPV-L1 comprises a PCR generating fragment encoding the L1 capsid protein of human papillomavirus (HPV), including HPV strains 16, 18, 31 and 33 cloned at the BamHI and EcoRI sites in pBabepuro 6.80 kb of plasmid. Such plasmids are useful for immunization against HPV infection and resulting cancer. DNA sequences are disclosed in GenBank M15781, which is incorporated herein by reference. Howley, P., 1990 Fields Virulogy, Volume 2, Eds., Incorporated herein by reference; Channock, R.M. et al. Chapter 58: 1625; and Shah, K. and P. Howley, 1990 Fiels Virology, Volume 2, Eds .; Channock, R.M. et al. Chapter 59].

Plasmid pBaHPV-L2 comprises a PCR generating fragment encoding the L2 capsid protein of human papillomavirus (HPV), including HPV strains 16, 18, 31 and 33 cloned at the BamHI and EcoRI sites in pBabepuro 6.80 kb plasmid. Such plasmids are useful for immunization against HPV infection and resulting cancer. DNA sequences are disclosed in GenBank M15781, which is incorporated herein by reference. Howley, P., 1990 Fields Virulogy, Volume 2, Eds., Incorporated herein by reference; Channock, R.M. et al. Chapter 58: 1625; and Shah, K. and P. Howley, 1990 Fiels Virology, Volume 2, Eds .; Channock, R.M. et al. Chapter 59].

Plasmid pBa.MNp7 is a 5.24 kb plasmid containing a PCR generated fragment encoding a p7 coding region, including an HIV MN gag (core protein) sequence cloned to the BamHI site in pBabepuro. Such plasmids containing these HIV viral genes encoding HIV target proteins are useful for immunization and treatment for HIV infection and AIDS. Reiz, M.S., 1992 AIDS Res., Incorporated herein by reference. Human Retro. 8: 1549. Such sequences are disclosed in GenBank M17449, which is incorporated herein by reference.

Plasmid pGA733-2 is a 6.3 kb plasmid containing GA733-2 tumor surface antigen cloned from colorectal cancer cell line SW948 to the BstXI site in the pCDM8 vector (Seed B. and A. Aruffo, 1987 Proc. Natl. Acad. Sci. USA 84: 3356, incorporated herein by reference. Tumor related target proteins are examples of target proteins useful for immunization and treatment for hyperproliferative diseases such as cancer. GA733-2 antigen is a useful target antigen for colon cancer. GA733 antigens are described in Szala, S. et al., 1990 Proc. Natl. Acad. Sci. USA 87: 3542-3546.

Plasmid pT4-pMV7 is a 11.5 kb plasmid containing cDNA encoding the human CD4 receptor cloned to the EcoRI site in the pMV7 vector. CD4 target proteins are useful for immunization and treatment for T cell lymphoma. Plasmid pT4-pMV7 is available from AIDS Inventory List 158.

Plasmid pDJGA733 is a 5.1 kb plasmid containing GA733 tumor surface antigen cloned at the BamHI site in pBabepuro. Tumor related target proteins are examples of target proteins useful for immunization and treatment for hyperproliferative diseases such as cancer. GA733 antigen is a useful target antigen for colon cancer.

Plasmid pBa.RAS is a 6.8 kb plasmid containing the ras coding region first subcloned from pZIPneoRAS and cloned to the BamHI site in pBabepuro. The ras target protein is an example of a molecule for cytoplasm signal. Cloning methods of ras are described in Weinberg, 1984, Mol. Cell. Biol. 4: 1577. Ras coding plasmids are useful for the immunization and treatment of cancer, in particular ras-related cancers such as bladder cancer, muscle cancer, lung cancer, brain tumors and bone cancer.

Plasmid pBa.MNp55 is a 6.38 kb plasmid containing a PCR generated fragment encoding a p55 coding region, including an HIV MN gag precursor (core protein) sequence cloned to the BamHI site in pBabepuro. Such plasmids containing these HIV viral genes encoding HIV target proteins are useful for immunization and treatment for HIV infection and AIDS. Reiz, M.S., 1992 AIDS Res., Incorporated herein by reference. Human Retro. 8: 1549. Such sequences are disclosed in GenBank M17449, which is incorporated herein by reference.

Plasmid pBa.MNp24 is a 5.78 kb plasmid containing PCR-induced fragments from the pMN-SF1 template encoding the p24 coding region, including the entire HIV MN gag coding region cloned into pBabepuro at the BamHI and EcoRI sites. Such plasmids comprising HIV viral genes encoding HIV target proteins are useful for immunization and treatment for HIV infection and AIDS. Reiz, M.S., 1992 AIDS Res., Incorporated herein by reference. Human Retro. 8: 1549. Such sequences are disclosed in GenBank M17449, which is incorporated herein by reference.

Plasmid pBa.MNp17 is a 5.5 kb plasmid containing PCR induced fragments encoding p17 coding regions including HIV MN gag (core protein) cloned into pBabepuro at the BamHI and EcoRI sites. Such plasmids comprising HIV viral genes encoding HIV target proteins are useful for immunization and treatment for HIV infection and AIDS. Reiz, M.S., 1992 AIDS Res., Incorporated herein by reference. Human Retro. 8: 1549. Such sequences are disclosed in GenBank M17449, which is incorporated herein by reference.

Plasmid pBa.SIVenv is a 7.8 kb plasmid containing a 2.71 kb PCR generated fragment amplified from a construct containing SIV 239 in pBR322 and cloned into the BamHI and EcoRI sites in pBabepuro. The plasmids are available from AIDS Research and Reference Regents Program (List No. 210).

Plasmid pcTSP / ATK.env is an 8.92 kb plasmid comprising PCR generated fragments encoding complete HTLV envelope coding regions from HTLV-1 / TSP and / ATK isolates subcloned into pcDNA1 / neo vectors. Plasmid pcTSP / ATK.env is described in the literature [1988, Proc. Natl. Acad. Sci. USA 85: 3599. HTLV envelope target proteins are useful for immunization and treatment for infection by T HTLV and T cell lymphoma.

Plasmid pBa.MNgp160 is a 7.9 kb plasmid containing a 2.8 kb PCR generated fragment amplified from a construct containing MNenv in pSP72 and cloned into the BamHI and EcoRI sites in pBabepuro. Reiz, M.S., 1992 AIDS Res., Incorporated herein by reference. Human Retro. 8: 1549. Such sequences are disclosed in GenBank M17449, which is incorporated herein by reference. Such plasmids containing these HIV viral genes encoding HIV target proteins are useful for immunization and treatment for HIV infection and AIDS.

Plasmid pC.MNp55 is a 11.8 kb plasmid containing a 1.4 kb PCR generated fragment amplified from the gag region of the MN isolate and cloned into the pCEP4 vector. Such plasmids containing these HIV viral genes encoding HIV target proteins are useful for immunization and treatment for HIV infection and AIDS. Reiz, M.S., 1992 AIDS Res. Human Retro. 8: 1549. Such sequences are disclosed in GenBank M17449, which is incorporated herein by reference.

Plasmid pC.Neu is a 14.2 kb plasmid containing a 3.8 kb DNA fragment containing the human neu oncogene coding region cut from the LTR-2 / erbB-2 construct and subcloned into the pCEP4 vector. pC.Neu plasmids are reported in DiFiore, 1987, Science 237: 178, which is incorporated herein by reference. neu oncogenic target proteins are examples of growth factor receptors useful as target proteins in the immunization and treatment of cancer, particularly hyperproliferative diseases such as colon cancer, breast cancer, lung cancer and brain cancer.

Plasmid pC.RAS is an 11.7 kb plasmid comprising a 1.4 kb DNA fragment containing a ras oncogene coding region, first subcloned from pZIPneoRAS and cloned into the BamHI site in pCEP4. pC.RAS plasmids are described in Weinberg, 1984, Mol. Cell. Biol. 4: 1577. The ras target protein is an example of a molecule for cytoplasm signal. Ras coding plasmids are useful for the immunization and treatment of cancer, in particular ras-related cancers such as bladder cancer, muscle cancer, lung cancer, brain tumors and bone cancer.

Plasmid pNLpuro is a 15 kb plasmid containing HIV gag / pol and SV40-puro inserts. Plasmids containing HIV viral genes encoding HIV target proteins are useful for immunization and treatment for HIV infection and AIDS.

<Example 4>

It may be important to specifically regulate the immune response to improve the vaccine. The type of immune response (Th1 vs Th2) has been reported to be predominant in models of various diseases including infectious diseases, autoimmune diseases, and allergies. In order to induce a strong and stable cell mediated immune response to HIV infection, the use of immunomodulators and immunomodulators such as cytokines in combination with immunization enhances cellular immune responses and promotes antigen-dependent immune responses of the Th2-Th1 type. To be adjustable.

To engineer the immune response in vivo, the human cytokine gene, IL-12 gene or GM-CSF gene was co-transported with the HIC construct. The immune response induced by the co-transportation of these three genes with HIV-1 DNA vaccine was verified and the induction of cellular immunization, specifically the antiviral CTL response, was studied.

Genes of IL-12 and GM-CSF were cloned individually into expression vectors under the control of the CMV promoter. The gene plasmid expression cassette was then injected into mice with a DNA vaccine cassette for HIV-1 (referenced in US Application No. 08/642045, filed May 6, 1996, incorporated herein by reference). The immunological effects of the concomitant immunization of the genetic adjuvant cassette on the magnitude of the antigen specific immune response were analyzed. Co-transportation of IL-12 was observed to decrease the humoral response, whereas IL-12 co-immunization was observed to mildly enhance the humoral response. Concomitant immunization with IL-12 or GM-CSF observed an increase in antigen specific T helper cell proliferation. It was important to observe meaningful CTL induction by co-administration of genes for IL-12 using direct CTL analysis. In contrast, the genes for GM-CSF in this study observed little effect on CTL induction. These results demonstrated the utility of DNA vaccines for the controlled production of certain immune responses. In addition, they demonstrated the utility of methods that reveal basic immunological functions with molecular specific trends.

<Materials and Methods>

Rat:

Bab / c female mice, 6-8 weeks old, were purchased from Harlan Sprague Dulissa (Indianapolis, Indiana). Mice were raised in a room where the temperature was controlled and the light cycled. They are under the leadership of the National Institutes of Health and the University of Pennsylvania.

Reagents:

DNA vaccine preparations pCMN160, pCGN160, pCGag / Pol were prepared. IL-12 and GM-CSF genes were cloned and inserted into expression vectors with CMV promoter present. Recombinant vaccinia (vMN462. VVK1, VV; gag and vSC8) was obtained from the National Institutes of Health AIDS Research and Reference Regents program.

DNA inoculation:                 

Easy DNA inoculation was used to increase protein expression levels in vivo from plasmid transported genes in vivo. Specifically, 100 μl of a solution containing 0.25% Bupivacaine-HCl (Sigma, St. Louis) was injected into a muscle of BALB / C mice using a 27 gauge needle. After 2 days, 50 g of the desired DNA construct in phosphate buffer was injected into the muscle at the same site as bupivacaine. Selected plasmids were mixed prior to injection for co-administration of various gene expression cassettes.

FACS analysis:

Cells (1 × 10 ⁵ ) were washed three times with FACS buffer (PBS containing 1% BSA and 0.1% sodium azide) and FITC and / or PE-conjugated for 30 minutes on ice under saturation conditions. Incubate with mAb. After washing three times with FACS buffer, cells were analyzed using FACScan (Becton Dickinson) ELISA.

ELISA:

2 μg / ml of gp120 or gp41 (Infracell, Cambridge, Mass.) In 0.1 M carbonate-bicarbonate buffer (pH 9.5) was adsorbed onto microtiter wells at 4 ° C. overnight as described above. Plates were washed with PBS-0.05% Tween-20, blocked with 3% BSA in PBS containing 0.05% Tween-20 for 1 hour at 37 ° C., and then HRP-conjugated goat IgG or IgA (Sigma) Incubation with dilution according to the manufacturer's instructions, St. Louis, Missouri. Plates were washed and developed with TM Blue Buffer (Sigma). OD 450 nm was read on a Dinatech MR5000 plate reader.

T cell proliferation assay:

Lymphocytes harvested from the spleen of mice were prepared. Isolated cell suspension was resuspended at a concentration of 1 × 10 ⁶ cells / ml. 100 μl aliquots containing 1 × 10 ⁵ cells were immediately added to each well of a flat bottom 96 well microtiter plate. 10 μl of protein was added to the wells in triplicate at 20 μg / ml. Cells were incubated at 37 ° C. in 5% CO ₂ for 3 days. After 1 μCi of ³ H thymidine was added to each well, the cells were incubated at 37 ° C. for 12-18 hours. The plates were harvested and the incorporated ³ H thymidine measured in a beta plate reader (Walaksa, Turku, Finland). To confirm that the cells are active, 10 g / ml PHA was used as a polyclonal promoter positive control.

Cytotoxic T Lymphocyte Assay:

CTL analysis releasing chromium ( ⁵¹ Cr) for 5 hours was performed. Lymphocytes were harvested from the spleen, red blood cells removed and washed several times with fresh medium to prepare effector cells. Vaccinia infected targets were prepared by infecting 3 × 10 ⁶ p815 cells at 37 ° C. for 16 hours. Target cells were labeled with 100 μCi / mL of Na ₂ ⁵¹ CrO ₄ for 90 minutes and the promoted spleen cells were used for incubation at 37 ° C. for 4-7 hours. CTLs were tested at effector: target (E: T) ratios ranging from 50: 1 to 12.5: 1. Supernatants were harvested and counted on an LKB clinygamma gamma counter. The insolubility in water was determined from the following formula.

100 × {(experimental-voluntary release) / (maximum release-voluntary release)}

Maximal release was determined by lysing the target cells in medium containing 1% Triton X-100. The assay is not considered valid if the value for the "voluntary release" factor exceeds 20% of the "maximum release".

<Result>

Phenotyping of the spleen of rats:

After the co-inoculation was performed, the spleen collected from each experimental group was observed to be different. Thus, spleens collected from all immunized animals were weighed and visually observed. The spleen mass of these animals is shown in Figure 1A. The spleen from mice injected with the single formulation control was similar to the mass of the non-immunized control mice (about 100 mg), whereas the spleen from mice injected with the Gag / Pol + IL-12 gene was about 3 times the control spleen mass. It was a ship. On the other hand, the spleen of Gag / Pol + GM-CSF immunized mice was not enlarged. It is interesting that the spleen does not significantly expand when immunized with Gag / Pol or IL-12 alone. Spleen enlargement results only when co-administered with the antigen and the IL-12 gene cassette suggest that this phenomenon is a combined effect of the two gene products. Furthermore, as shown in FIG. 1B, the number of lymphocytes derived from Gag / Pol + IL-12 spleens exceeded three times that of control spleens. In other words, the spleens of Gag / Pol and GM-CSF immunized mice did not cause any significant increase in lymphocyte counts above the control splenic cell count. Representative spleen photographs are shown in FIG. 2. Depending on their mass, the antigen + IL-12 spleen was observed to be several times larger than the other spleens. It has been reported that the p35 chain of IL-12 is constitutively expressed in several cell types. Thus, only a single p40 chain and DNA immunogen can exert an effect. To test this, each of the two IL-12 heterodimer genes (p35 and p40) was coadministered with Gag / Pol. As shown in FIG. 3, no enlargement of the spleen size was observed in any case. The data indicated that co-administration of the DNA vaccine and two p35 and p40 IL-12 genes increased the size of the spleen and correspondingly increased the number of spleen cells. This data demonstrates that the plasmids are injected into the same cells in vivo and regulate the transcription of all three components, namely the p35 chain and the p40 chain, and the specific antigen, leading to the observed biological changes.

-FACS method:

To determine the cell composition of the expanded spleen, FACS analysis was performed. Table 3 shows FACS results obtained by bistaining spleen cells with antibodies to CD3 and antibodies to B220, CD4 and CD8. As described, B220 in the group immunized with invelop + IL-12 or Gag / Pol + IL-12 constructs (17.46% and 21.62%, respectively) relative to the percentage of B cells in unimmunized control spleen (25.43%). A slight decrease in the positive B cell population density was observed. In addition, the percentage of CD8 + T cells in the group immunized with Invelop + IL-12 or Gag / Pol + IL-12 constructs (21.72% and 16.88%, respectively) compared to the percentage in the non-immunized group (13.69%). This somewhat increased.

-Humoral reaction                 

Antisera from immunized mice were recovered and analyzed for specific antibody response to HIV-1 antigen by ELISA. 4 shows ELISA results from samples collected 28 days after immunization. At a dilution ratio of 1: 100, serum from the group immunized with pCEnv + pCGM-CSF showed an antibody response to HIV-1 gp120 protein that exceeded the value in the group immunized with pCEnv alone. On the other hand, the group immunized with pCEnv + pCIL-12 showed significantly lower humoral response during the same period. In repeated experiments, IL-12 generally inhibits specific antibody responses by 1-20%, while GM-CSF has the opposite effect. This humoral response can be associated with observed changes in B cell numbers in spleen cells, as revealed by FACS analysis.

T cell proliferation

Activation and proliferation of T helper lymphocytes play an important role in inducing both humoral immune responses through an increase in antigen-activated B cells and cellular immune responses through an increase in CD8 + cytotoxic T lymphocytes. Two weeks after DNA immunization, spleens were harvested from immunized mice and their lymphocytes isolated. These cells were then tested for T cell proliferation as described above. 5 shows the results of proliferation assays for mice immunized with DNA vaccines encoding HIV-1 gag / pol (pCGag / Pol) and mice immunized with pCGag / Pol and IL-12 or GM-CSF. Lectin PHA of 20 g / ml recombinant p55 protein was used as a polyclonal promoter positive control. As shown, low basal levels of proliferation were observed in controls from spleens of natural mice at a palpation index of 1.2 at a dilution of 1: 2 and immunized with pCGag / Pol only at palpation index 9.2 at a 1: 2 dilution. Proper levels of growth were observed in the groups. In the group immunized with pCGag / Pol and IL-12 genes, a dramatic increase in proliferation was observed with a facilitation index of 17.1, and in the group immunized with pCGag / PoL and GM-CSF genes, a dramatic increase in growth was observed with a facilitation index of 15.6.

CTL assays not promoted in vitro

In order to observe the enhancement of cell activity, a direct CTL assay was performed. CTL assays were performed on spleen cells harvested from immunized mice as described above, without in vitro palpation induced on spleen cells. The assay was performed by measuring chromium release from targets infected with specific and nonspecific vaccinia on the day of spleen harvesting. A dramatic increase in specific CTL activity from Gag / PoL + IL-12 immunized spleen cells was observed (FIG. 6). Controls immunized with the IL-12 gene cassette alone did not show cost-effectiveness of target cells above the basal level. In addition, a low level (3%) of non-solubility was observed when the effector: target ratio was immunized with Gag / Pol only at 50: 1. In contrast, 62% of the nasal infiltrates were observed with Gag / Pol + IL-12 at 50: 1 effector: target ratio and 9% at 12.5: 1 effector: target ratio. . In contrast, immunization with Gag / Pol and GM-CSF plasmids showed no detectable CTL activity. Similar results were observed from mice immunized with HIV-1 envelope constructs and cytokine genes (FIG. 7). Colonies immunized with envelope alone and with envelope + GM-CSF showed low levels of specific CTLs at 4% and 1%, respectively. On the other hand, with Envelop + IL-12, a dramatic increase in CTL activity was observed with 59% dissolution. For concomitant administration of Gag / Pol and envelope (FIGS. 6 and 7), the same CTL analysis performed on targets made with inappropriate antigen-expressing vaccinia did not result in a significant CTL response. Thus, dramatic enhancement of CTL activity from antigen and IL-12 DNA immunization results was not attributable to NK activity as in antigen specific results.

<Discussion>

Evoking an immune response in vivo using DNA inoculation has been reported to use different therapeutic targets and transport methods.

 Induction of cell mediated immunization can be an important feature for various vaccines. For example, during spontaneous infection, anti-HIV-1 CTL responses appear transiently very early in connection with the establishment of the time of viral infection. CTL T cells play an important role in removing viruses by targeting and destroying virus infected cells. The generation of specific CTL responses modulates the immune response to viral proteins to induce a wide range of immune responses against multiple antigen targets in the virus. CTL activity against the virus is more easily measured in healthy infected patients than AIDS patients, and specific CTLs have been reported to decrease with a clear increase in relevant CTL responses in clinical conditions in which disease pathology is desirable. In this regard, macaques with high specific CTL and low antibody response are protected against chimeric SIV / HIV (SHIV) antigen administration, while animals with low CTL and high antibody response are resistant to viral replication. Controlled but not fully defended. Specific CTL responses have been shown to contribute to maintaining the asymptomatic stage of HIV-1 infection. Thus, induction of strong HIV-1 specific CTL in vivo through DNA immunization may play an important role in ultimate defense of the progression of HIV infection in the host.

Potential enhancement of the immune response, particularly the CTL response, from DNA vaccines against HIV-1 via the co-transportation of the IL-12 and GM-CSF genes as genetic aids was investigated. Genes for IL-12 and GM-CSF were cloned into expression vectors and injected into mice with DNA vaccine cassettes for HIV-1. Concomitant immunization of the plasmid encoding IL-12 with DNA vaccines against HIV-1 dramatically increased antigen specific CTL responses. GM-CSF co-transportation enhanced humoral response, while IL-12 co-transportation suppressed humoral response about 20%.

The concomitant transport of DNA vaccines and cytokine genetic adjuvant against HIV-1 has many significant immunological effects. First, the size and mass of the spleen from mice injected with the DNA vaccine and IL-12 gene were nearly three times that of the control spleen. In addition, the leukocyte cell number from the spleen was twice that of the control spleen. These results were consistent with previous findings that in vivo administration of recombinant IL-12 in mice results in splenic enlargement. Carr et al. Found that IL-12 injection increased the spleen mass by 5 times in wild-type mice. IL-12 inducible changes in wild type mice were associated with significantly increased INF-gamma serum levels. However, IL-12 administration also induced a qualitatively similar (two-fold normal) increase in spleen size in IFN-gamma receptor deficient mice. This previous study reported splenic enlargement following injection of IL-12 protein. Small amounts of IL-12 gene transported in vivo induced splenic expansion to levels comparable to published experiments using recombinant IL-12 proteins. In vivo injection of recombinant IL-12 into mice has been reported to produce toxic effects such as weight loss and severe death in rats injected. It is important that the co-administration of the IL-12 gene induced spleen enlargement without causing any visible reverse change in the administered mice. This suggests that natural similar processing and sustained low levels of transport through plasmid inoculation are clinically beneficial. DNA transport to induce a meaningful systemic immune response has been demonstrated to be non-toxic here.

In addition to induction of splenic enlargement, the specific immune response from Th2 to Th1 can be manipulated through the coadministration of IL-12 with DNA vaccines. In this regard, the co-transportation of the DNA vaccine and IL-12 gene reduces the specific antibody response, while co-injection of the GM-CSF gene enhances the specific antibody response. These results were consistent with previous reports that IL-12 is an important cytokine in regulating the immune response from Th2 to Th1 type. These antibody results were also consistent with splenic cell FACS data, where a decrease in the proportion of B220 + B cells was observed in mice immunized with immunogen (HIV-1 envelope or GaG / Pol) and IL-12 gene.

To further elucidate the T cell response to co-administration of DNA, direct CTL analysis was performed without in vitro palpation of harvested spleen cells. Enhancement of the CTL response is important evidence demonstrating the ability to modulate the immune response from Th2 to Th1 type responses via DNA immunogens. A dramatic increase in specific CTL responses was observed from the co-immunized group of DNA vaccines and IL-12 genes. In summary, we observed co-administration of immunogens and genes encoding IL-12, a cytokine important for generating Th1 type immune responses, and enhancing cellular immune responses measured by T cell proliferation and CTL analysis. It was.

To modulate the type and direction of the immune response, for example to direct the response from Th2 to Th1, co-transportation of genes of immunologically important molecules can be used to generate a clinically more effective immune response. By concomitant administration of DNA immunogen and IL-12 gene. Humoral responses were mildly suppressed and CTL responses increased dramatically. In addition, spleen enlargement, which is characteristic of in vivo administration of recombinant protein IL-12, occurred in mice. Thus, DNA transport in vivo has been demonstrated for new methods of making vaccines more effective and for assaying molecules of mechanisms of immune function.

<Example 5>

In addition, in order to modulate the immune response in vivo, the induction and regulation of immune response was observed from concomitant transport of a wide range of cytokine genes with HIV-1 DNA immunogen constructs. Since cytokines play a major role in regulation and signal function in immunization, cytokine gene co-transport was chosen. In addition to the immune system, although cytokines are released by various cells, cytokines produced by lymphocytes are of particular interest because of their ability to regulate cells in the immune system. For example, the presence of IL-2, IFN-γ and IL-12 activates Th0 precursor cells to become Th1 inflammatory T cells. On the other hand, release of IL-4, IL-5 or IL-10 results in Th0 precursor, resulting in inorganicized Th2 helper cells. In addition, pro-inflammatory cytokines such as IL-1, TNF-α and TNF-β play an important role in initiating the inflammatory response.                 

Pro-inflammatory cytokines (IL-1α, TNF-α and TNF-β), Th1 cytokines (IL-2, IL-15 and IL-18), and Th2 cytokines (IL-4, IL-5 and IL -10) function was investigated. Specifically, the genes of these inflammatory Th1 and Th2 cytokines were cloned into expression vectors, respectively, under the control of the cytomegalovirus (CMV) promoter. Gene plasmid expression cassettes were injected with the DNA vaccine cassette for HIV-1 as described above. The immunological effects of infusion with these genetic adjuvant cassettes were analyzed for the direction and size of the antigen specific immune response.

Antigen specific immune responses can be regulated by co-injecting cytokine genes with DNA immunogen cassettes. More specifically, it demonstrated the potency of concomitant transport of immunologically important genes as a vehicle for the development of DNA vaccines with enhanced potential in clinical efficacy and utility.

<Materials and Methods>

DNA plasmid:

DNA vaccine constructs expressing HIV-1 envelope protein (pCEnv) and gag / pol protein (pCGag / Pol) were prepared using standard methods and readily available starting materials. Genes for human IL-1α, IL-2, IL-5, IL-10, IL-15, TNF-α, TNF-β, and IL-4 and IL-18 in rats are standard and readily available Starting material was cloned into the pCDNA3 expression vector (Introgen, San Diego, CA). Human IL-1α, IL-2, IL-5, IL-10, IL-15, TNF-α, TNF-β have been reported to be rat cellet activity. Plasmid DNA was generated in bacteria and purified using the Qiagen Maxim Prep Kit (Qiagen, Santa Clara, CA).

Reagents and cell lines:

Human Rabdomio sarcoma (RD) and mastocytosis P815 cell lines of mice were obtained from ATCC (Rockville, MD). Recombinant vaccinia expressing HIV-1 envelope (vMN462), gag / pol (vVK1), and galactosidase (vSC8) were obtained from the National Institutes of Health AIDS Research and Reference Regents program. HIV-1 envelope peptide (RIHIGPGRAFYTTKN) was synthesized using standard techniques and readily available starting materials. Recombinant Pr55 or gp120 protein was obtained from Quality Biological (Gaithersburg, Maryland). Recombinant P24 protein was purchased from Intracell (Cambridge, MI). Antibodies to IL-1α, IL-2, IL-5, IL-10, IL-15, TNF-α and TNF-β were obtained from R & D Systems (Minneapolis, Minnesota).

Expression of cytokine gene constructs:

Expression of cytokine constructs was verified by immunoprecipitation or cytokine ELISA after transfection into RD cells. Cells were washed twice with PBS and incubated for 1 hour in nutrients deficient in serum, methionine and cysteine for 1 hour, followed by 200 μCi / ml (1200 Ci / mmolmol) of ³⁵ S protein labeling mixture (NEN / Dupont). Labeled. Labeled cells were lysed in 0.5 ml of RIPA buffer (50 mM Tris HCl, pH 7.6; 150 mM NaCl; 0.2% Triton X-100; 0.2% deoxycholic acid; 0.1% SDK and 1 mM PMSF) on ice and then 15000. Purification by centrifugation at RPM for 10 minutes. The cleared lysates were incubated for 90 minutes with the appropriate antibody (R & D) on ice. Protein A Sepharose was added to the antigen-antibody complex and mixed by shaking at 4 ° C. for 90 minutes. Protein pellets were resuspended in 50 μl 1 × sample buffer, washed vigorously in buffer containing large amounts of salt and BSA and then heated to 100 ° C. for 3-5 minutes. Fractions of protein samples were analyzed by SDS 12% -PAGE. For fluorography, the gel was immersed in 1M sodium salicylate containing 10% glycerol for 15 minutes, dried and autoradiographed using a Kodak X-Omart-AR film. Supernatants from transfected RD cells were harvested and tested for expression using the cytokine ELISA kit (Parmingen, San Diego, CA).

Rat DNA Inoculation:

The muscles of 6-8 week old BALB / C mice (Harlan Sprague Dulsa) were injected with 50 g of the desired DNA construct and 0.25% Bupivacaine (Sigma, St. Louis, MO) in phosphate buffer. Selected plasmids were mixed to a total of 100 μg per injection prior to injection for co-administration of various gene expression cassettes.

ELISA:

50 μl of p24 or gp120 protein diluted at a concentration of 2 μg / ml in 0.1 M carbonate-bicarbonate buffer (pH 9.5) was adsorbed onto microtiter wells at 4 ° C. overnight. Plates were washed with PBS-0.05% Tween-20 and blocked with 3% BSA in PBS containing 0.05% Tween-20 for 1 hour at 37 ° C. The antiserum of mice was diluted with 0.05% Tween-20 and incubated at 37 ° C. for 1 hour, followed by incubation with HRP-conjugated goat's mouse IgG or IgA (Sigma, St. Louis, MO). Plates were washed and developed with 3'3'5'5 'TMB buffer (Sigma). Optical density at 450 nm was read on a Dinatech MR5000 plate reader.

T helper cell proliferation assay:

Lymphocytes were harvested from the spleen, red blood cells removed and washed several times with fresh medium to prepare effector cells. Isolated cell suspension was resuspended at a concentration of 5 × 10 ⁶ cells / ml. 100 μl aliquots containing 5 × 10 ⁵ cells were immediately added to each well of a flat bottom 96 well microtiter plate. Recombinant Pr55 or gp120 protein was added to the wells in triplicates at the highest concentrations of 5 μg / ml and 1 μg / ml. Cells were incubated at 37 ° C. in 5% CO ₂ for 3 days. After 1 μCi of ³ H thymidine was added to each well, the cells were incubated at 37 ° C. for 12-18 hours. The plates were harvested and the incorporated ³ H thymidine measured in a beta plate reader (Walaksa, Turku, Finland). A facilitation index was determined from the following formula.

Acceleration Index (SI) = (Coefficient / Voluntary Coefficient)

Spontaneous counting wells contain 10% calf serum to serve as an unrelated protein control. In addition, the SI value for Pr55 protein is typically 1 in pCEnv or control immunized animals. Similarly, the SI value for the gp120 protein is typically 1 in pCGag / poL or the control. To confirm that the cells are active, PHA or con A (Sigma) was used as a polyclonal promoter positive control. PHA or conA control samples have SI values of 20-40.                 

Cytotoxic T Lymphocyte Assay:

CTL analysis was performed to release chromium ( ⁵¹ Cr) for 5 hours using vaccinia infected target or peptide treated target. Assays were performed in the presence and absence of in vitro effector palpation. In in vitro palpation assays, effectors are promoted by the desired vaccinia infected cells (vMN462 for envelope, vVK1 for gag / pol), and 0.1% glutaraldehyde or envelope specific peptide (RIHIGPGRAFYTTKN) at a 1M concentration. 4 to 5 days at 5 × 10 ⁶ cells per ml in CTL medium. CTL medium was 1: 1 in the presence of 10% calf serum 1640 and 10% RAT-T-STIM (Gibco-BRL, Grand Island, NY) and in the absence of Con A (Becton Dickinson Labware, Badford, Mass.). 1, consisting of an improved Dulbecco media (Gibco-BRL) and Hank's balanced salt solution (Gibco-BRL). Vaccinia infected targets were prepared by infecting 3 × 10 ⁶ PB15 cells at 37 ° C. for 5-12 hours at 10-20 multiple infections (MOI). Peptide treated targets were prepared by incubating P815 cells with 1 μM concentration of peptide. Standard chromium release assays were performed. Here, target cells were labeled with 100 μCi / ml of Na ₂ ⁵¹ CrO ₄ for 60 to 120 minutes and used to incubate with promoted effector spleen cells at 37 ° C. for 4 to 6 hours. CTL dissolution was tested at effector: target (E: T) ratios ranging from 50: 1 to 12.5: 1. Supernatants were harvested and counted on an LKB clinygamma gamma counter. The insolubility in water was determined from the following formula.

100 × {(experimental-voluntary release) / (maximum release-voluntary release)}                 

Maximal release was determined by lysing the target cells in medium containing 1% Triton X-100. The assay is not considered valid if the value for the "voluntary release" factor exceeds 20% of the "maximum release".

Complement lysis of CD8 + T cells

CD8 + T cells were treated with anti-CD8 monoclonal antibody (Pharmingen) to remove them from splenocytes and then incubated with rabbit's complement (Sigma) at 37 ° C. for 45 minutes.

<Result>

Expression of the cytokine gene cassette

Cytokine genes were cloned into pCDNA3 plasmid expression vector (FIG. 10). Cytokine expression cassettes were identified by sequencing of the entire insert (both 5 'and 3'). In addition, this cytokine gene was transfected into RD cells in vitro and expression of this construct was confirmed by immunoprecipitation or cytokine ELISA with appropriate antibodies as described in Materials and Methods.

Humoral response after coadministration of cytokine genes:

Antisera from pCGag / pol immunized mice were harvested and analyzed for specific antibody response to HIV-1 antigen by ELISA.

In this experiment, 50 μg of each DNA was coadministered intramuscularly on days 0 and 14. Prior to injection and 28 days after injection, mice (4 per colony) were bled and serum was harvested. Serial dilution is 1: 100. 1: 200, 1: 400, 1: 800, 1: 1600, and 1: 3200. Baseline optical density for ELISA was less than 0.015. This experiment was repeated with similar results. As shown in the data, the endpoint of antibody titers for the immunized group was determined using ELISA for p24 gag protein and the endpoint for antibody titers for the immunized group was determined using ELISA for gp120 envelope protein. It was. The data below is for gag / pol-specific antibody titers from serum harvested 28 days after DNA immunization.

Anti-p24 antibody titers

pCGag / pol 400

pCGag / pol + IL-1α 1600

pCGag / pol + TNF-α 1600

pCGag / pol + TNF-β 1600

pCGag / pol + 1L-2 3200

pCGag / pol + 1L-15 800

pCGag / pol + 1L-18 3200

pCGag / pol + 1L-4 3200

pCGag / pol + 1L-5 3200

pCGag / pol + 1L-10 600

Higher titer endpoints were observed when using serum from the IL-2, IL-4, IL-5 and IL-18 co-administered groups. In addition, dramatic enhancement of humoral response was observed with the co-administration group using IL-1α, TNF-α, TNF-β, and IL-10 compared to the group immunized with pCGag / pol alone. Similar results were obtained in the group immunized with pCEnv.

Anti-gp120 antibody titers

pCEnv 200

pCEnv + IL-1α 800

pCEnv + TNF-α 800

pCEnv + TNF-β 400

pCEnv + 1L-2 800

pCEnv + 1L-15 400

pCEnv + 1L-18 800

pCEnv + 1L-4 1600

pCEnv + 1L-5 1600

pCEnv + 1L-10 1600

In addition, high titer endpoints were observed when using serum from the IL-4, IL-5 and IL-10 coadministration groups.

Generation of T helper cells

Activation and proliferation of T helper lymphocytes play an important role in inducing both humoral immune responses through B cells and cellular immune responses through CD8 + cytotoxic T cells. Mice immunized with two DNA (50 μg each) were isolated after 2 weeks. One week after reinoculation, mice were sacrificed, spleens were harvested, lymphocytes isolated and tested for T helper cells.

Concomitant administration of pro-inflammatory cytokines                 

Proliferation assays were performed with spleen cells from mice administered with pCEnv or pCGag / pol and the pro-inflammatory cytokines IL-1α, TNF-α and TNF-β.

In the experiment of measuring the T helper cell proliferation response after co-administration with the pro-inflammatory cytokines IL-1α, TNF-α, and TNF-β, the method is as follows. Two weeks after the first DNA immunization (50 μg each) with pCEnv, mice (4 per colony) were re-inoculated at the same dose. After a further week, the spleens were harvested from immunized mice, their lymphocytes isolated and tested for recombinant gp120 protein (final concentrations of 5 and 1 μg / ml). Two weeks after co-administration of the first DNA with pCGag / pol (50 μg each), mice (4 per colony) were re-inoculated at the same dose. After a further week, the spleens were harvested from immunized mice, their lymphocytes were isolated and tested for T cell proliferation response to Pr55 protein (final concentrations of 5 and 1 μg / ml). This experiment was repeated twice with similar results. The following data were obtained.

Antigen-Specific T Cell Proliferative Responses

gp120 protein

                       5 μg / ml 1 μg / ml

pCEnv 2.2 1.3

pCEnv + IL-1α 2.3 0.1

pCEnv + TNF-α 6.1 2.7

pCEnv + TNF-β 3.1 1.8

Control 0.5 0.2

p24 protein

                       5 μg / ml 1 μg / ml

pCGag / pol 2.4 0.8

pCGag / pol + IL-1α 2.8 1.4

pCGag / pol + TNF-α 12.4 2.2

pCGag / pol + TNF-β 4.0 1.9

Control 0.6 0.8

The data indicate that basal levels of proliferation were observed in the control and moderate levels of proliferation were observed in the groups immunized with pCEnv or pCGag / pol only. Although T helper cell proliferation did not increase at all with pCEnv or pCGag / pol immunization in the co-administered group with IL-1α, T helper with a facilitation index of 3.1 at gp120 protein concentration of 5 μg / ml in the co-administered group of pCEnv + TNF-β. Significant enhancement of cell proliferation was shown. Similarly, the co-administered group of pCGag / pol + TNF-β showed a significant enhancement of the palpation index 4.0 T helper cell proliferation at a Pr55 protein concentration of 5 μg / ml. Co-administration with pCEnv + TNF-α and pCGag / pol + TNF-α showed high levels of T helper cell proliferation with palpation indices of 6.1 and 12.4 (each protein concentration 5 μg / ml), respectively.

-With Th1 cytokine

The co-administration effect of Th1 cytokines, IL-2, IL-15, and IL-18, was investigated. Co-administered groups of pCEnv + IL-18 and pCGag / pol + IL-18 resulted in facilitation indices 4.4 and 10.0, respectively (at protein concentrations of 5 μg / ml, respectively). In the experiment of measuring the T helper cell proliferation response by performing co-administration with INF-γ which induces Th1 cytokines IL-2 and IL-18, the method is as follows. Two weeks after the first DNA immunization (50 μg each) with pCEnv, mice (4 per colony) were re-inoculated at the same dose. After a further week, the spleens were harvested from immunized mice, their lymphocytes isolated and tested for recombinant gp120 protein (final concentrations of 5 and 1 μg / ml). Two weeks after co-administration of the first DNA with pCGag / pol (50 μg each), mice (4 per colony) were re-inoculated at the same dose. After a further week, the spleens were harvested from immunized mice, their lymphocytes were isolated and tested for T cell proliferation response to Pr55 protein (final concentrations of 5 and 1 μg / ml). This experiment was repeated twice with similar results. The following data were obtained.

gp120 protein

                       5 μg / ml 1 μg / ml

pCEnv 2.2 1.3

pCEnv + IL-18 4.4 0.8

pCEnv + IL-12 7.8 3.8

Control 0.5 0.2

p24 protein

                       5 μg / ml 1 μg / ml

pCGag / pol 2.4 0.8

pCGag / pol + IL-18 10.0 2.1

pCGag / pol + IL-12 12.0 3.8                 

Control 0.6 0.8

In addition, pCEnv + IL-2 and pCGag / pol + IL-2 coadministration resulted in facilitation indices 6.0 and 12.0, respectively (at a protein concentration of 5 μg / ml, respectively). However, co-administration of IL-15 resulted in some increase in T helper cell proliferation. In order to measure the T helper cell proliferation response according to the co-administration effect of IL-2 dependent Th1 cytokines, IL-2 and IL-15, the following method was used. Two weeks after the first DNA immunization (50 μg each) with pCEnv, mice (4 per colony) were re-inoculated at the same dose. After a further week, the spleens were harvested from immunized mice, their lymphocytes isolated and tested for recombinant gp120 protein (final concentrations of 5 and 1 μg / ml). Two weeks after co-administration of the first DNA with pCGag / pol (50 μg each), mice (4 per colony) were re-inoculated at the same dose. After a further week, the spleens were harvested from immunized mice, their lymphocytes were isolated and tested for T cell proliferation response to Pr55 protein (final concentrations of 5 and 1 μg / ml). This experiment was repeated twice with similar results. The following data were obtained.

gp120 protein

                        5 μg / ml 1 μg / ml

pCEnv 2.2 1.3

pCEnv + IL-2 6.0 2.1

pCEnv + IL-15 2.3 2.0

Control 0.5 0.2

p24 protein

                         5 μg / ml 1 μg / ml

pCGag / pol 2.4 0.8

pCGag / pol + IL-2 12.0 2.5

pCGag / pol + IL-15 2.6 0.6

Control 0.6 0.8

-With Th2 cytokine
In addition to validation of pre-inflammatory and Th1 cytokine co-administration, the effects of co-administration with pCEnv and pCGag / pol and the Th2 cytokines IL-4, IL-5 and IL-10 were also investigated. Both co-administration groups with IL-4 or IL-5 showed some increase in T helper cell proliferation compared to immunization with pCEnv or pCGag / pol only. In order to measure the T helper cell proliferation response according to the co-administration effect of Th2 cytokines IL-5 and IL-10, the following method was used. Two weeks after the first DNA immunization (50 μg each) with pCEnv, mice (4 per colony) were re-inoculated at the same dose. After a further week, the spleens were harvested from immunized mice, their lymphocytes isolated and tested for recombinant gp120 protein (final concentrations of 5 and 1 μg / ml). Two weeks after co-administration of the first DNA with pCGag / pol (50 μg each), mice (4 per colony) were re-inoculated at the same dose. After a further week, the spleens were harvested from immunized mice, their lymphocytes were isolated and tested for T cell proliferation response to Pr55 protein (final concentrations of 5 and 1 μg / ml). This experiment was repeated twice with similar results. The following data were obtained.

gp120 protein

                      5 μg / ml 1 μg / ml                 

pCEnv 2.2 1.3

pCEnv + IL-4 3.8 2.9

pCEnv + IL-5 2.4 1.8

pCEnv + IL-10 4.5 2.3

Control 0.5 0.2

p24 protein

                       5 μg / ml 1 μg / ml

pCGag / pol 2.4 0.8

pCGag / pol + IL-4 5.0 3.1

pCGag / pol + IL-5 3.1 2.1

pCGag / pol + IL-10 8.0 2.4

Control 0.6 0.8

Concomitant transport of IL-10 (each protein concentration is 5 μg / ml) dramatically increased T helper cell proliferation such that the facilitating indices were 4.5 and 8.0, respectively.

Production of cytotoxic T lymphocytes

To observe the enhancement of cellular immunity, cytotoxic T lymphocyte (CTL) analysis was performed using spleen cells of mice immunized with pCEnv and pCGag / pol. Mice immunized with two DNA (50 μg each) were isolated after 2 weeks. One week after reinoculation, mice were sacrificed, spleen harvested, lymphocytes isolated and tested for CTL response. The assay was performed in the presence and absence of in vitro palpation of effector spleen cells before measuring chromium release from targets infected with specific and nonspecific vaccinia or targets treated with peptides. The solubility of nonspecific (vSC8 infected) targets was subtracted from the solubility of specific (vMN462 or vVK1 infected) targets in order to calculate the nasal solubility of the target.

CTL reactions with in vitro palpation of effectors

Data on cytotoxic T lymphocytes following various cytokines were obtained. The data are as follows.

Antigen-Specific CTL Reactions

                       50: 1 25: 1 12.5: 1

pCEnv 10% 4% 3%

pCEnv + IL-1α 14% 10% 6%

pCEnv + TNF-α 30% 23% 18%

pCEnv + TNF-β 20% 16% 13%

pCEnv + 1L-2 22% 16% 4%

pCEnv + 1L-15 46% 28% 10%

pCEnv + 1L-12 35% 24% 19%

pCEnv + 1L-18 22% 16% 13%

pCEnv + 1L-4 4% 4% 7%

pCEnv + 1L-5 13% 11% 9%

pCEnv + 1L-10 13% 8% 3%

Control 3% 2.0% 3.5%                 

                        50: 1 25: 1 12.5: 1

pCGag / pol 12% 11% 7%

pCGag / pol + IL-1α 16% 8% 1%

pCGag / pol + TNF-α 29% 20% 7%

pCGag / pol + TNF-β 12% 13% 3%

pCGag / pol + 1L-2 14% 11% 10%

pCGag / pol + 1L-15 30% 21% 7%

pCGag / pol + 1L-12 38% 24% 15%

pCGag / pol + 1L-18 19% 15% 4%

pCGag / pol + 1L-4 2% 2% 2%

pCGag / pol + 1L-5 0% 3% 2%

pCGag / pol + 1L-10 6% 6% 0%

Control 3.3% 2.8% 4.5%

delete

In experiments performed to measure cytotoxic T lymphocyte responses by conducting co-administration with pro-inflammatory cytokines, namely IL-1α, TNF-α and TNF-β, co-administration of first DNA with pCGag / pol ( Two weeks after each 50 μg) mice (4 per colony) were re-inoculated at the same dose. A further week later, the spleens were harvested from immunized mice, their lymphocytes isolated and tested for CTL response using target cells infected with specific (vVK1) and nonspecific vaccinia (vSC8). Two weeks after the first DNA immunization (50 μg each) with pCEnv, mice (4 per colony) were re-inoculated at the same dose. After a further week, the spleens were harvested from immunized mice, their lymphocytes isolated and tested for CTL responses using target cells infected with specific (vMN462) and nonspecific vaccinia (vSC8). This experiment was repeated twice with similar results.

In experiments conducted to measure cytotoxic T lymphocyte responses by conducting coadministration with Th1 cytokines, namely IFN-γ, which induces IL-12 and IL-18, coadministration of first DNA with pCGag / pol ( Two weeks after each 50 μg) mice (4 per colony) were re-inoculated at the same dose. A further week later, the spleens were harvested from immunized mice, their lymphocytes isolated and tested for CTL response using target cells infected with specific (vVK1) and nonspecific vaccinia (vSC8). Two weeks after the first DNA immunization (50 μg each) with pCEnv, mice (4 per colony) were re-inoculated at the same dose. After a further week, the spleens were harvested from immunized mice, their lymphocytes isolated and tested for CTL responses using target cells infected with specific (vMN462) and nonspecific vaccinia (vSC8). This experiment was repeated twice with similar results.

In experiments conducted to measure cytotoxic T lymphocyte responses by performing co-administration with IL-2 receptor dependent Th1 cytokines, namely IL-2 and IL-15, co-administration of first DNA with pCGag / pol (each Two weeks after 50 μg) mice (4 per colony) were re-inoculated at the same dose. A further week later, the spleens were harvested from immunized mice, their lymphocytes isolated and tested for CTL response using target cells infected with specific (vVK1) and nonspecific vaccinia (vSC8). Two weeks after the first DNA immunization (50 μg each) with pCEnv, mice (4 per colony) were re-inoculated at the same dose. After a further week, the spleens were harvested from immunized mice, their lymphocytes isolated and tested for CTL responses using target cells infected with specific (vMN462) and nonspecific vaccinia (vSC8). This experiment was repeated twice with similar results.

In experiments conducted to measure cytotoxic T lymphocyte response by co-administration with Th2 cytokines, namely IL-5 and IL-10, co-administration of the first DNA with pCGag / pol (50 μg each) Two weeks after Hanji, mice (4 per colony) were re-inoculated at the same dose. A further week later, the spleens were harvested from immunized mice, their lymphocytes isolated and tested for CTL response using target cells infected with specific (vVK1) and nonspecific vaccinia (vSC8). Two weeks after the first DNA immunization (50 μg each) with pCEnv, mice (4 per colony) were re-inoculated at the same dose. After a further week, the spleens were harvested from immunized mice, their lymphocytes isolated and tested for CTL responses using target cells infected with specific (vMN462) and nonspecific vaccinia (vSC8). This experiment was repeated twice with similar results.

Concomitant administration of pro-inflammatory cytokines

Regarding data from the results of CTL analysis for mice administered with pCEnv or pCGag / pol and pro-inflammatory cytokines IL-1α, TNF-α and TNF-β, specific killing was observed at baseline levels in control animals. In contrast, animals immunized with pCEnv alone showed low levels of CTL responses. Co-administration with pCEnv + IL-1α or pCGag / pol + TNF-β slightly increased CTL activity. On the other hand, after coadministration with pCEnv + TNF-α, specific killing of targets infected with vaccinia (vMN462) expressing HIV-1 envelope was observed to increase dramatically. After co-administration with pCEnv + TNF-α at a ratio of effector to target (E: T) of 50: 1, greater than 30% nasolacrimality was observed in the target cells. Similarly, mice immunized with pCGag / pol + TNF-α result in significant enhancement of antigen specific CTL lysis of targets infected with vaccinia (vVK1) expressing HIV-1 gag / pol (E: T Ratio of 50: 1 to 20% solubility), co-administration to pCGag / pol + IL-1α or pCGag / pol + TNF-β slightly increased the CTL response.

-With Th1 cytokine

 The effect of co-transportation of DNA vaccine constructs and Th1 cytokines was investigated. In CTL assays from mice immunized with pCEnv and from mice co-administered with Th1 cytokines IL-12 and IL-18, co-administration of IL-18, unlike co-administration of IL-12, resulted in a CTL response. Increased somewhat. In addition, co-administration of IL-2 slightly increased the CTL response. On the other hand, after pCEnv + IL-15 immunization, a more dramatic increase in CTL response was observed with a cost of 46%. Similarly, mice immunized with pCGag / pol + IL-15 resulted in a significant enhancement (approximately 30%) of antigen specific CTL solubility.

-With Th2 cytokine

In addition to the effects from pre-inflammatory and Th1 cytokine co-administration, the effects of co-administration of Th2 cytokines IL-4, IL-5 and IL-10 on the level of CTL response were studied. Although coadministration with these cytokines increased T helper cell proliferative responses, coadministration of either pCEnv or pCGag / pol and IL-4, IL-5 or IL-10 did not specifically increase the CTL response .

Determination of MHC Class I Restrictions in CTL Reactions

To determine whether enhancement of the CTL response through co-administration with TNF-α and IL-15 was due to CD8 + MHC class I restricted promotion, specific epitopes for MHC class I restricted CTL in balb / c mice were identified. CTL analysis was performed using HIV-1 envelope peptide (RIHIGPGRAFYTTKN). Mice were immunized twice at two week intervals with 50 μg of each DNA construct and harvested one week after the second immunization of their spleen. CTL analysis was performed on splenocytes in the presence of in vitro palpation using envelope specific peptides. Significant enhancement of the CTL response was observed after co-administration with IL-15 and TNF-α (FIGS. 11A-11E) with 25% and 32% specific killing at an E: T ratio of 50: 1. This observation was confirmed by the removal of CD8 + T cells from effector cell population by complement lysis followed by measurement of CTL activity. Mice were immunized twice with 50 μg of each plasmid at the same interval as above. One group of effector cells was treated as above and CTL analysis was performed in a manner that eliminates CD8 + T cells from the second group. As shown in FIGS. 11F-11O, removal of CD8 + T cells inhibited the antigen specific CTL enhancement observed after coadministration with IL-15 and TNF-α. These results indicate that enhancement of cytotoxic activity is antigen specific, class I limited and CD8 + T cell dependent.

CTL direct reaction (without in vitro promotion of effectors)

Since a high and constant level of CTL response was observed from two co-administration groups (in the presence of in vitro palpation), the level of direct CTL response induced by co-administration with TNF-α or IL-15 was observed. Chromium release assay was performed on the day the splenocytes were isolated. Unlike direct CTL administered with IL-12, no induction of direct CTL activity was observed after coadministration with pCEnv and TNF-α or IL-15 (FIG. 12).

<Discussion>

The overall goal of any immunization is to induce an effective and sustained pathogen specific immune response using a minimal number of immunizations. However, this defense relationship changes with pathogens, and the resulting improvement can be obtained by driving the immune response. For example, high levels of specific antibody responses are believed to be important in defending against infections of Hepatis B virus, but protection from lymphoma coriomeningitis virus (LCMV) infection is primarily mediated by T cell mediated responses. The design of DNA vaccination can be improved by directing and controlling the extent of the immune response induced to be associated with defense against each target pathogen.

With new immunizations, it has been demonstrated that nucleic acid immunization can elicit both antigen specific humoral and cellular immune responses in vivo in various animal models. More clinically effective vaccines can be produced using methods that modulate the direction and extent of the immune response. Fine regulation and immunotherapy of specific types and directions of immune responses from vaccines can be established by the co-transportation of genes for immunologically important molecules such as cytokines and promoter molecules.

Cytokines play an important role in immune and inflammatory responses as initiators and regulators of the immune network. Based on their specific function in the immune system, cytokines can be further subdivided into pro-inflammatory cytokines, Th1 cytokines and Th2 cytokines. The pro-inflammatory cytokines IL-1, TNF-α and TNF-β play important roles as initiators of host responses to injury and infection. IL-1 indirectly activates T cells by induction of IL-2 production and upregulation of IL-2R on cells. IL-1 also affects B cells by inducing differentiation, proliferation, and synthesis of IgG. IL-1 exists in at least two forms, designated IL-1α and IL-1β, and exhibits similar activity. TNF-α and TNF-β are analogous related proteins (with about 30% amino acid homology) that bind to the same cell surface receptor. TNF-α is produced by activated macrophages and monocytes, neutrophils, and activated NK cells, while TNF-β is produced by lymphocytes. TNF-α and TNF-β are also involved in septic shock and rheumatoid arthritis accompanied by infection by Gram-negative bacteria. In addition, TNF-α has been suggested to play an important role in regulating the synthesis of other pro-inflammatory cytokines.

Th1 cytokines are associated with cellular or T cell mediated mineralization of the immune response. IFN- [gamma], ie cytokine of the circular Th1 type, is produced by Th1, CD8 + and NK cells and is known to have antiviral effects and immunomodulatory effects such as upregulation of MHC class I and II antigens. Novel cytokine IFN-γ inducing factor (IGIF) or IL-18 has been shown to enhance the production of IFN-γ, while inhibiting the production of IL-10 in promoted PBMCs. In addition, IL-18 enhances natural killer (NK) cell activity in culture of human peripheral blood mononuclear cells (PBMC), similarly to structurally unrelated cytokine IL-12. IL-12 is produced mainly by T cells activated by external stimuli and is important for proliferation and polyclonal expansion of antigen specific T cells. IL-2 plays an important role in activating T cells by interacting with a receptor system consisting of three chains, α, β and γc chains. IL-15 is a newly discovered homologue of IL-2 and is a polymorphic cytokine with T cell promoting activity similar to IL-2.

Th2 cytokines regulate humoral or antibody mediated mineralization of the immune response. IL-5 is a dimeric cytokine that regulates the differentiation of B cells into antibody producing plasma cells. IL-5 has been shown to induce antigen specific IgA production by rat and human B cells. In addition, IL-5 promotes the growth and proliferation of eosinophils. Although IL-10 has been found to be produced early by Th2 T cell clones, they are also produced by B cells and single cells. IL-10, a circular Th2 type cytokine, inhibits the production of cytokines such as IL-1α, IL-6, IL-8 and TNF-α by mitogen activated single cells, and activates TFN-γ macrophages It has been shown to suppress the effect. Possible roles of IL-10 in HIV-1 infection have been reported and IL-10 mRNA is upregulated and PBMCs from individuals with asymptomatic HIV positive increased levels of IL-10 compared to PBMCs from uninfected individuals. Was observed from. In addition, IL-10 has been observed to reduce viral replication in vitro in human macrophages.

Expression cassettes of pro-inflammatory Th1 and Th2 cytokines have been developed to analyze their function as in vivo regulators of immune responses induced by DNA vaccines. Cytokine genes are co-transported into the muscles of mice with DNA immunogen constructs and their effects on the direction and extent of the induced immune response were analyzed. A dramatic increase in antibody response was observed when co-administered with IL-2, IL-4, IL-5, IL-10 and IL-18. Coadministration with TNF-α, TNF-β, IL-2, IL-10, and IL-18 results in dramatic enhancement of the T helper proliferative response, while coadministration with IL-5 and IL-15 results in T helper proliferation. Resulted in a mild increase. Moreover, only TNF-α and IL-15 co-administration of all co-administration combinations resulted in CTL enhancement at levels similar to those of IL-12 co-administration (more than 30% in cost). Coadministration with TNF-β, IL-2 and IL-18 mildly increased the CTL response to the group immunized with DNA immunogens only. As a result of observation of the co-administration of IL-12 or CD86, the enhancement of the CTL response observed from the co-administration of TNF-α and IL-15 is limited by MHC class I and CD8 + T cells.

IL-18 is reported to share similar activity with IL-12. For example, IL-18 in culture of human peripheral blood mononuclear cells (PBMC) enhances natural killer (NK) cell activity, similar to structurally unrelated cytokine IL-12. In addition, IL-18 enhances the production of IFN-γ, while inhibiting the production of IL-10 in Concanavalin A (con A) promoted PBMCs. IL-18 was observed to induce IFN via the IL-12 dependent pathway. In the case of IL-12 co-transportation, no significant similar effect to IL-18 co-transportation was observed, although significant enhancement of the T cell mediated response and some reduction in humoral response occurred. A significant increase in antibody titer was observed when using IL-18 co-administration. Furthermore, unlike IL-12 co-transportation, which leads to dramatic enhancement of CTLs, IL-18 co-administration did not induce similar levels of CTL enhancement. The immunomodulatory properties of IL-18 appear to be similar to IL-10.

In addition to the fractional effects of the co-administration of IL-12 and IL-18 in vivo, co-administration of IL-2 and IL-5 also resulted in different directions and degrees of immune response. IL-2 and IL-15 have been reported to share the γ chain of the IL-2 receptor and have similar bioactivity, including signaling of T cell promotion. IL-2 co-administration dramatically increases antibody and T helper cell proliferative responses, while IL-15 co-administration results in enhancement of meaningful CTL responses. This difference can be explained by the properties of IL-15. For example, IL-15 has been reported to induce significant TNF-α production through activation of synovial T cells in rheumatoid arthritis. In contrast, IL-2 induces significantly lower levels of TNF-α. This in vivo data suggests that signaling is activated differently by the involvement of two molecules.

Th2 cytokines can be used to enhance Th2 type immune responses without affecting T cell mediated responses. IL-4, IL-5 and IL-10 are reported to be potent Th2 cytokines. Significant increases in antibody response levels and T helper proliferation due to IL-4, IL-5 and IL-10 co-transportation are significantly increased. In contrast, no increase in CTL response levels was observed. These results demonstrated that Th2 type of response can be engineered with IL-4, IL-5 or IL-10 co-administration.

Dramatic observations of the function of TNF-α and IL-15 as multifunctional immune modulators were made.

The functions of various cytokines that are immunologically important in inducing a host's immune response from DNA immunization were investigated. As shown in FIG. 13, induction of specific mineralization of the immune response can be engineered using co-administration of cytokine genes. The cytokine adjuvant network is under a new level of regulation that induces specific immune responses to regulate the vaccination program to more closely correlate with varying defenses against disease. Such fine regulation of vaccines and immunotherapy has not been attainable previously. As a result, the control of the degree and direction of the immune response may have advantages in various vaccine methods. For example, if the T cell mediated response is excellent but the humoral response is no longer necessary or even detrimental, the IL-12 gene can be selected as an immune modulator to be transported with a specific DNA immunogen. On the other hand, to produce a vaccine against the extracellular bacteria of the target, for example, the IL-4, IL-5 or IL-10 gene may be co-administered. Moreover, where CD4 + T helper cells and antibodies play an important role in defense, GM-CSF and IL-2 can be transported in tandem. Finally, where three mineralizations of all immune responses are important, TNF-α may be coadministered to result in combined enhancement of antibodies, T helper cells, and CTL responses.

<Example 6>

Using the PCR reaction, the insert named BL1 was cloned and ligated into the PCR3 eukaryotic expression vector and vector pBBKan using the appropriate restriction enzymes shown in FIG. 15. BL1 constructs were co-administered with different HIV-1 antigens and immunostimulating effects were measured in mice. The results are shown in FIGS. 16 and 17A, 17B, 17C and 17D, indicating that the DNA in BL1 enhances the immune response when coimmunized with the HIV-1 antigen. Differently observed effects are an increase in spleen size and an increase in antibody and CTL responses.

Picornavirus Genus Renovirus: (Medical) Associated with about 50% of colds Heteroviruses: Human enteroviruses, such as (medical) polioviruses, coxsackieviruses, echoviruses, and infected A viruses. Aptovirus: (veterinary) foot and mouth disease virus Target antigen VP1, VP2, VP3, VP4, VPG Calcivirus genus Norwalk Group of viruses: (Medicine) These viruses are the major causative agents of infectious gastroenteritis. Togavirus genus Alphaviruses: (medical and veterinary) eg Seniles virus, Ross River virus, Eastern and Western encephalitis Reovirus: Rubella Virus Flaviviridae                                          Examples: (medicine) dengue fever, yellow fever, Japanese encephalitis, St. Louis encephalitis and tick-borne encephalitis virus Hepatitis C virus                                          (Medicine) These viruses have not yet been identified as family, but are thought to be either togaviruses or Flavibarus family. The most similar is the Togavirus family. Coronavirus                                          (Medical and veterinary) infectious bronchitis virus (poultry), swine-transferable gastrointestinal virus (pig), swine hemagglutinin encephalomyelitis virus (pig), feline infectious peritonitis virus (cat), feline intestinal coronavirus (cat), dog coronavirus ( Dogs), the human respiratory coronavirus has about 40 common colds. Causes 224E, 0C43. Coronaviruses can cause non-A, B, and C hepatitis. Target antigen Called E1-M or Matrix Protein Called E2-S or Spike Protein Also called E3-HE or hemagglutin-eltorose glycoprotein (not in all coronaviruses) N-nucleocapsid Rhabdovirus genus Besiliovirus Lisa virus: (medical and veterinary) rabies Target antigen G protein N protein Filoviridue                                          Hemorrhagic fever viruses such as Marburg and Ebola virus Paramyxovirus genus Paramyxoviruses: (medical and veterinary) Mumps virus, Newcastle disease virus (an important pathogen for chickens) Morbilili Virus: (Medical and Veterinary) Measles, Gadstamper Pneumovirus: (Medical and Veterinary) Respiratory Polyps Orthomyxovirus (Medicine) Influenza Virus Bungavirus genus Bunga Virus: (Medical) California Encephalitis, LA Cross Flebovirus: (Medicine) Rift Valley Hantavirus: Puremala is a hemahagin fever virus. Nair virus (veterinary) Nairobi sheep disease Many unspecified murine viruses besides Arenavirus                                          (Medicine) LCM, Lhasa Fever Virus

Reovirus genus Reovirus: Possible Human Pathogens Rotavirus: Acute Gastroenteritis in Children Orbivirus: (Medicine and Veterinary) Colorado tick fever, Rebobo (People) speech encephalopathy, hearing sickness Retrovirus subfamily Oncorivirinal: (veterinary) (medicine) feline leukemia virus, HTLVI and HTLVII Lentivirinal: (medical and veterinary) HIV, feline immunodeficiency virus, equine infection, anemia virus Spumavirinal Papovavirus subfamily Polyomaviruses: (medical) BKU and JCU viruses subfamily Papillomaviruses: (Medical) Many types of viruses associated with malignant progression of cancer or papilloma Adenovirus                                          (Medicine) EX AD7, ARD., O.B. Adenoviruses, such as 275, cause enteritis. Parvovirus                                          Veterinary Medicine Feline parvovirus: causes cat enteritis Feline panycopenia virus Canine parvovirus Porcine Parvovirus Herpesvirus subfamily Alpha Herpes Bere Dew genus Simplex Virus (Medicine) HSVI, HSVII Varicellovirus: (Medicine-Veterinary) Rabies-Varicella zoster subfamily Beta Herpes Bere Dew genus Cytomegalovirus (Medicine) HCMV Muromegalovirus subfamily Gamma Herpes Bere Dew genus Lymphocytovirus (Medicine) EBV-(Burkitz Lympho) Radinovirus Poxvirus subfamily Cordofoxbyredude (Medicine-Veterinary) genus Barriola (Natural Doo) Vaccinia (Via) Parapoxvirus-Veterinary Oypoxvirus-Veterinary Capripoxvirus Repolipoxvirus Siopoxvirus subfamily Entemo Fox Buddy Dew Hepadnavirus Hepatitis B Virus Unclassified Hepatitis Delta Virus

Bacterial pathogens Pathogenic gram positive cocci include pneumococcal, staphylococcal, and streptococcal, and pathogen gram negative cocci include meningococcal, and gonococcal. Contains Pathogenic intestinal gram-negative bacilli include enterobacteriaceae, pseudomonas, acinetobacteria and eikenella, melioidosis, salmonella, and shigellosis ( shigellosis, hemophilus, chancroid, brucellosis, turalemia, ersinia (pasteurella), streptobacillus monoliformis (streptobacillus moniliformis) and spirillum, listeria monocytogenes, erysipelothrix rhusiopathiae, diphtheria, cholera, anthrano, anthrano Includes donovanosis, granuloma inguinale and bartonellosis. Pathogenic anaerobic bacteria include tetanus, botulism, other Clostridia, Mycobacterium tuberculosis, leprosy and other mycobacteria; pathogenic spirochetal diseases include syphilis, treponematose ( treponematoses, yaws, pinta and endemic syphilis, and leptospirosis. Infections by pathogenic bacteria and fungi include actinomycosis, nocardiosis, cryptococcosis, blastomycosis, histoplasmosis and cosidiodomico Coccidiodomycosis, candidasis, aspergillosis, and mucormycosis, sporotrichosis, paracoccidiodomycosis, petriellidiosis ), Toulolososis, mycetoma and chromomycosis and dermatophytosis. Rickettsial infections include rickettsial and rickettsioses Mycoplasma and chlamydial infections include mycoplasma pneumoniaes, lymphogranuloma venerum, psittacosis, and perinatal chlamydial infections. Eukaryotic pathogens Protozoa and parasites and infectious agents include amebiasis, malaria, leishmaniasis, trypanosimiasis, toxoplasmosis, pneumocystis, carinii , Babesiosis, giardiasis, trichinosis, filariasis, schistosomiasis, nematodes, trematode or fluke, And nematode (worm) infections

Claims (69)

delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete a) a nucleotide sequence encoding an IL-12 p35 subunit operably linked to a first promoter; b) a second nucleotide sequence encoding an IL-12 p40 subunit operably linked to a second promoter; And c) a third nucleotide sequence encoding an immunogen operably linked to a regulatory sequence capable of functioning in a mammalian cell A DNA vaccine composition comprising a plasmid, wherein said nucleotide sequence is operably linked to a regulatory sequence capable of functioning in a mammalian cell. The DNA vaccine composition of claim 42, further comprising a nucleotide sequence encoding IL-15 operably linked to a regulatory sequence capable of functioning in a mammalian cell. A composition comprising a first plasmid and a second plasmid, (a) an IL-12 p35 subunit operably linked to a first promoter and an IL-12 p35 subunit operably linked to a first promoter operably linked to a regulatory sequence capable of functioning in a mammalian cell A nucleotide sequence encoding a -12 p40 subunit; (b) a DNA vaccine composition wherein the nucleotide sequence of said second plasmid operably linked to a regulatory sequence capable of functioning in a mammalian cell comprises a nucleotide sequence encoding an immunogen. A composition comprising a first plasmid and a second plasmid, (a) the nucleotide sequence of said first plasmid operably linked to a regulatory sequence capable of functioning in a mammalian cell comprises a nucleotide sequence encoding a subunit of IL-12 operably linked to a first promoter; (b) the nucleotide sequence of said second plasmid operably linked to a regulatory sequence capable of functioning in a mammalian cell comprises a nucleotide sequence encoding another subunit of IL-12 operably linked to an immunogen and a second promoter. DNA vaccine composition. 46. The DNA vaccine composition of claim 44 or 45, further comprising a third plasmid comprising a nucleotide sequence encoding IL-15 operably linked to a regulatory sequence capable of functioning in mammalian cells. 46. The DNA of claim 44 or 45, wherein said first plasmid or second plasmid further comprises a nucleotide sequence encoding IL-15 operably linked to a regulatory sequence capable of functioning in mammalian cells. Vaccine composition. 46. The DNA of claim 44 or 45, wherein said first and second plasmids further comprise a nucleotide sequence encoding IL-15 operably linked to a regulatory sequence capable of functioning in mammalian cells. Vaccine composition. A composition comprising a first plasmid, a second plasmid and a third plasmid, (a) the nucleotide sequence of said first plasmid operably linked to a regulatory sequence capable of functioning in a mammalian cell comprises a nucleotide sequence encoding a subunit of IL-12 operably linked to a first promoter; (b) the nucleotide sequence of said second plasmid operably linked to a regulatory sequence capable of functioning in a mammalian cell comprises a nucleotide sequence encoding another subunit of IL-12 operably linked to a second promoter; (c) the nucleotide sequence of said third plasmid operably linked to a regulatory sequence capable of functioning in a mammalian cell comprises a nucleotide sequence encoding an immunogen. The method of claim 49, wherein at least one of the first plasmid, second plasmid, or third plasmid further comprises a nucleotide sequence encoding IL-15 operably linked to a regulatory sequence capable of functioning in mammalian cells. DNA vaccine composition. A composition comprising a first plasmid, a second plasmid and a third plasmid, (a) the nucleotide sequence of said first plasmid operably linked to a regulatory sequence capable of functioning in a mammalian cell comprises a nucleotide sequence encoding a human IL-12 p35 subunit operably linked to a first promoter; (b) the nucleotide sequence of said second plasmid operably linked to a regulatory sequence capable of functioning in a mammalian cell comprises a nucleotide sequence encoding a human IL-12 p40 subunit operably linked to a second promoter; (c) the nucleotide sequence of said third plasmid operably linked to a regulatory sequence capable of functioning in a mammalian cell comprises a nucleotide sequence encoding an immunogen. 52. The method of claim 51, wherein at least one of said first plasmid, second plasmid or third plasmid further comprises a nucleotide sequence encoding IL-15 operably linked to a regulatory sequence capable of functioning in mammalian cells. DNA vaccine composition. 52. The DNA of any one of claims 42, 44, 45, 49 and 51, wherein the immunogen is a target protein that encodes an antigen associated with a pathogen antigen, cancer associated antigen or autoimmune disease related cell. Vaccine composition. 52. The DNA vaccine composition of any one of claims 42, 44, 45, 49 and 51 wherein the immunogen is an HIV-1 antigen. 52. An individual for a pathogen, cancer cell or autoimmune disease related cell comprising the DNA vaccine composition of any one of claims 42, 44, 45, 49 and 51 and a pharmaceutically acceptable carrier. A pharmaceutical composition for immunizing. a) a first nucleotide sequence encoding an IL-12 p35 subunit operably linked to a first promoter; b) a second nucleotide sequence encoding an IL-12 p40 subunit operably linked to a second promoter; And c) a third nucleotide sequence encoding an immunogen operably linked to a regulatory sequence capable of functioning in a mammalian cell Recombinant DNA vaccine comprising a. 59. The recombinant DNA vaccine of claim 56, wherein said immunogen is selected from the group consisting of pathogen antigens, cancer associated antigens and antigens associated with autoimmune disease related cells. The recombinant DNA vaccine of claim 56, wherein said immunogen is an HIV-1 antigen. The recombinant DNA vaccine of claim 56, further comprising a nucleotide sequence encoding IL-15 operably linked to a regulatory sequence capable of functioning in mammalian cells. 59. The recombinant DNA vaccine of claim 56, wherein said vaccine is a recombinant vacinia vaccine. delete delete delete delete A pharmaceutical composition for immunizing an individual against a pathogen, cancer cell or autoimmune disease related cell, comprising the recombinant DNA vaccine of claim 56 and a pharmaceutically acceptable salt. A composition comprising a first plasmid and a second plasmid, (a) an IL-12 p35 subunit operably linked to a first promoter and an IL-12 p35 subunit operably linked to a first promoter operably linked to a regulatory sequence capable of functioning in a mammalian cell A nucleotide sequence encoding a -12 p40 subunit; (b) a DNA vaccine composition wherein the nucleotide sequence of said second plasmid operably linked to a regulatory sequence capable of functioning in a mammalian cell comprises a nucleotide sequence encoding an HIV-1 antigen. A composition comprising a first plasmid and a second plasmid, (a) the nucleotide sequence of said first plasmid operably linked to a regulatory sequence capable of functioning in a mammalian cell comprises a nucleotide sequence encoding a subunit of IL-12 operably linked to a first promoter; (b) the nucleotide sequence of said second plasmid operably linked to a regulatory sequence capable of functioning in a mammalian cell encodes a nucleotide that encodes another subunit of IL-12 operably linked to an HIV-1 antigen and a second promoter DNA vaccine composition comprising a sequence. The DNA vaccine composition of claim 66 or 67, further comprising a third plasmid comprising a nucleotide sequence encoding IL-15 operably linked to a regulatory sequence capable of functioning in a mammalian cell. 68. The DNA of claim 66 or 67, wherein said first plasmid or second plasmid further comprises a nucleotide sequence encoding IL-15 operably linked to a regulatory sequence capable of functioning in mammalian cells. Vaccine composition.

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