CN1257976C

CN1257976C - Immunotherapy and improved vaccines

Info

Publication number: CN1257976C
Application number: CNB97180897XA
Authority: CN
Inventors: J·J·基姆; 王宾; J·D·博耶; D·B·韦纳; V·阿亚沃
Original assignee: University of Pennsylvania Penn
Current assignee: University of Pennsylvania Penn
Priority date: 1996-10-23
Filing date: 1997-10-23
Publication date: 2006-05-31
Anticipated expiration: 2017-10-23
Also published as: AU5002297A; BR9712852A; CN1242045A; CA2745736A1; CA2269074A1; KR20050053796A; WO1998017799A1; EP0958364A4; CA2751712A1; KR100629835B1; AU729579B2; KR20050008860A; JP2008194046A; CN1868545A; KR20070108418A; KR100780158B1; CA2751712C; CA2745736C; EP0958364A1; JP2009232860A

Abstract

Improved vaccines which include a nucleotide sequence that encodes IL-12, GM-CSF, IL-1, TNF- alpha , TNF- beta , IL-2, IL-4, IL-5, IL-10, IL-15, IL-18, and/or BL-1 operably linked to regulatory elements are disclosed. The improved vaccines include DNA vaccines, recombinant vaccines for delivering foreign antigen and live attenuated vaccines. Methods of immunizing individuals are disclosed. Pharmaceutical compositions comprising nucleic acid molecules that encode one or more immunomodulatory proteins selected from the group consisting of IL-12, GM-CSF, IL-1, TNF- alpha , TNF- beta , IL-2, IL-4, IL-5, IL-10, IL-15, IL-18, and BL-1 are disclosed. An immunomodulatory protein, BL-1, and nucleic acid molecules that encode BL-1 are disclosed. Methods of making and using BL-1 are disclosed.

Description

The improvement vaccine

Invention field

The present invention relates to immunotherapy composition and method, and the protectiveness and the therapeutic vaccine of improvement, also relate to and be used for preventative and/or therapeutic is induced improving one's methods of anti-antigen immune response.

Background of invention

Vaccine can be used for immune body with the opposing target antigen, for example pathogenicity bo antigen or with the class disease in the cell related antigen that relates to.The antigen relevant with participating in the human diseases cell comprises cancer dependency tumour antigen and participates in the cell related antigen of autoimmune disease.

The total target of various immunization strategies all is the immune response of inducing specific, and the lifelong protection to certain given cause of disease by the immune is given in such replying.For a main difficult problem that reaches this purpose is, can change because of the variation of infectious factor by the mutual relationship of a certain cause of disease and the provide protection that causes thereof.So a more effective clinically vaccine should be induced its target cause of disease is produced the stronger immunne response of specificity.According to the design of the mutual relationship needs of known provide protection and specific pathogen with the immunization strategy always " focusing " in specific cause of disease.

State in design and have been found that in the process of vaccine that the vaccine that can produce target antigen in the individual cell of inoculation is the effective vaccine of induction of immunity system cells immunity.Specifically, the recombiant vaccine of attenuated live vaccine, employing non-virulent carrier and dna vaccination can both produce antigen in the cell individual by inoculation, thus the cellular immunization of induction of immunity system.On the other hand, only comprise proteinic subunit vaccine and vaccine dead or deactivation, inductive is the humoral response reaction, but can not induce the good cell immune response.

In order to treat pathogen infection, cancer or autoimmune disease, usually needing cellullar immunologic response to react provides the protection and the effective immune-mediated treatment of opposing pathogen infection.So, be preferably the vaccine that can in the cell of immunization individuality, produce target antigen, for example the recombiant vaccine of attenuated live vaccine, employing no pathogenicity carrier and dna vaccination.

Nucleic acid immunization is a kind of new inoculation technique, and its certain specific immunogenic DNA construction of will encoding is delivered to the host.Dna vaccination is except simultaneously inducing antigen-specific cell and humoral immunoresponse(HI), and it also may handle the immune response that is produced by the common transmission of important molecule on the immunology.

Though above-mentioned vaccine is by preventative or therapeutic immunization is individual often can resist pathogen infection or human body diseases effectively, but still needs improved vaccine, still need to produce the composition and the method for enhanced immunne response.

Summary of the invention

The present invention relates to gene constructs, it comprises the nucleotide sequence of the immune modulator of encoding, and described adjusting albumen can give accepting individuality preventative or therapeutic inoculation or therapeutic immunization adjusting therapy.These immune modulators comprise human body protein IL-12, GM-CSF, IL-1, TNF-α, TNF-β, IL-2, IL-4, IL-5, IL-10, IL-15, IL-18 and a kind of new molecule BL-1.

The gene constructs that the present invention relates to comprises: the nucleotide sequence of coding IL-12, GM-CSF, IL-1, TNF-α, TNF-β, IL-2, IL-4, IL-5, IL-10, IL-15, IL-18 or BL-1; Perhaps encode and be no less than two nucleotide sequence among IL-12, GM-CSF, IL-1, TNF-α, TNF-β, IL-2, IL-4, IL-5, IL-10, IL-15, IL-18 or the BL-1.The present invention wishes that gene constructs can comprise many parts of copies of same nucleotide sequence.

The present invention relates to the method for individual immunity inoculation, promptly lead people's immunogen and unite and lead the people's gene construction by giving individual vaccine composition, this gene constructs comprises one or more immune modulators of coding, thus the nucleotide sequence of immunne response that can be enhanced and/or better.And the invention still further relates to the method that the adjusting individual immunity is replied, promptly comprise the gene constructs of one or more immune modulator coding nucleotide sequences.For the adjusting of immunne response can be a step in the vaccination program: wherein with vaccine composition with promote a kind of immune modulator of immunne response form to give the patient jointly, give the patient with vaccine composition jointly with the immune modulator that promotes another kind of immunne response form then and carry out booster immunization, immunne response with the patient is transformed into Th2 by being mainly Th1 thus, or changes Th1 into by Th2.

Vaccine composition is preferably directly led the intravital plasmid of people.Similarly, also plasmid preferably of the gene constructs that comprises one or more immune modulator coding nucleotide sequences.

The present invention relates to a kind of plasmid, it comprises the nucleotide sequence of coding people nksf protein, this sequence is connected with the necessary controlling element operability of expression in eukaryotic cell, the nucleotide sequence that also comprises coding immunogenicity target antigen, this sequence is connected with the necessary controlling element operability of expression in eukaryotic cell.At some preferably in the embodiment, described immunogenicity target antigen is pathogenicity bo antigen, cancer associated antigen or the antigen that is connected with the autoimmune disease relevant cell.In certain embodiments, this plasmid comprises the nucleotide sequence of one section coding strand people nksf protein, and this sequence is connected with the necessary controlling element operability of expression in eukaryotic cell.Strand IL-12 albumen is a kind of monomer (single) albumen, and it is encoded by single encoding sequence, and comprises a joint that connects two subunits.Joint has enough sizes and snappiness, is enough to make this monomeric protein can be folded into the original shown biological activity conformation of functional two subunits.

The present invention relates to a kind of individual interior method of inducing at certain antigenic immunne response, it comprises the step that gives individual certain plasmid, described plasmid comprises the nucleotide sequence of coding people nksf protein, this sequence be connected in the necessary controlling element operability of this individual cell inner expression, the nucleotide sequence that also comprises the target antigen of encoding, this sequence be connected in the necessary controlling element operability of this individual cell inner expression.At some preferably in the embodiment, target antigen is pathogenicity bo antigen, cancer associated antigen or the antigen that is connected with the autoimmune disease relevant cell.In embodiment preferably; inductive at the immunne response of target antigen for treatment infection, disease, disorder and some illness (with anti-antigen immune react at albumen relevant) effect is provided; and/or; induce the protective immunological reaction of generation at cause of disease or some cell, the immunne response that albumen that these cells are contained and antigen produce has cross reaction.In some embodiment, people's nksf protein is a kind of strand IL-12 albumen.

The present invention relates to a kind of composition, it comprises a plurality of plasmids, and they have gathered the nucleotide sequence of coding people IL-12 two subunits and target antigen, and each encoding sequence is connected with the essential controlling element operability of genetic expression respectively.In certain embodiments, composition comprises two kinds of plasmids: first kind of plasmid comprises the proteic nucleotide sequence of coding IL-12, and it is connected with the necessary controlling element operability of expression in eukaryotic cell; Second kind of plasmid comprises the nucleotide sequence of coding immunogenicity target antigen, and it is connected with the necessary controlling element operability of expression in eukaryotic cell.In certain embodiments, a plasmid comprises the nucleotide sequence of a coding immunogenicity target antigen and a subunit of people IL-12, and second plasmid then comprises the nucleotide sequence of another subunit of coding IL-12.In certain embodiments, 3 kinds of different plasmids are provided: one comprises the nucleotide sequence of coding immunogenicity target protein, its two nucleotide sequence that comprises the p35 subunit of coding people IL-12, another then comprises the nucleotide sequence of the p40 subunit of coding people IL-12.In some embodiment, said composition comprises two kinds of plasmids, first plasmid comprises the proteic nucleotide sequence of coding strand IL-12, this sequence is connected with the necessary controlling element operability of expression in eukaryotic cell, second plasmid comprises the nucleotide sequence of coding immunogenicity target antigen, and this sequence also is connected with the necessary controlling element operability of expression in eukaryotic cell.

The present invention relates in individuality, induce method at certain antigenic immunne response, it comprises the step that gives the group of individuals compound, this composition contains a plurality of plasmids, these plasmids have been gathered the nucleotide sequence of coding people's IL-12 two subunits and target antigen, and each encoding sequence all must the controlling element operability be connected with genetic expression.In certain embodiments, two or three above-mentioned plasmid comprises the nucleotide sequence of coding people's IL-12 two subunits and immunogenicity target protein jointly, the nucleotide sequence of coded protein be connected in the necessary controlling element operability of the cell inner expression of individuality.In embodiment preferably, by target antigen inductive immunne response and pathogenicity bo antigen, cancer associated antigen or the antigen generation cross reaction that is connected with the autoimmune disease relevant cell.The present invention relates to the method for immunization individuality with opposing cause of disease, cancer or autoimmune disease.In embodiment preferably, target antigen is pathogenicity bo antigen, cancer associated antigen or the antigen that is connected with the autoimmune disease relevant cell.In some embodiment, people IL-12 is a strand IL-12 albumen.

The present invention relates to a kind of plasmid, one or more in its contained nucleotide sequence coded human GM-CSF, IL-1 α, TNF-α, TNF-β, IL-2, IL-15, IL-18, IL-4, the IL-5h IL-10 albumen, described nucleotide sequence be connected in the necessary controlling element operability of the cell inner expression of individuality, the nucleotide sequence that also comprises the target antigen of encoding, this sequence be connected in the necessary controlling element operability of the cell inner expression of individuality.At some preferably in the embodiment, the immunogenicity target antigen is a kind of pathogenicity bo antigen, cancer associated antigen or the antigen that is connected with the autoimmune disease relevant cell.At some preferably among the embodiment, the immunogenicity target antigen is relevant anti-source of pathogenicity bo antigen, cancer or the anti-source that is connected with the autoimmune disease relevant cell.

The present invention relates in individuality, induce method at the immunne response in certain anti-source, it comprises the step that gives individual a kind of plasmid, one or more in the contained nucleotide sequence coded human GM-CSF of this plasmid, IL-12, INF-α, INF-β, IL-2, IL-15, IL-18, IL-4, IL-5 or the IL-10 albumen, this nucleotide sequence with in individual cells, express necessary controlling element operability and be connected.It also comprises the nucleotide sequence that gives the anti-source of individual coding target, and this sequence is connected with the necessary controlling element operability of expression in an individual cell.In some preferred embodiments, this target antigen is a pathogenicity bo antigen, cancer associated antigen or the antigen that is connected with the autoimmune disease relevant cell.In embodiment preferably; institute's inductive at the immunne response of target antigen be infection, disease, disorder and some illness (with anti-antigen immune reaction at albumen relevant) result of treatment is provided; and/or; induce the protective immune response that has produced at cause of disease or some cell, the contained albumen of these cells has cross reaction with the immunne response at target antigen that is produced.

The present invention relates to a kind of composition, it comprises a plurality of plasmids, comprising two kinds of plasmids: one or more in nucleotide sequence coded human GM-CSF, IL-1 α, TNF-α, TNF-β, IL-2, IL-15, IL-18, IL-4, IL-5 or the IL-10 albumen that first kind of plasmid comprises, these sequences be connected in the necessary controlling element operability of the cell inner expression of individuality, second plasmid comprises the nucleotide sequence of coding immunogenicity target antigen, this sequence be connected in the necessary controlling element operability of the cell inner expression of individuality.Said composition comprises three kinds of plasmids in some embodiment, the 3rd plasmid wherein comprises one or more nucleotide sequence in coding GM-CSF, IL-1 α, TNF-α, TNF-β, IL-2, IL-15, IL-18, IL-4, IL-5 or the IL-10 albumen, and these sequences are connected with the necessary controlling element operability of expression in eukaryotic cell.In some embodiment, said composition comprises 4 kinds of plasmids, wherein the 3rd plasmid comprises one or more nucleotide sequence in coding human GM-CSF, IL-1 α, TNF-α, TNF-β, IL-2, IL-15, IL-18, IL-4, IL-5 or the IL-10 albumen, and these sequences are connected with the necessary controlling element operability of eukaryotic cell expression; And the 4th plasmid comprise one or more nucleotide sequence in coding GM-CSF, IL-1 α, TNF-α, TNF-β, IL-2, IL-15, IL-18, IL-4, IL-5 or the IL-10 albumen, these sequences are connected with the necessary controlling element operability of expression in eukaryotic cell.

The present invention relates in individuality, induce method at certain antigenic immunne response, it comprises and gives individual a kind of composition, comprise a plurality of plasmids in the said composition, comprising two kinds of plasmids: first kind of plasmid comprises one or more nucleotide sequence in coding human GM-CSF, IL-1 α, TNF-α, TNF-β, IL-2, IL-15, IL-18, IL-4, IL-5 or the IL-10 albumen, and these sequences are connected with necessary for gene expression controlling element operability; Second kind of plasmid comprises the nucleotide sequence of coding immunogenicity target antigen, and this sequence is connected with the necessary controlling element operability of expression.In embodiment preferably, by target antigen inductive immunne response and pathogenicity bo antigen, cancer associated antigen or the antigen generation cross reaction that is connected with the autoimmune disease relevant cell.The present invention relates to the method for immunization individuality with opposing cause of disease, cancer or autoimmune disease.In embodiment preferably, target antigen is pathogenicity bo antigen, cancer associated antigen or the antigen that is connected with the autoimmune disease relevant cell.In some embodiment, described method comprises and gives certain composition, said composition comprises 3 kinds of plasmids, the 3rd plasmid wherein comprises one or more nucleotide sequence in coding human GM-CSF, IL-1 α, TNF-α, TNF-β, IL-2, IL-15, IL-18, IL-4, IL-5 or the IL-10 albumen, and these sequences are connected with the necessary controlling element operability of expression in eukaryotic cell.In some embodiment, described method comprises and gives certain composition, said composition comprises 4 kinds of plasmids, wherein the third plasmid comprises the coding human GM-CSF, IL-1 α, TNF-α, TNF-β, IL-2, IL-15, IL-18, IL-4, one or more nucleotide sequence in IL-5 or the IL-10 albumen, these sequences are connected with the necessary controlling element operability of eukaryotic cell expression, and the 4th kind of plasmid, it comprises the coding human GM-CSF, IL-1 α, TNF-α, TNF-β, IL-2, IL-15, IL-18, IL-4, one or more nucleotide sequence in IL-5 or the IL-10 albumen, these sequences are connected with the necessary controlling element operability of expression in eukaryotic cell.

The present invention relates to a kind of recombinant vaccine vector of improvement, it comprises one or more nucleotide sequence in coding people IL-12, GM-CSF, IL-1 α, TNF-α, TNF-β, IL-2, IL-15, IL-18, IL-4, IL-5, IL-10 or the BL-1 albumen, these sequences are connected with the necessary controlling element operability of expression in eukaryotic cell, the nucleotide sequence that also comprises the target antigen of encoding, this sequence also are connected with the necessary controlling element operability of expression in eukaryotic cell.In some embodiment, the IL-2 of the proteic encoding gene coding of human IL-2 single chain protein form.In embodiment preferably, target antigen is pathogenicity bo antigen, cancer associated antigen or the antigen that is connected with the autoimmune disease relevant cell.

The present invention relates to a kind of immunization individuality with the opposing cause of disease, the method of cancer or autoimmune disease, it comprises the step that gives individual a kind of recombinant vectors, this vector encoded people IL-12, GM-CSF, IL-1 α, TNF-α, TNF-β, IL-2, IL-15, IL-18, IL-4, IL-5, one or more nucleotide sequence in IL-10 or the BL-1 albumen, these sequences be connected in the necessary controlling element operability of the cell inner expression of individuality, the nucleotide sequence that also comprises the target antigen of encoding, this sequence also with in the necessary controlling element operability of the cell inner expression of individuality is connected, and target antigen wherein is a pathogenicity bo antigen, cancer associated antigen or the antigen that is connected with the autoimmune disease relevant cell.

The attenuated live vaccine that the present invention relates to improve, it comprises one or more nucleotide sequence in coding people IL-12, GM-CSF, IL-1 α, TNF-α, TNF-β, IL-2, IL-15, IL-18, IL-4, IL-5, IL-10 or the BL-1 albumen, and these sequences are connected with the necessary regulating and controlling sequence operability of expression in eukaryotic cell.

The present invention relates to the method for a kind of immunization individuality with opposing cause of disease, cancer or autoimmune disease, it comprises the step that gives individual attenuated vaccine, this vaccine comprises one or more nucleotide sequence in coding people IL-12, GM-CSF, IL-1 α, TNF-α, TNF-β, IL-2, IL-15, IL-18, IL-4, IL-5, IL-10 or the BL-1 albumen, these sequences be connected in the necessary regulating and controlling sequence operability of the cell inner expression of described individuality.

The present invention relates to a kind of plasmid, it comprises one or more nucleotide sequence in coding people IL-12, GM-CSF, IL-1 α, TNF-α, TNF-β, IL-2, IL-15, IL-18, IL-4, IL-5, IL-10 or the BL-1 albumen, and these sequences are connected with the necessary regulating and controlling sequence operability of expression in eukaryotic cell.

The present invention relates to a pair of plasmid, one of them plasmid contains the nucleotide sequence of coding people IL-12 albumen p35 subunit, this sequence is connected with the necessary regulating and controlling sequence operability of expression in eukaryotic cell, another plasmid contains the nucleotide sequence of coding people IL-12 albumen p40 subunit, and this sequence is connected with the necessary regulating and controlling sequence operability of expression in eukaryotic cell.

The present invention relates to a kind of plasmid, it contains the proteic nucleotide sequence of coding strand people IL-12, this sequence is connected with the necessary regulating and controlling sequence operability of expression in eukaryotic cell, wherein strand people IL-12 albumen is a kind of monomeric protein, its p35 subunit and p40 subunit are connected to each other by a joint sequence, when being expressed, this single chain protein can form the IL-12 molecule of biologically active.

The present invention relates to a kind of pharmaceutical composition, it comprises a kind of plasmid and pharmaceutically acceptable carrier or thinner, this plasmid contains one or more nucleotide sequence in coding people IL-12, GM-CSF, IL-1 α, TNF-α, TNF-β, IL-2, IL-15, IL-18, IL-4, IL-5, IL-10 or the BL-1 albumen, and these sequences are connected with the necessary regulating and controlling sequence operability of expression in eukaryotic cell.

The present invention relates to a kind of pharmaceutical composition, it comprises a pair of plasmid and pharmaceutically acceptable carrier or thinner, a plasmid contains the nucleotide sequence of coding people IL-12 albumen p35 subunit, this sequence is connected with the necessary regulating and controlling sequence operability of expression in eukaryotic cell, another plasmid contains the nucleotide sequence of coding people IL-12 albumen p40 subunit, and this sequence is connected with the necessary regulating and controlling sequence operability of expression in eukaryotic cell.

The present invention relates to a kind of pharmaceutical composition, it comprises a kind of plasmid and pharmaceutically acceptable carrier or thinner, this plasmid contains the proteic nucleotide sequence of coding strand people IL-12, this sequence is connected with the necessary regulating and controlling sequence operability of expression in eukaryotic cell, wherein strand people IL-12 albumen is a kind of monomeric protein, its p35 subunit and p40 subunit are connected to each other by a joint sequence, and when being expressed, this single chain protein can form the IL-12 molecule of biologically active.

The present invention relates to treat suffer from anaphylaxis, the methods of treatment of pathogen infection, cancer or autoimmune disease individuality, comprise the step that gives a kind of plasmid of patient, this plasmid contains coding IL-12 proteic nucleotide sequence, and this sequence be connected in the necessary regulating and controlling sequence operability of the cell inner expression of individuality.

The present invention relates to treat suffer from anaphylaxis, the methods of treatment of pathogen infection, cancer or autoimmune disease individuality, comprise the step that gives a pair of plasmid of patient, one of them plasmid contains the nucleotide sequence of coding people IL-12 albumen p35 subunit, this sequence is connected with the necessary regulating and controlling sequence operability of expression in eukaryotic cell, another plasmid contains the nucleotide sequence of coding people IL-12 albumen p40 subunit, and this sequence is connected with the necessary regulating and controlling sequence operability of expression in eukaryotic cell.

The present invention relates to the methods of treatment that treatment suffers from anaphylaxis, pathogen infection, cancer or autoimmune disease individuality, comprise the step that gives a kind of plasmid of patient, this plasmid contains the proteic nucleotide sequence of coding strand people IL-12, this sequence is connected with the necessary regulating and controlling sequence operability of expression in eukaryotic cell, described strand people IL-12 albumen is a kind of monomeric protein, its p35 subunit and p40 subunit are connected to each other by a joint sequence, when being expressed, this single chain protein can form the IL-12 molecule of biologically active.

The present invention relates to strengthen or promote the method that individual interior immunne response changes to Th1 type immunne response, it comprises the step that gives individual a kind of plasmid, this plasmid contains the nucleotide sequence of coding people nksf protein, this sequence be connected in the necessary controlling element operability of the cell inner expression of individuality.

The present invention relates to a kind of strengthen individual in TH1 type immunne response or promote the method that intraindividual immunne response changes to the TH1 type, it comprises the step that gives individual a pair of plasmid, one of them plasmid contains the nucleotide sequence of coding people IL-12 albumen p35 subunit, this sequence is connected with the necessary regulating and controlling sequence operability of expression in eukaryotic cell, another plasmid contains the nucleotide sequence of coding people IL-12 albumen p40 subunit, and this sequence is connected with the necessary regulating and controlling sequence operability of expression in eukaryotic cell.

The present invention relates to a kind of strengthen individual in TH1 type immunne response or promote the method that intraindividual immunne response changes to the TH1 type, it comprises the step that gives individual a kind of plasmid, this plasmid contains the proteic nucleotide sequence of coding strand people IL-12, this sequence is connected with the necessary regulating and controlling sequence operability of expression in eukaryotic cell, wherein strand people IL-12 albumen is a kind of monomeric protein, its p35 subunit and p40 subunit are connected to each other by a joint sequence, when being expressed, this single chain protein can form the IL-12 molecule of biologically active.

The present invention relates to a kind of recombinant vectors, it comprises the proteic nucleotide sequence of coding people IL-12, and this sequence is connected with the necessary controlling element operability of expression in eukaryotic cell.

The present invention relates to a kind of recombinant vectors, it comprises the proteic nucleotide sequence of coding strand people IL-12, this sequence is connected with the necessary controlling element operability of expression in eukaryotic cell, wherein strand people IL-12 albumen is a kind of monomeric protein, its p35 and p40 subunit are connected to each other by a joint sequence, when expressing, this single chain protein can form biological activity IL-12 molecule.

The present invention relates to a kind of pharmaceutical composition, it comprises a kind of recombinant vectors and pharmaceutically acceptable carrier or thinner, described recombinant vectors contains one or more nucleotide sequence in coding people IL-12, GM-CSF, IL-1 α, TNF-α, TNF-β, IL-2, IL-15, IL-18, IL-4, IL-5, IL-10 or the BL-1 albumen, and these sequences are connected with the necessary regulating and controlling sequence operability of expression in eukaryotic cell.

The present invention relates to a kind of pharmaceutical composition, it comprises a kind of recombinant vectors and pharmaceutically acceptable vehicle or thinner, described recombinant vectors contains the proteic nucleotide sequence of coding strand people IL-12, this sequence is connected with the necessary controlling element operability of expression in eukaryotic cell, wherein strand people IL-12 albumen is a kind of monomeric protein, its p35 and p40 subunit are connected to each other by a joint sequence, and when expressing, this single chain protein can form biological activity IL-12 molecule.

The present invention relates to treat suffer from anaphylaxis, the methods of treatment of pathogen infection, cancer or autoimmune disease individuality, comprise the step that gives a kind of recombinant vectors of patient, described recombinant vectors contains the proteic nucleotide sequence of coding people IL-12, and this sequence is connected with the necessary regulating and controlling sequence operability of expression in eukaryotic cell.

The present invention relates to treat suffer from anaphylaxis, the methods of treatment of pathogen infection, cancer or autoimmune disease individuality, comprise the step that gives a kind of recombinant vectors of patient, described recombinant vectors contains the proteic nucleotide sequence of coding strand people IL-12, this sequence is connected with the necessary regulating and controlling sequence operability of expression in eukaryotic cell, wherein strand people IL-12 albumen is a kind of monomeric protein, its p35 subunit and p40 subunit are connected to each other by a joint sequence, when being expressed, this single chain protein can form the IL-12 molecule of biologically active.

The present invention relates to a kind of strengthen individual in Th1 type immunne response or promote the method that immunne response changes to Th1 type immunne response, it comprises the step that gives individual a kind of recombinant vectors, this recombinant vectors contains the nucleotide sequence of coding people nksf protein, and this sequence is connected with the necessary controlling element operability of expression in eukaryotic cell.

The present invention relates to a kind of strengthen individual in TH1 type immunne response or promote the method that intraindividual immunne response changes to the TH1 type, it comprises the step that gives individual a kind of recombinant vectors, described recombinant vectors contains the proteic nucleotide sequence of coding strand people IL-12, this sequence is connected with the necessary regulating and controlling sequence operability of expression in eukaryotic cell, wherein said strand people IL-12 albumen is a kind of monomeric protein, its p35 subunit and p40 subunit are connected to each other by a joint sequence, when being expressed, single chain protein can form the IL-12 molecule of biologically active.

The invention provides composition, they contain the preceding inflammatory cytokine of one or more people of coding: (IL-1 α, TNF-α and TNF-β), Th1 cytokine (IL-12, IL-15 and IL-18), with the proteic nucleotide sequence of Th2 cytokine (IL-4, IL-5 and IL-10), with this as major ingredient, also relate to the method that promotes or guide immunne response with said composition, promptly give individual above-mentioned nucleic acid molecule.The present invention relates to strengthen the method for antigen-specific humoral response, promptly give IL-5 or IL-8 and vaccine simultaneously, this vaccine has been introduced the such target immunogen of dna vaccination construction.The present invention relates to strengthen the method for antigen specific T helper propagation, promptly give IL-2, IL-5, IL-10, IL-18, TNF-α or TNF-β and vaccine simultaneously, this vaccine such target immunogen of dna vaccination construction that induced one.The invention still further relates to the method for strengthening cytotoxic response, promptly give TNF-α or IL-5 gene and vaccine simultaneously, this vaccine such target immunogen of dna vaccination construction that induces one.

The invention provides composition, they comprise the nucleic acid molecule of the human GM-CSF of encoding, and the invention still further relates to the method for inducing and regulating immunne response, promptly give or the gene constructs of the GM-CSF that encodes simultaneously.The present invention relates to reply method with the t helper cell proliferative response by injecting GM-CSF gene and dna vaccination construction enhancement antigen specific antibody simultaneously.

The present invention relates to pure substantially BL-1 and immunomodulatory fragment thereof.

The present invention relates to isolated nucleic acid molecule, their coding BL1 and immunomodulatory fragments thereof.

The present invention relates to nucleic acid probe and primer, they are specifically at coding BL1 or the segmental nucleic acid molecule of its immunomodulatory.

The present invention relates to oligonucleotide molecules, they are made of the complementary nucleotide sequence of the specificity part of BL1 coding nucleotide sequence.

The present invention relates to comprise the carrier of coding BL1 or the segmental nucleic acid molecule of its immunomodulatory.

The present invention relates to recombinant expression vector, they comprise coding BL1 or the segmental nucleotide sequence of its immunomodulatory.

The present invention relates to comprise the host cell of recombinant expression vector, described carrier comprises coding BL1 or the segmental nucleotide sequence of its immunomodulatory.The present invention relates to the gene therapy carrier, it comprises coding BL1 or the segmental nucleic acid molecule of its immunomodulatory.

The present invention relates to isolated antibody, the specificity epitope on this antibodies BL1.

The present invention relates to make the segmental method of BL1 and immunomodulatory thereof.

The present invention relates to regulate the method that individual immunity is replied, comprise giving individual BL1 albumen or its immunomodulatory fragment, or contain the carrier of coding BL1 albumen or the segmental nucleotide sequence of its immunomodulatory.According to the present invention, the carrier that contains the BL1 keying sequence is enough to regulate this immunne response.

The present invention relates to the method strengthening and guide individual immunity to reply, comprise that giving individuality is used for premunition former and BL1 albumen or the segmental a kind of vaccine composition of its immunomodulatory, or contain the carrier of coding BL1 albumen or the segmental nucleotide sequence of its immunomodulatory.According to the present invention, the carrier that contains the BL1 keying sequence is enough to regulate immunne response.

The accompanying drawing summary

What Figure 1A and 1B showed is the data that embodiment 4 draws.In Figure 1A, at each cDNA expression cassette of first day intramuscularly 50 μ g.Immunity back the 14th day is collected all and is accepted the spleen of immune animal and weigh.Give the negative control animal immune.Only inject the spleen similar to the spleen weight of non-immune control mice (about 100mg) of Gag/Pol and the mouse of only injecting IL-12.But its weight of spleen of the mouse of injection Gag/Pol+IL-12 almost is three times of control mice spleen.On the contrary, the spleen of Gag/Pol+GM-CSD immune mouse does not increase.In Figure 1B, from each spleen preparation and purifying white corpuscle.Directly corresponding with their spleen weight difference, the cell quantity of Gag/Pol+IL-12 immunity spleen surpasses more than three times of spleen of contrast.The lymphocyte number of Gag/Pol+GM-CSF immune mouse spleen is not compared with the lymphocyte number of contrast spleen obviously to be increased.

Among Fig. 2, at each cDNA expression cassette of first day intramuscularly 50 μ g.Immunity back the 14th day, results spleen and photography.The apparent size of spleen is directly corresponding with its weight, and the spleen of having inoculated immunogen+IL-12 is obviously greater than nonvaccinated contrast spleen.Group: (one) does not inoculate; The IL-12 inoculation; Empty carrier+IL-12 inoculation; The Gag/Pol+IL-12 inoculation.

Among Fig. 3, give in IL-12 two chains simultaneously.Each plasmid uses 50 μ g respectively.P35 and p40 subunit and Gag/Pol are that the spleen increase is necessary.

Among Fig. 4, at each cDNA expression cassette of first day intramuscularly 50 μ g.Collect the antiserum(antisera) of immune mouse, by the antigenic specific antibody reaction of elisa assay anti-HIV-1.That show is the ELISA result of the sample of collection in the 28th day.When extent of dilution was 1: 100, the antiserum(antisera) of empty carrier+GM-CSF immune group showed the proteic antibody response of anti-HIV-1 gp120, and this reaction is better than the group of only using the empty carrier immunity.On the other hand, after the same time, show obvious more weak humoral response with the group of empty carrier+IL-12 immunity.

Among Fig. 5, the activation of t helper cell and propagation play an important role in inducing humoral immunization (amplification of the B cell by antigen activates) reaction, play an important role in inducing cell immunity (by the amplification of CD8+ cytotoxic T cell) reaction simultaneously.Each cDNA expression cassette of first day intramuscularly 50 μ g.Collect spleen cell, the propagation of test T cell.In each hole of test panel, add the reorganization p55 albumen of 20 μ g/ml, be used to stimulate T cell proliferation.10 μ g/ml lectin PHA are used as polyclone stimulator positive control.Stimulation index is the record level of the radioactivity level of specific protein stimulated cells divided by cell in the medium.The stimulation index of PHA stimulated control is 58.8.

Among Fig. 6, each cDNA expression cassette of first day intramuscularly 50 μ g.The results spleen, the preparation cell does not have the CTL test of stimulated in vitro with these cells.Control group is only used the immunity of IL-12 box gene, and the target cell SL is no more than background level as a result.In addition, only use Gag/Pol immunity, effect at 50: 1: target than the time observed the SL of low-level (3%).On the contrary, give the sample of Gag/Pol+IL-12 simultaneously, effect: can see target at this moment 62% specificity cracking, when effect: when the target ratio is 12.5: 1, be 9% through titration at 50: 1.With Gag/Pol+GM-CSF plasmid immunization, the CTL activity that the result can not the survey level.With the target cell of non-correlation antigen presentation Smallpox Vaccine preparation, carry out same CTL test, the result is without any tangible ctl response.

Among Fig. 7, each cDNA expression cassette of first day intramuscularly 50 μ g.The results spleen, the preparation cell does not have the CTL test of stimulated in vitro with these cells.Effect at 50: 1: target than the time, only immune and low-level specific CTL is arranged with the animal groups of empty carrier+GM-CSF immunity with empty carrier, be respectively 4% and 1%.On the other hand,, find the active significantly enhancing of its CTL, reach 59% with the group of empty carrier+IL-12 immunity.With the target cell of non-correlation antigen presentation Smallpox Vaccine preparation, carry out same CTL test, the result is without any tangible ctl response.

Fig. 8 A, 8B and 8C show the used plasmid of the present invention.The plasmid that Fig. 8 A shows comprises the encoding sequence of IL-12 single chain protein.The plasmid that Fig. 8 B and Fig. 8 C show contains the encoding sequence of two subunits separately respectively.

Fig. 9 indicator gauge 3.

Among Figure 10, various cytokine genes are cloned in the expression plasmid, be under the control of CMV promotor, and in-vitro transfection are in the RD cell.Various cytokine expression confirm with immuno-precipitation or cytokine ELISA.

Figure 11 A-11O shows the result of the CTL test that detects I class MHC restriction.Shown in Figure 11 A-E, DNA and pCEnv inject (each 50 μ g) 2 weeks of back jointly for the first time, with same dosage mouse (4 1 group) are carried out booster immunization.Spent for 1 week again, gather the spleen of inoculation mouse, separate its lymphocyte, detect ctl response with target cell, this target cell it is reported that with empty carrier-particular peptide (RIHIGPGRAFYTTKN) preparation this particular peptide is that I class MHC is restrictive in the balb/c mouse.Shown in Figure 11 F-110, DNA and pCGag/pol inject (each 50 μ g) 2 weeks of back jointly for the first time, with same dosage mouse (4 1 group) are carried out booster immunization.Spent for 1 week again, gather the spleen of immune mouse, isolate lymphocyte, the test ctl response.Remove the CD8+T cell by the complement dissolving and carry out the CTL test.(figure below) preparation effector cell behind (last figure) in the presence of the CD8+T cell and removal CD8+T cell.More than experiment repeats twice, and the result is similar.

Figure 12 shows evaluation direct antigen-specific CTL result of experiment (not having the vitro effect cytositimulation).DNA and pCEnv inject (each 50 μ g) 2 weeks of back jointly for the first time, with same dosage mouse (4 1 group) are carried out booster immunization.Spent for 1 week again, gather the spleen of immune mouse, isolate lymphocyte, test ctl response with the target cell that specificity (vMN462) and non-specific (vSC8) Smallpox Vaccine infect.More than experiment repeats, and the result is similar.

Figure 13 has summarized and has given various cytokine antagonists (Y-axis) jointly, the effect of t helper cell (X-axis) and cytotoxic T lymphocyte reaction (Z axle).Various cytokines are plotted on 3 dimension coordinates according to its effect to three kinds of immune response patterns.

Figure 14 shows the nucleotide sequence of BL1 and the aminoacid sequence of inferring.

Figure 15 shows that BL1 is connected to process and the pBBKan carrier in the PCR3 carrier for expression of eukaryon.

Figure 16 shows the result of ELISA test.This test has been compared and has been given simultaneously and when not giving BL1 simultaneously, at the anti-HIV antigen-reactive of HIV antigen Nef.

Figure 17 A, 17B, 17C and 17D show test-results, and this test has been compared and given simultaneously and when not giving BL1 simultaneously, at the anti-HIV antigen immune reaction of HIV antigen Gag/Pol.

Detailed description of the present invention

Term used herein " immune modulator " refers to one of people IL-12, GM-CSF, IL-1 α, TNF-α, TNF-β, IL-2, IL-15, IL-18, IL-4, IL-5 and IL-10 and new BL-1 molecule.

Term used herein " IL-12 gene constructs " refers to following plasmid: comprise the coded sequence of one or two subunit of encoding human IL-12 albumen and/or immunogenicity target protein, these coded sequences with in eukaryotic, express required controlling element operability and be connected. The DNA expression regulation element comprises promoter and polyadenylation signal. In addition, other element such as Kozak district also can be included in this gene constructs. Initial signal and termination signal are essential controlling elements, and they often are considered to belong to coded sequence. The coded sequence of gene constructs of the present invention comprises functional initial signal and termination signal.

Term used herein " required IL-12 albumen " refers to one or two subunit of people IL-12, comprising strand IL-12 albumen, two subunits are encoded by a coded sequence in this albumen, and are expressed as the single chain protein that is connected two subunits by a joint sequence.

Term used herein " required albumen " refers to that described vaccine comprises the nucleic acid molecules of coding immunogenicity target protein by the immunogenicity target protein of the coded sequence coding of vaccine.

Term used herein " single chain protein " and " strand IL-12 albumen " refer to following single chain protein, wherein the p35 subunit of IL-12 and p40 subunit are connected to each other by one section amino acid joint, this joint long enough and enough pliable and tough, allow two subunits of this single chain protein partly to interact, form the biologically active complex conformation, namely IL-12. The IL-12 that the function of strand IL-12 and p35 and p40 form is basic identical. Present invention resides in all occasions that uses IL-12 and use strand IL-12. This single chain protein is nucleotide sequence coded by one.

Interleukin 12 (IL-12) is a kind of heterodimer cell factor, is mainly produced by macrophage and B cell. IL-12 is made of two different subunits that are called p35 and p40, (Podlaski, F.J. etc., (1991) Arch.Biochem.Biophys.294 (1): 230-237, this paper receives it as a reference).

Different immune responses relates to different T cell masses. Specifically, two kinds of dissimilar T cells are arranged, I type t helper cell (Th1) and 2 type T cells (Th2), their differences each other are the cell factor that produces. Finding that IL-12 plays an important role in the Th1 immune response, mainly is to induce to produce Th1 correlation interferon-γ (IFN-γ). It activates natural killer cell (NK) and T cell by inducing and discharging the cytokine profiles that comprises IFN-γ.

The nucleic acid molecules that some aspects of the present invention relate to encoding human IL-12 albumen is used as immunomodulator. The nucleic acid molecules of encoding human IL-12 albumen can be used as main active ingredient and transmits, and namely as gene therapy or as the part of vaccine combination or be incorporated into wherein, for example comprises the vaccine of coding immunogenicity target protein nucleic acid molecules.

The purposes of the nucleic acid molecules of relevant encoding human IL-12 albumen as Main Ingredients and Appearance the invention provides and promotes immune response to the development of Th1 immune response or strengthen this composition of replying and method, namely gives the nucleic acid molecules of individual encoding human IL-12 albumen. According to some aspects of the present invention, anaphylactic disease, pathogen infection, cancer or Serum of Patients With Autoimmune Diseases can be treated by giving following nucleic acid molecules, this nucleic acid molecules comprises the nucleotide sequence of encoding human IL-12, this sequence be connected in the necessary controlling element operability of patient's cell inner expression. This nucleic acid molecules is taken the photograph the people by cell, and the nucleotide sequence of encoding human IL-12 is expressed. The people IL-12 that is so produced by cell has biologically active, and its activity inducement and/or strengthened the immune response that the patient produces. Preferably in the embodiment, the nucleic acid molecules of encoding human IL-12 albumen is plasmid at some.

The present invention includes the application of the nucleic acid molecules of coding granulocyte-macrophage colony stimutaing factor (GM-CSF), GM-CSF is a kind of hemopoieticgrowth factor, and it stimulates the formation of neutrophil leucocyte, monocyte/macrophage and acidophic cell colony. It also induces propagation and the differentiation of erythron and megacaryocyte CFU-GM. GM-CSF also improves the cytotoxicity of the antibody dependent cellular mediation of neutrophil leucocyte, acidophic cell and macrophage, but it is reported that it does not have direct effect to the generation that CTL replys in vivo. The invention provides the composition and the method that promote immune response, namely give the gene constructs of the nucleotide sequence of individual coding GM-CSF. This nucleic acid molecules is taken the photograph the people by cell, and the nucleotide sequence of coding GM-CSF is thus by cellular expression and generation. The GM-CSF that produces has biologically active, this activity inducement and/or strengthened individual immune response. Preferably in the embodiment, the nucleic acid molecules of coding GM-CSF is plasmid at some.

The present invention includes as inflammatory cytokine before the encoding human of Main Ingredients and Appearance (IL-1 α, TNF-α and TNF-β), Th1 cell factor (IL-2, IL-15 and IL-18) and the purposes of the nucleic acid molecules of Th2 cell factor (IL-4, IL-5 and IL-10) albumen. The invention provides composition and method that promotion immune response direction or booster immunization are replied, namely give the front inflammatory cytokine (IL-1 α, TNF-α and TNF-β) of individual encoding human, Th1 cell factor (IL-2, IL-15 and IL-18) and the nucleic acid molecules of Th2 cell factor (IL-4, IL-5 and IL-10). According to partial content of the present invention, patient's acceptance comprises the treatment of the nucleic acid molecules of following nucleotide sequence, inflammatory cytokine (IL-1 α, TNF-α and TNF-β), Th1 cell factor (IL-2 before these nucleotide sequence coded people, IL-15 and IL-18) and Th2 cell factor (IL-4, IL-5 and IL-10), described nucleotide sequence be connected in the necessary controlling element operability of patient's cell inner expression. These nucleic acid molecules are taken the photograph the people by cell, and the nucleotide sequence of the above-mentioned albumen of encoding is expressed: the human protein that is so produced by cell has biologically active, this activity inducement and/or strengthened the immune response of individual generation. Preferably in the embodiment, described nucleic acid molecules is plasmid at some.

The purposes of the nucleic acid molecules of the part of relevant nucleic acid molecules as coding immunogenicity target protein or encoding human IL-12 albumen that are attached thereto, namely form the immune response of inducing anti-immunogenic protein as vaccine, the nucleic acid molecules of encoding human IL-12 albumen can be one of composition of vaccine, described vaccine comprises the nucleic acid molecules of coding immunogenicity target protein, it can be one of the composition that comprises the vaccine of immunogenicity target antigen, or independent composition, the vaccine of said composition and the nucleic acid molecules that contains coding immunogenicity target protein or comprise that the vaccine of immunogenicity target gives simultaneously. Preferably in the embodiment, the nucleic acid molecules of encoding human IL-12 albumen is plasmid at some, and vaccine is the dna vaccination that comprises coding immunogenicity target protein plasmid. Preferably in the embodiment, described dna vaccination comprises the plasmid of coding immunogenicity target protein and people IL-12 albumen at some.

IL-12 can be referring to disclosed PCT application on May 17 nineteen ninety WO 90/05147, and this paper receives it and is reference. Wolf, S.F. etc., 1991, J.Immnol.146 (9): 3074-3081, this paper receive it and are reference, have disclosed the nucleotide sequence of cDNA of coding IL-12 and the predetermined amino acid sequence of IL-12 albumen. Natural human IL-12 albumen is made of p35 and p40 two subunits. These two subunits form has bioactive different dimeric complexes.

According to some embodiment of the present invention, the nucleotide sequence of coding IL-12 two subunits can be positioned on a plasmid, non-plasmid nucleic acid molecules or virus or the bacterial genomes, and the nucleotide sequence of two subunits of wherein encoding is under the control of complete controlling element separately. Preferably in the embodiment, the coded sequence of IL-12 two subunits is positioned on the plasmid at some; Each coded sequence is connected with separately complete controlling element operability. In some embodiment, the coded sequence of certain immunogenicity target protein is connected with the controlling element operability, is positioned on the same plasmid with the coded sequence of two subunits. In some embodiment, the coded sequence of certain immunogenicity target protein is connected with the controlling element operability, is positioned on another plasmid separately, and namely not on the plasmid that contains two subunit coded sequences, these two kinds of plasmids all pass to same patient.

According to part embodiment of the present invention, the nucleotide sequence of coding p35 subunit is positioned on first plasmid, and the nucleotide sequence of coding p40 subunit is positioned on second plasmid, and two plasmids are injected people patient's same position simultaneously. In some embodiment, the coded sequence of the immunogenicity target protein that is connected with the controlling element operability and the coded sequence of p35 subunit are positioned on the same plasmid. In some embodiment, the coded sequence of the immunogenicity target protein that is connected with the controlling element operability and the coded sequence of p40 subunit are positioned on the same plasmid. In some embodiment, the coded sequence of certain immunogenicity target protein is connected with the controlling element operability, is positioned on another plasmid separately, and namely not on the plasmid that contains two subunit coded sequences, these three kinds of plasmids give same patient simultaneously.

IL-12 albumen and coding nucleotide sequence thereof can be modified, so that two subunits are nucleotide sequence coded by one, and are expressed as strand (fusion) protein molecule. According to the present invention, one section joint amino acid sequence is provided, it mainly connects two subunits, the different piece of single chain protein can be compounded to form and have bioactive protein but its pliability is enough to. Fig. 8 A has shown a routine single chain protein, and wherein the coded sequence of single chain protein is under the regulation and control of human cytomegalovirus promoter. The coded sequence from 5 ' to 3 ' of this single chain protein comprises: the coded sequence of p35 subunit, and the coded sequence of the coded sequence of joint and p40 subunit, they are as a coded sequence. In the another kind of considering arranged, the coded sequence of single chain protein comprised the coded sequence of p40 subunit, the coded sequence of the coded sequence of joint and p35 subunit, and they are as a coded sequence. Joint must have enough length and toughness, can make two parts of monomeric protein relatively arrange to become and have bioactive complex.

According to part embodiment of the present invention, the nucleotide sequence of strand IL-12 albumen (two subunits connect into monomeric protein by joint) of will encoding mixes in plasmid, non-plasmid nucleic acid molecules or virus or the bacterial genomes, and with in eukaryotic, express necessary controlling element operability and be connected. In embodiment preferably, the nucleotide sequence of coding strand IL-12 albumen (two subunits connect into monomeric protein by joint) mixes in the plasmid. In some embodiment, the coded sequence of immunogenicity target antigen is connected with the controlling element operability, is positioned on the same plasmid with the coded sequence of strand IL-12 albumen. In some embodiments, the coded sequence of immunogenicity target antigen is connected with the controlling element operability, is positioned on another plasmid separately, and namely not on the plasmid that contains the single chain protein coded sequence, these two kinds of plasmids all give same patient.

According to partial content of the present invention, relate to improving one's methods and composition of vaccine inoculation (especially dna vaccination), the DNA of the immunogenicity of wherein encoding target protein is given the patient, also expresses in this DNA people's patient body being shot, produces for this immunogenic immune response. The content according to the present invention; the DNA of various immune modulators of encoding is passed to the patient simultaneously; the expression of this DNA produces immune modulator; the degree of this protein regulation immune response and direction; with the inducing specific immune response so that immunity inoculation after improving with because of the relation of the different protective effect of various diseases closer.

Have been found that in inoculating individual cell of expressing target antigen, producing simultaneously IL-12 albumen and unexpectedly strengthened immune response for target antigen. The nucleotide sequence of the coding IL-12 albumen by but expression-form is provided, the vaccine that can work at the individual cell inner expression target antigen of inoculation, for example dna vaccination, recombinant vector vaccine and attenuated vaccine are improved.

In producing the cell of antigen, produce simultaneously IL-12 and strengthened cellular immunity for antigen. So, the invention provides a kind of improvement vaccine, the nucleotide sequence of one section coding IL-12 namely is provided, and this sequence is connected with the necessary controlling element operability of expression in the inoculator, and this sequence is as the part such as dna vaccination, nontoxic recombinant vector vaccine and attenuated live vaccine.

The present invention induces and has regulated immune response by the gene constructs that transmits simultaneously coding GM-CSF. GM-CSF gene and dna vaccination construction are injected jointly, strengthened antigen-specific antibodies and replied and the t helper cell breeder reaction.

The present invention is by transmitting simultaneously the front inflammatory cytokine (IL-1 α, TNF-α and TNF-β) of coding, immune response is induced and regulated to the gene constructs of Th1 cell factor (IL-2, IL-15 and IL-18) and Th2 cell factor (IL-4, IL-5 and IL-10).

Some part of the present invention has significantly been strengthened the antigentic specificity HI by transmitting simultaneously IL-5 or IL-18.

Some part of the present invention has been strengthened the propagation of antigen specific T auxiliary cell by transmitting simultaneously IL-2, IL-15, IL-10, IL-18, TNF-α or TNF-β.

Some part of the present invention is by transmitting simultaneously TNF-α or the IL-15 gene has been strengthened cell-cytotoxic reaction.

So when cell-mediated the replying of T is primary replying, and humoral response does not need or even when unfavorable, then the IL-12 gene should be sent with the specific DNA immunogene simultaneously as immunological regulation person. On the other hand, in order for example to make up the vaccine for the outer bacterium of born of the same parents, can inject simultaneously with IL-4, IL-5 or IL-10 gene. And, when CD4+T auxiliary cell and antibody all play the even more important time spent of doing in protective effect, can send simultaneously GM-CSF and IL-12. At last, when three kinds of immune responses are all very important, can inject simultaneously TNF-α, reply comprehensively to add powerful antibody, t helper cell and CTL.

Nucleotides and the amino acid sequence of people IL-1 α are well-known, can be referring to Telford etc., (1986) Nucl. Acids.Res.14:9955-9963, Furutani etc., (1985) Nucl.Acids.Res.14:3167-3179, March etc., (1985) Nature 315:641-647 and accession number Swissprot PO1583, this paper receive it and are reference.

Human IL-2's nucleotides and amino acid sequence are well-known, can be referring to (1984) such as Holbrook, Proc.Natl.Acad.Sci.USA 81:1634-1638, Fujita etc. (1983), Proc.Natl.Acad.Sci. USA 80:7437-7441, Fuse etc. (1984), Nucl.Acids.Res.12:9323-9331, Taniguchi etc. (1983), Nature 302:305-310, Maede etc. (1983) Biochem.Biophys.Res.Comm. 115:1040-1047, (1983) Nucl.Acids.Res.11:4307-4323 such as Devos and accession number Swissprot PO1585, this paper receive it and are reference.

The nucleotides of people IL-4 and amino acid sequence are well-known, can be referring to (1989) such as Arai, J. Immunol.142:274-282, Otsuka etc. (1987), Nucl.Acids.Res.15:333-344, Yokota etc. (1986), Proc.Natl.Acad.Sci.USA 83:5894-5898, Noma etc. (1984), Nature 319:640-646, Lee etc. (1986), Proc.Natl.Acad.Sci.USA 83:2061-2063 and accession number Swissprot 05112 (accession number of mouse IL-4 is Swissprot 07750), this paper receive it and are reference.

The nucleotides of people IL-5 and amino acid sequence are well-known, can be referring to (1987) such as Campbell, Proc.Natl.Acad.Sci.USA 84:6629-6633, Tanabe etc. (1987), J.Biol.Chem. 262:16580-16584, Campbell etc. (1988), Eur.J.BioChem.171:345-352, Azuma etc. (1986), Nucl.Acids.Res.14:9149-9158, Yokota etc. (1986), Proc.Natl.Acad. Sci.USA 84:7388-7392 and accession number Swissprot PO5113, this paper receive it and are reference.

The nucleotides of hIL-10 and amino acid sequence are well-known, can be referring to (1991) such as Viera, and Proc. Natl.Acad.Sci.USA 88:1172-1176 and accession number Swissprot P22301.

The nucleotides of human IL-15 and amino acid sequence are well-known, can be referring to (1994) such as Grabstein, and Science 264:965-968 and accession number Swissprot U03099, this paper receive it and are reference.

The nucleotides of human il-18 and amino acid sequence are well-known, can be referring to (1996) J. Immunol.156:4274-4279 and accession number Swissprot D49950 such as Ushio, and this paper receives it and is reference.

HumanTNF-α's nucleotides and amino acid sequence are well-known, can be referring to Pennica (1984) Nature 312:724-729 and accession number Swissprot PO1375, and this paper receives it and is reference.

Nucleotides and the amino acid sequence of people TNF-β are well-known, can be referring to Gray (1984) Nature 312:721-724 and accession number Swissprot PO1374, and this paper receives it and is reference.

The relative dna vaccine can be referring to United States Patent (USP) 5,593,972, United States Patent (USP) 5,589,466, PCT/US90/01515, PCT/US93/02338, PCT/US93/048131 and PCT/US94/00899, and each patent and promulgated the priority application that patent application is quoted, these are all received by this paper and are reference. Except the transmission method described in the above patent application, other method of transmitting DAN can be referring to United States Patent (USP) 4,945, and 050 and 5,036,006,, this paper receives it and is reference.

An improvement of the present invention relates to except producing the target antigen by the dna vaccination nucleic acid sequence encoding, also contains the genetic material that produces simultaneously immune modulator.

The present invention relates to inhereditary material is introduced the method for individual cells, to induce for the coded albumen of this inhereditary material or the immune response of peptide. Described method comprises that giving described individuality organizes single nucleic acid molecule or composition, described single nucleic acid molecule comprises the nucleotide sequence of the target protein of encoding and the nucleotide sequence of coding immune modulator, described composition comprises two kinds of nucleic acid molecules, a kind of nucleotide sequence that comprises the target protein of encoding, another kind comprises the nucleotide sequence of the immune modulator of encoding. The nucleic acid molecules that provides can be the inhereditary material part in DNA, recombinant vector nucleic acid molecules or the attenuated vaccine.

According to the present invention, composition and the method that gives individual preventative and/or therapeutic immunization that provide is to resist certain cause of disease or unusual disease association cell. The target protein of inhereditary material coding is peptide or the protein that has at least an identical epi-position with the cause of disease of target or certain immunogenic protein on the cell, and, the inhereditary material immune modulator of also encoding. Inhereditary material is by the cellular expression of individuality, and causes immune response for it as the immunogenicity target. The immune response that causes and the cause of disease of target or cell effect, and have widely immune-base: not only cause humoral immunity, and the input and output branch of trigger cell immune response. Method of the present invention can be used for giving preventative or therapeutic immunization power. So immunization method comprises the individual method of avoiding the cause of disease attack or avoiding special cells to occur or breed of protection, and comprises the method for the treatment of pathogen infection, super proliferative diseases or Serum of Patients With Autoimmune Diseases.

Term used herein " target protein " refers to that they play the target protein effect that causes immune response by the peptides and proteins of gene constructs coding of the present invention. " target protein " refers to cause the immunoreactive albumen for it. Target protein is immunogenic protein, it with immune response for cause of disease or the albumen of bad cell type (for example cancer cell or with autoimmunity disease relevant cell) have at least an identical epi-position. Immune response for target protein will provide protection and treat to resist specific infection or the disease relevant with target protein for individuality. Target protein not necessarily with required immune response for albumen identical. But, target protein must be able to induce with required immune response for the immune response of albumen generation cross reaction.

The present invention can be used for causing the extensive immune response for certain target protein (i.e. the albumen relevant with " unusually " cell-specific of pathogen or individuality self). The present invention can be used for immune body with opposing pathogenicity material and biology, thus, provides the protective immunity of resisting this cause of disease for the immune response of certain cause of disease albumen. The present invention can be used for resisting super proliferative diseases and disorder (for example cancer), namely causes the immune response for the target protein relevant with super proliferative cell specificity. The present invention can be used for resisting autoimmunity disease and disorder, namely causes the immune response for the target protein relevant with the cell-specific that participates in autoimmune state.

According to the present invention, the DNA of coding target protein and immune modulator or RNA are introduced in the individual histocyte, and express, and produce thus target protein. The DNA of coding target protein and immune modulator or RNA sequence be connected at the necessary controlling element of the cell inner expression of individuality. DNA expresses required controlling element and comprises promoter and polyadenylation signal. In addition, also can comprise other element such as the Kozak district in this gene constructs.

Term used herein " but expression-form " refers to such gene constructs, and it comprises the essential controlling element that is connected with target protein or immune modulator coded sequence operability, thereby can be at the cell inner expression coded sequence of individuality.

Term used herein " has an identical epi-position " and refers to some protein to contain at least one epi-position identical or basic simlarity of epi-position with another albumen.

Term used herein " basic simlarity epi-position " is incomplete same in certain epi-position that this represents structure that described epi-position has and certain albumen, but can cause cell or humoral immune reaction with this albumen generation cross reaction.

Gene constructs comprises one section nucleotide sequence, its coding target protein and/or immune modulator, and the required controlling element operability of this sequence and gene expression is connected. According to the present invention, also provide the combination of gene constructs, but namely one of them comprises the nucleotide sequence of the coding target protein of expression-form, but another construction then comprises the nucleotide sequence of the coding immune modulator of expression-form. DNA or the RNA molecule that will comprise the gene constructs combination are introduced living cells, and DNA or RNA are expressed, and produce target protein and immune modulator. The result produces and replys for the booster immunization of target protein.

When genetic constructs during by cellular uptake, they are stayed in the cell as functional extrachromosomal molecule, and/or are incorporated in the chromosomal DNA of cell. DNA can be introduced cell with the plasmid form as independent inhereditary material. Perhaps, can be with the cell that can induce one with the linear DNA of chromosomal integration. When DNA is induced one cell, can add and promote DNA to be incorporated into the reaction reagent in the chromosome. Also can in dna molecular, comprise the dna sequence dna that promotes integration. Perhaps, RNA can be introduced cell. Also can consider provides a kind of linear microchromosome, and it has centromere, telomere and replication origin. Gene constructs can be used as inhereditary material and partly is deposited in and moves in intracellular attenuated live microorganism or the recombinant microorganism carrier. Gene constructs can be the genome part of recombinant viral vaccine, and at this moment, inhereditary material or be incorporated in the cell chromosome perhaps is deposited in outside the chromosome.

Genetic constructs comprises the necessary controlling element of nucleic acid molecules gene expression. These elements comprise: promoter, initiation codon, terminator codon and polyadenylation signal. In addition, in order to express the gene of coding target protein or immune modulator, often need to add hadron. These elements must be connected with the series of operations of coding desirable proteins, and these controlling elements must be in the individuality that gives effectively.

Initiation codon and terminator codon be it is generally acknowledged in the nucleotide sequence that should be included in the coding desirable proteins. But these elements have function the individual planted agent who gives gene constructs. Initiation codon and terminator codon must with coded sequence in same reading frame.

Used promotor and polyadenylation signal must have function in the cell of individuality.

Can be used for implementing the present invention, include but not limited to the promotor of simian virus 40 (SV40) in particular for the promotor example that produces the human gene vaccine, the promotor of mouse mammary tumour virus (MMTV), human immunodeficiency virus's (HIV) promotor such as HIV long-terminal repeat promoter, the Moloney viral promotors, the ALV promotor, the promotor of scavenger cell virus (CMV) is early promoter among the CMV for example, the promotor of Epstein-Barr virus, the promotor of sieve silk sarcoma virus (RSV) and for example human actin, human myoglobulin, human hemoglobin, people's creatine, the promotor of people's genes such as human metal thioalbumen.

Can be used for implementing the present invention, include but not limited to SV40 polyadenylation signal and LTR polyadenylation signal in particular for the example of the polyadenylation signal of producing the human gene vaccine.Be pointed out that what the present invention used is the SV40 polyadenylation signal that is positioned in the pCEP4 plasmid (Invitrogen, San Diego CA), is called the SV40 polyadenylation signal in the literary composition.

Except DNA expresses essential controlling element, can comprise other element in the dna molecular.These other elements comprise and add hadron.Adding hadron can be selected from but be not limited to: human actin, human myoglobulin, human hemoglobin, people's creatine and such as the hadron that adds of viruses such as CMV, RSV and EBV.

In order to make construction be retained in karyomit(e) many parts of copies outer and generation construction in cell, can provide genetic constructs with Mammals replication origin.The plasmid pCEP4 of Invitrogen (San Diego CA) and pREP4 contain the zone of Epstein-Barr virus replication origin and coding nuclear antigen EBNA-1, and they produce, and high copy is free to duplicate product and unconformability.

In the better embodiment that relevant immunization is used, the nucleic acid molecule that is transmitted comprises coding target protein, IL-12 albumen and further strengthens the nucleotide sequence of other proteic gene of anti-target protein immunne response.The gene of the Codocyte factor and lymphokine for example, the described factor is interferon-alpha, IFN-, PDGF (PDGF), G-CSF, GM-CSF, TNF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-8, IL-10 and B7.2 for example.In some embodiment, the genetic constructs that is used for the immunization composition should comprise the gene of GM-CSF.

If need eliminate the cell of accepting this genetic constructs, can add other element as the cell dissociation target for certain reason.But bleb thymidine kinase (tk) gene that can in genetic constructs, comprise expression-form.Can give individual drugs gangcyclovir, this medicine will optionally be killed all cells that produce tk, provide selective destruction to have the method for the cell of genetic constructs thus.

In order to enlarge proteinic production as far as possible, can select regulating and controlling sequence to make them be suitable for giving the cell inner expression gene of construction.And, can select for use and in cell, transcribe most effective codon.Persons skilled in the art can be manufactured on the DNA construction that has function in the cell.

The example of two class skeletons comprises: a class is used for two pUC pUCs, and a class is used for simple substance grain system.In two pUC pUCs, but plasmid has the target protein encoding sequence of expression-form, but another plasmid has the IL-12 encoding sequence of expression-form.In simple substance grain system, but same plasmid has comprised the target protein encoding sequence and the IL-12 encoding sequence of expression-form simultaneously.

The step of the inventive method comprises the tissue that nucleic acid molecule is given individuality.At some preferably in the embodiment, the route of administration of nucleic acid molecule is in the intramuscular, nose, intraperitoneal, subcutaneous, intradermal, intravenously, give lung tissue or use or lavation gives mucosal tissue by the part by the aerosol approach, and described mucosal tissue is selected vagina, rectum, cheek, urethra oral cavity and hypogloeeis.

In some embodiment, nucleic acid molecule passes to cell with promotor (facilitator).Promotor claims polynucleotide function to add hadron or gene vaccine promotor again.Relevant promotor can be referring to the U.S. Patent application of submitting on January 26th, 1,993 08/008,342, the U.S. Patent application 08/029 that on March 11st, 1993 submitted to, 336, the United States Patent (USP) 5 that on January 14th, 1997 submitted to, International Patent Application PCT/the US94/00899 that submitted on January 26th, 593,972 and 1994, more than all received and be reference by this paper.In addition, promotor also can be referring to PCT/US95/12502 that submits to September 28 nineteen ninety-five and the PCT/US95/04071 that submits to March 30 nineteen ninety-five, more than all received and be reference by this paper.Can mix to give with nucleic acid molecule with the promotor of nucleic acid molecule coupling, perhaps when giving nucleic acid molecule, before or after separately give.In addition, have transfection agents and/or duplicate agent and/or other material of short scorching agent effect, with can comprise somatomedin being with or without other material that gives simultaneously in the presence of the promotor, cytokine and lymphokine, interferon-alpha for example, IFN-, PDGF (PDGF), G-CSF, GM-CSF, TNF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 and B7.2, and fibroblast growth factor, immunostimulating mixture (ISCOMS) and so on tensio-active agent, Freund's incomplete adjuvant, LPS congener (comprising monophosphoryl lipid A), muramylpeptides, quinone congener and the vesicle such as squalene and squalene, and hyaluronic acid.

In embodiment preferably, genetic constructs of the present invention and the formulated together or coupling of promotor that is selected from following group: benzoic ether, amidine, aethylis carbamas and hydrochloride thereof, for example those of local anesthetic family.

Promotor in the part preferred embodiment can be the compound with one of following structure:

Ar-R ¹-O-R ²-R ³

Or Ar-N-R ¹-R ²-R ³

Or R ⁴-N-R ⁵-R ⁶

Or R ⁴-O-R ¹-N-R ⁷

Wherein, Ar is benzene, p-aminophenyl, m-aminophenyl, adjacent amino-benzene, substituted benzene, replacement p-aminophenyl, replaces m-aminophenyl, replaces adjacent amino-benzene, and the amino in the amino-benzene compound can be amino, C ₁-C ₅Alkylamine, C ₁-C ₅, C ₁-C ₅Dialkylamine, the substituting group in the substitution compound can be halogen, C ₁-C ₅Alkyl and C ₁-C ₅Alkoxyl group;

R ¹Be C=O;

R ²Be C ₁-C ₁₀Alkyl comprises branched-chain alkyl;

R ³Be hydrogen atom, amine, C ₁-C ₅Alkylamine, C ₁-C ₅, C ₁-C ₅Dialkylamine;

R ²May with R ³Form a cycloalkyl, a C ₁-C ₁₀The cycloalkyl that alkyl replaces, cycloaliphatic amine, C ₁-C ₁₀Cycloaliphatic amine, heterocycle, C that alkyl replaces ₁-C ₁₀The heterocycle that alkyl replaces (comprises C ₁-C ₁₀The heterocycle that alkyl N-replaces);

R ⁴Be Ar, R ²Or C ₁-C ₅Alkoxyl group, cycloalkyl, C ₁-C ₁₀The cycloalkyl that alkyl replaces, cycloaliphatic amine, C ₁-C ₁₀Cycloaliphatic amine, heterocycle, C that alkyl replaces ₁-C ₁₀Heterocycle and C that alkyl replaces ₁-C ₁₀The heterocycle that alkoxyl group replaces (comprises C ₁-C ₁₀The heterocycle that alkyl N-replaces);

R ⁵Be C=NH;

R ⁶Be Ar, R ²Or C ₁-C ₅Alkoxyl group, cycloalkyl, C ₁-C ₁₀The cycloalkyl that alkyl replaces, cycloaliphatic amine, C ₁-C ₁₀Cycloaliphatic amine, heterocycle, C that alkyl replaces ₁-C ₁₀Heterocycle and C that alkyl replaces ₁-C ₁₀The heterocycle that alkoxyl group replaces (comprises C ₁-C ₁₀The heterocycle that alkyl N-replaces);

R ⁷Be Ar, R ²Or C ₁-C ₅Alkoxyl group, cycloalkyl, C ₁-C ₁₀The cycloalkyl that alkyl replaces, cycloaliphatic amine, C ₁-C ₁₀Cycloaliphatic amine, heterocycle, C that alkyl replaces ₁-C ₁₀Heterocycle and C that alkyl replaces ₁-C ₁₀The heterocycle that alkoxyl group replaces (comprises C ₁-C ₁₀The heterocycle that alkyl N-replaces);

The example of ester comprises benzoic ether, for example piperocaine, meprylcaine and Isobucaine; The para-amino benzoic acid ester comprises PROCAINE HCL, PHARMA GRADE, tetracaine, tanacain, propoxycaine and chloroprocaine; Gavaculine ester, for example Metabuthamine and primacaine; With paraethoxybenxoic acid ester, for example parethoxycaine.The example of aniline comprises lignocaine, etidocaine, mepivacaine, Bupivacaine, pyrrocaine and prilocaine.Other example of this compounds comprises Dibucaine, Benzocaine, dyclonine, Pramoxine, Proparacaine, its cacaine of cloth, oxybuprocaine, Polocaine, methyl Bupivacaine, Butasin picrate, phenacaine, Diothan, Luccaine, Intracaine, Nupercaine, Metabutoxycaine, piridocaine, xenysalate and plant-sourced bicyclic compound, for example Cocaine, cinnamylcocaine, tell former times woods and cocaethlene, and the mixture of all above compounds and hydrochloric acid.

In preferred embodiments, promotor is Bupivacaine.The difference of Bupivacaine and mepivacaine is to have replaced with the N-butyl N-methyl of mepivacaine.Compound may have C in the N position ₁-C ₁₀Compound can replace by halogen, for example PROCAINE HCL, PHARMA GRADE and chloroprocaine.Be preferably aniline.

Promotor before gene constructs, simultaneously or give afterwards.Promotor and genetic constructs can be formulated in the same composition.

The chemical name of Bupivacaine-hydrochloric acid is 2-piperidyl urea (2-piperidinecarboxamide), 1-butyl-N-(2, the 6-dimethyl benzene)-one hydrochloric acid, monohydrate, its market wide material sources, comprise AstraPharmaceutical Product Inc. (Westboro, MA) and Sanofi Winthrop Pharmaceuticals (New York, NY), Eastman Kodak (Rochester, NY), be used to pharmacy.Commercially available Bupivacaine is formulated together with para methyl paraben and suprarenin sometimes, sometimes then not.Above-described preparation all can use.Purchasing the concentration that is used for pharmacy is 0.25%, 0.5% and 0.75%, and these can be used for the present invention.Can make other concentration, the especially concentration between 0.05% to 1.0% that causes required effect as needs.According to the present invention, about 250g to 10mg Bupivacaine.In some embodiment, give 250g to 7.5mg.In some embodiment, give 0.05mg to 5.0mg.In some embodiment, give 0.5mg to 3.0mg.In some embodiment, give 5 to 50g.For example, in some embodiment, before giving vaccine, simultaneously or afterwards, be formulated at same position with first-class and ooze 50 μ l to 2ml in the pharmaceutical carrier, be preferably 50 μ l to 1500 μ l, be more preferably the 0.25-0.50% solution and 0.1 para methyl paraben of about 1ml Bupivacaine-hydrochloric acid.In some embodiment, before giving vaccine, simultaneously or afterwards, the 50 μ l to 2ml that ooze in the pharmaceutical carrier such as be formulated at same position, be preferably 50 μ l to 1500 μ l, be more preferably the 0.25-0.50% solution of about 1ml Bupivacaine-hydrochloric acid.Can give those compounds in Bupivacaine and any other effect similar compounds, especially Xiang Guan local anesthetic family, its concentration should be able to effectively promote cell to take in genetic constructs.

In some embodiment of the present invention, before giving genetic constructs, individuality is accepted the promotor injection earlier.For example, the about 1 thoughtful 10 day time before giving genetic constructs, give individual injection promotor earlier.In some embodiment, giving genetic constructs before about 1 to 5 day, be before 24 hours sometimes and give individual injection promotor afterwards.Perhaps, as long as be used, promotor can be when giving genetic constructs, before the several minutes or give afterwards.So, promotor and genetic constructs can be incorporated in the pharmaceutical composition.

In some embodiment, give genetic constructs and do not give promotor, promptly use the preparation that does not contain promotor, used medication is not for genetic constructs and promotor coupling.

The nucleic acid molecule that passes to cell among the present invention can be used as proteinic hereditary template, and described albumen is as preventative and/or therapeutic immunization agent.In embodiment preferably, nucleic acid molecule is included in transcribes and translates the essential regulating and controlling sequence of described protein-coding region in the zooblast.

The present invention can be used for the immunization individuality with the opposing cause of disease, for example virus, prokaryotic organism and pathogenicity bo eukaryote, for example unicellular causal organism and many cells parasite.The present invention is specially adapted to cause of disease and nothing bag quilt (not encapculated) cause of disease, for example prokaryotic organism such as virus, gonorrhoea, Listeria and Shigella of immunization individuality with the opposing cells infected.In addition, the present invention also can be used for the immunization individuality with the opposing pathogenic agent, and these pathogenic agent have a stage in its life cycle be cause of disease in the born of the same parents.Term used herein " intra-cellular pathogens " refers to such virus or causal organism, is present in the host cell to small part in its breeding or the life cycle, and produces thus or cause producing cause of disease albumen.Table 1 has been listed some Viraceae and the genus that can prepare the specific aim vaccine according to the present invention.Can use such DNA construction in vaccine, the epi-position during the polypeptide of their contained dna sequence encodings has at least one epi-position and shows on the pathogen antigen is identical or similar substantially.And the present invention also can be used for immune body to resist other pathogenic agent, comprises the protozoon cause of disease of protokaryon and eucaryon, and the many cells parasite, for example listed those of table 2.

In order to produce the gene vaccine that disease-resistant former infection provide protection is provided, must in gene constructs, comprise the gene coded sequence of coding immunogenic protein (as target protein), described albumen can cause the protective immune response at it.No matter cause of disease is that infection (to this, the present invention is especially suitable) still is that born of the same parents infect in the born of the same parents, can not all cause protective response by all pathogenicity bo antigen outward.Because DNA and RNA are less, and be easier to produce, the present invention also provides the additional advantage of multivalence pathogenicity bo antigen immune inoculation.The gene constructs that is used for gene vaccine can comprise the antigenic genetic stew of the multiple pathogenicity bo of coding.For example, several virogenes can be included in the construction, multiple target is provided thus.

Table 1 and table 2 have been listed the part pathogenicity bo factor or biological list, can prepare at their gene vaccine and avoid being infected by them to protect individuality.In some preferred embodiment, the method for immune body opposing cause of disease at be HIV, HTLV or HBV.

Another aspect of the present invention content provides that a kind of to give that broad-based protective immune response resists with super proliferative disease be the method for the super proliferative cell of feature, also relates to the method for the treatment of super proliferative disease patient.Term " super proliferative disease " refers to that the super propagation with cell is the disease or the disorder of feature.The example of super proliferative disease comprises various forms of cancers and psoriasis.

Have been found that the gene constructs that will comprise coding immunogenicity " super proliferative cell " the associated protein nucleotide sequence individual cells that induces one, the result individuality be subjected to generated these albumen in the immunocyte.Term " super proliferative albumen " refers to the albumen relevant with super proliferative disease.In order to be used for anti-super proliferative disease immunization, need give the gene constructs that individuality comprises the super proliferative disease associated protein nucleotide sequence of encoding.

In order to make super propagation associated protein become effective target immunogen, this albumen must only produce in super proliferative cell, or the generation level in super proliferative cell is higher than normal cell.Target antigen comprises such albumen, its fragment and peptide, comprises an epi-position in the above-mentioned albumen at least.Sometimes, super propagation associated protein is the product that certain proteic group of coding is undergone mutation.Mutator gene encoded protein and normal protein have only produced the epi-position that does not have on the normal protein because of trickle aminoacid sequence difference much at one.Such target protein comprises by such as myb, myc, fyn and so on oncogene and transposable genetic bcr/abl, ras, src, p53, neu trk encoded protein and EGRF.Except oncoprotein as target antigen; the target protein that is used for the treatment of anticancer therapy and protectiveness also comprises the variable region of the TXi Baoshouti of the variable region of the antibody that B cell lymphoma produces and t cell lymphoma, and they also treat autoimmune disease as target antigen in certain embodiments.Can also use other tumor correlated albumen as target protein,, comprise the albumen and the folic acid-binding protein of monoclonal antibody 17-1A identification for example at the very high albumen of tumour cell intensive amount.

Though the present invention can be used for immune body to resist the cancer of one or more forms, the present invention is particularly useful for individuality is carried out preventative immunization, and described individuality has the tendency that develops into certain cancer or once suffered from cancer thereby suspected recurrence.Genetics and gene engineering and EPDML development make determines that the individual possibility that cancer takes place becomes possibility with the risk assessment of carrying out this respect.Adopt genetic screening and/or family's health history, might predict any possibility in certain individual several cancer of generation.

Similar, those suffered from the individuality of cancer and receive treatment remove cancer or be in paracmastic individuality and especially be easy to recurrence.As the integral part of methods of treatment,, can carry out immunization to individuality at the cancer of having made a definite diagnosis in order to defeat recurrence.Like this, once suffered from certain cancer and the danger of recurrence is arranged in case learn certain individuality, it is any with the cancer that occurs to resist that they can make immunity system get ready through immunization.

The invention provides a kind of method for the treatment of super proliferative disease patient.In this method, introduce gene constructs and play a part immunotherapy, promptly guide and promote the super proliferative cell of individual immunity system opposing generation target protein.

The invention provides treatment autoimmunization patient's method, broad-based protective immune response promptly is provided, resist the target antigen relevant with autoimmunization, it comprises cell receptor and the cell that produces at (self-derected) antibody of " self ".

The cell-mediated autoimmune disease of T comprises rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren syndrome, sarcoidosis, insulin-dependent diabetes (IDDM), autoimmunization thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wei Genashi granulomatosis, Crohn disease and ulcerative colitis.The feature of above-mentioned disease all is TXi Baoshouti in conjunction with endogenous antigen, causes the inflammatory cascade reaction relevant with autoimmune disease thus.At the immunization of T cell variable region initiation is comprised the immunne response of CTL, eliminate those T cells.

With regard to RA, characteristic description several participate in the specificity variable region of the TXi Baoshouti (TCR) of this disease.These TCR comprise V β-3, V β-14, V β-17 and V α-17.Like this, with at least more than one proteic DNA construction immunization of coding, with the immunne response that causes at the T cell that participates in RA.Referring to: Howell, M.D. etc., 1991Proc.Natl.Acad.Sci.USA 88:10921-10925; Paliard, X. etc., 1991Science 253,325-329; Williams.W.C. wait 1992.J.Clin.Invest.90:326-333; Below all received and be reference by this paper.

With regard to MS, characteristic description several participate in the specificity variable region of this disease TCR.These TCR comprise V β-7 and V α-10.Like this, with at least more than one proteic DNA construction immunization of coding, with the immunne response that causes at the T cell that participates in MS.Referring to: Wucherpfennig, K.W. etc., 1990Science 248:1016-1019; Oksenberg, J.R. etc., 1990Nature 345:344-346; Below all received and be reference by this paper.

With regard to scleroderma, characteristic description several participate in the specificity variable region of this disease TCR.These TCR comprise V β-6, V β-8, V β-14 and V α-16, V α-3C, V α-7, V α-14, V α-15, V α-16, V α-28 and V α-12.Like this, with at least more than one proteic DNA construction immunization of coding, will cause at the immunne response that participates in sclerodermatous T cell.

In order to treat the cell-mediated autoimmunization patient of T, especially the TCR variable region waits the disease patient of specificity analysis, can carry out the synovia biopsy.Can extract the sample that contains the T cell, identify the variable region of its TCR with standard technique.Utilize above information just can prepare gene vaccine.

The cell-mediated autoimmune disease of B comprises lupus (SLE), Graves disease, myasthenia gravis, autoimmune hemolytic anemia, autoimmunization thrombocytopenia, asthma, sclerosis of cryoglobulinemia primary bile duct and pernicious anemia.The feature of above-mentioned disease all is the endogenous antigen of antibodies, and the initiation inflammatory cascade reaction relevant with autoimmune disease.Use at the variable region vaccine immunity of these antibody initiation is comprised the immunne response of CTL, eliminate the B cell that produces these antibody thus.

In order to treat the cell-mediated autoimmunization patient of B, must identify the variable region that participates in active those antibody of autoimmunization.Can carry out biopsy, obtain the antibody sample of inflammation part.Identify the variable region of these antibody with standard technique.Utilize this information just can prepare gene vaccine.

With SLE is example, and certain antigen is considered to DNA.Like this, can prepare the vaccine that comprises the DNA construction of anti-DNA antibody variable region in the coding serum then to the patient's that accepts anti-SLE immunity the anti-DAN antibody of serum screening.

The general constitutional features of the variable region of TCR and antibody is well-known.General available known technology finds the dna sequence dna of coding specific T CR or antibody, described technology can be referring to Kabat etc., 1987, Sequenceof Proteins of Immunological Interest U.S.Department of Health and Human Services, Bethesda MD, this paper receive it and are reference.In addition, the common method of the functional variable region of clonal antibody can be referring to Chaudhary, V.K. etc., and 1990, Proc.Natl.Acad.Sci.USA 87:1066, this paper receive it and are reference.

, the invention still further relates to improved attenuated live vaccine and utilize recombinant vectors to transmit the improvement vaccine of the foreign gene of coding for antigens except that but the immune modulator encoding sequence that utilizes expression-form improves the gene vaccine.Attenuated live vaccine and utilize recombinant vectors to transmit the example of the vaccine of exogenous antigen can be referring to United States Patent (USP): 4,722,848; 5,017,487; 5,077,044; 5,110,587; 5,112,749; 5,174,993; 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, more than all received and be reference by this paper.Gene constructs provided by the invention comprises the nucleotide sequence of the immune modulator of encoding, described sequence with can be in being subjected to inoculator's body the performance function be connected with the regulating and controlling sequence operability of effective expression.Gene constructs is mixed in people's attenuated live vaccine and the recombiant vaccine, produced improvement vaccine of the present invention.

The invention provides a kind of modification method of immunization individuality, it comprises gene constructs is passed to individual cell as the integral part of vaccine composition that described vaccine composition comprises dna vaccination, attenuated live vaccine and recombiant vaccine.Gene constructs comprise the coding immune modulator and with can be in being subjected to immune's body the performance function regulating and controlling sequence operability that causes expression be connected.The vaccine of improvement can produce the cell immune response of reinforcement.

In partial content of the present invention, the nucleic acid molecule of coding people immune modulator transmits as therapeutic gene, does not promptly give the nucleic acid molecule of target immunogen or coding immunogenicity target protein simultaneously.This gene therapy is owing to the function of the immune modulator that promotes immunne response causes.

The gene constructs that transmits as therapeutic gene is the nucleic acid molecule of coding people IL-12, and such gene constructs is good with plasmid.Also can consider other carrier based on nucleic acid, for example recombinant virus, recombinant microorganism and linear nucleic acid molecule, these are all known for persons skilled in the art.Being used for giving individuality, is well-known with the plasmid of taking in individual tissue and express.The recombinant vectors of considering comprises: recombinant vaccinia virus vector, recombinant adenoviral vector and recombinant retroviral vector.The recombinant microorganism of considering comprises recombinant BCG carrier and reorganization salmonella carrier.Linear nucleic acid molecule and plasmid DNA molecule can be wrapped in microsphere and the liposome.

In some preferred embodiment, the plasmid of coding people immune modulator is foregoing gene constructs.In essence, same composition and method can be used for gene therapy, (described genes encoding people immune modulator), group immune composition and method as the gene constructs that comprises coding people immune modulator are described, and its difference is that gene therapy compositions does not comprise the encoding sequence of immunogenic target protein.Content disclosed by the invention is intended to the gene therapy with reference to said gene construction explanation coding people immune modulator.Will be with above-mentioned gene constructs Benq because of treatment, described construction comprises the nucleotide sequence of coding people immune modulator, this sequence is connected with the necessary controlling element operability of expression in individual.Such construction can be given individuality changes and/or promotes immunne response.

For example, IL-12 be can transmit and allergy, cancer, autoimmune disease or infected patient treated.The coding IL-12 gene constructs with or do not give with promotor.In certain embodiments, this gene constructs and one or more aforementioned promotor couplings.In the part embodiment, this gene constructs transmits separately, does not contain any promotor.In some preferred embodiment, the coding IL-12 gene not with any promotor together.In the part embodiment, this gene constructs utilizes needleless injector to give.In certain embodiments, the gene of coding IL-12 utilizes particulate to transmit.In the part embodiment, the gene of coding IL-12 does not have any solids when transmitting.

Pharmaceutical composition of the present invention, i.e. gene vaccine or gene therapy compositions comprise the DNA of 1ng to 1000 μ g.In some preferred embodiment, said composition comprises the DNA of 10ng to about 800 μ g.In some preferred embodiment, said composition comprises the DNA of 0.1 to 500 μ g.In the part preferred embodiment, said composition comprises the DNA of 1 to 350 μ g.In some preferred embodiment, said composition comprises the DAN of 25 to 250 μ g.In some preferred embodiment, said composition comprises the DNA of about 100 μ g.

Pharmaceutical composition of the present invention is prepared according to administering mode.Persons skilled in the art can make the pharmaceutical composition that comprises gene constructs easily.If select for use intramuscularly to come administration, should use etc. and to ooze preparation.In general, the additive that is used to regulate osmotic pressure comprises sodium-chlor, glucose, N.F,USP MANNITOL, sorbyl alcohol and lactose.Sometimes, be advisable with the isotonic solution that uses phosphate buffered saline buffer and so on.Stablizer has gelatin and albumin.In certain embodiments, be added with vasoconstrictor in the preparation.Aseptic and the pyrogen-free of pharmaceutical preparation of the present invention.

The inventive method can be used for people and beast.So, the present invention relates to genetic immunization to Mammals, birds and fish.The inventive method is specially adapted to comprise the Mammals of people, ox, sheep, pig, horse, dog and cat.

The present invention has disclosed a kind of new immunomodulator, people BL1.The aminoacid sequence of its dna sequence dna and expectation is seen Figure 14.This albumen and fragment thereof and immunogen introduced to reply by booster immunization when individual vaccine composition uses simultaneously.Term " immunomodulatory fragment " refers to the fragment of BL1, and it is shorter than the full length sequence among Figure 14, but has kept the immunoregulatory activity of total length compound.According to the present invention, the transmission of BL1 gene order will produce immunoregulation effect.In some instances, though do not record protein expression, immunne response still is subjected to the influence of common transmission, and this shows that transmitting BL1 DNA is the committed step of regulating and guide immunne response.As previously mentioned, the disclosure of invention is intended to disclose BL1 DNA in the immunomodulatory composition of not considering the protein generation and the purposes in the method.Therefore, the disclosure of invention relates to the carrier that adopts BL1 DNA and comprise it as immunomodulator, with in immune regulation composite as main active medicine or as with the assistant agent of the immunogen or the immunogenic DNA coupling of encoding, be used for handling and the guiding immunne response.

The albumen of BL1 dna encoding can separate and purifying; Can produce hybridoma in conjunction with this protein antibodies; Encode this proteic cDNA separated earlier, check order, mix the people in the carrier that comprises expression vector, these carriers are introduced into host cell recombinant expression protein then.To encode immunogenic carrier and above-mentioned carrier gives altogether with anti-this immunogenic immunne response of reinforcement.

BL1 is found to be design and uses reinforcement, promotes and guide the vaccine scheme of immunne response that means are provided.

The cDNA that separates coding BL1 can be used as parent material and makes up and can produce BL1 or the segmental recombinant expression vector of its immunomodulatory.This cDNA is imported in the carrier that comprises expression vector, with the carrier host cell that induces one, recombinant expressed then this albumen.Nucleic acid molecule or its fragment can be used as the existence that probe detects the BL1 encoding sequence.This class probe specificity ground and the hybridization of BL1 encoding sequence.Term " specific b L1 sequence " refers to the distinctive sequence of BL1 at this.The cDNA specific fragment complementary nucleic acid molecule of contained nucleotide sequence and coding BL1 can be used as antisense molecule and primer suppresses the transcribing of mRNA and the genetic sequence that increases respectively.

BL1 is encoded by the cDNA among Figure 14, and has the predetermined amino acid sequence among Figure 14.The BL1 encoding sequence can be conventional synthetic, and BL1 albumen can be produced or synthesizes with the standard protein synthetic technology with recombinant DNA method.

The parent material that adopts standard technique and obtain easily can be prepared the nucleic acid molecule of the BL1 that encodes.The present invention relates to comprise the isolated nucleic acid molecule of coding BL1 nucleotide sequence.The present invention relates to comprise the isolated nucleic acid molecule of aminoacid sequence among coding Figure 14 or the segmental nucleotide sequence of its immunomodulatory.In the part embodiment, this nucleic acid molecule is made of the nucleotide sequence of coding BL1.In the part embodiment, this nucleic acid molecule comprises the nucleotide sequence of Figure 14 encoding sequence.In certain embodiments, this nucleic acid molecule is made of the nucleotide sequence among Figure 14.Isolated nucleic acid molecule of the present invention can be used for preparing construction and recombinant expression vector.

The specific probe of BL1 or primer have at least 16 Nucleotide, are advisable with at least 24.These probes or primer are used to screen the cDNA library by the standard hybridization technique.

The cDNA of coding BL1 can be used to design the PCR primer, is used for amplifying nucleic acid sequence.Round pcr is used always by persons skilled in the art, and its application aspect diagnosis is well-known and has obtained approval.The method of implementing round pcr is referring to " PCR Protocols:A Guide to Methods andApplication ", Innis, and editors such as M.A., Academic Press Inc., San Diego, CA (1990), this paper receive it and are reference.The application of round pcr is referring to " polymerase chain reaction " Erlich, editors such as H.A., and Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), this paper receive it and are reference.

Some simple rule helps to design effective primer.The typical long 18-28 of a primer Nucleotide, the g+c composition accounts for 50% to 60%.The sequence complementation best, that whole primer all must be hybridized with it with it.Be preferably, primer produces the PCR product of 100 to 200 base pairs.But, might produce 50 base pairs to 10kd or longer product.

Round pcr can produce many parts of copies of nucleotide sequence rapidly, it be by provide with a nucleic acid molecule in the 5 ' end and 3 ' of sequence hybridization hold primer, and provide free nucleotide and enzyme, this enzyme is filled out nucleotide sequence complementary base between people and two primers with free nucleotide, produces the complementary strand of DNA thus.This enzyme will be filled the complementary sequence that primer adjoins.If the primer of 5 ' end and 3 ' end all with the same fragment complementation chain of nucleic acid molecule on nucleotide sequence hybridization, the result is that the index of specific double-stranded product increases.If have only a primer and making nucleic acid molecular hybridization, linear amplification will produce the single stranded product of different lengths.

The present invention relates to comprise the carrier or the recombinant expression vector of coding BL1 nucleotide sequence, BL1 has the aminoacid sequence of Figure 14.Term " recombinant expression vector " refers to such plasmid, phage, virion or other carrier at this, and they contain and are introduced into after the appropriate host guiding BL1 encoding sequence and express necessary genetic elements.To the encode nucleic acid molecule of BL1 of persons skilled in the art available standards technology and the parent material that obtains easily inserts in the expression vector, and this encoding sequence is connected with essential regulating and controlling sequence operability.Expression vector is buied as everyone knows and easily.The example of expression vector comprises plasmid, phage, virus vector and other nucleic acid molecule or contains the nucleic acid molecule that can be used for transformed host cell and promote the medium that encoding sequence is expressed.In the part embodiment, this recombinant expression vector comprises the nucleotide sequence among Figure 14.Recombinant expression vector of the present invention is plasmid preferably.

The present invention relates to comprise the host cell of recombinant expression vector, described carrier comprises the nucleotide sequence of the BL1 that encodes.In the part embodiment, host cell comprises a recombinant expression vector, and this carrier comprises the nucleotide sequence of Figure 14.The host cell that is used to produce the protein recombinant expression system is buied as everyone knows and easily.The example of these host cells comprises the bacterial cell such as intestinal bacteria, with S.cerevisiae is the yeast cell of example, with S.frugiperda is the insect cell of example, be the non-human mammal tissue culture cells of example and be people's tissue culture cells of example with Chinese hamster ovary cell (CHO) with the Hela cell.

In the part embodiment, persons skilled in the art can utilize the technology of knowing that the DAN molecule is inserted the commercially available expression vector that is used for expression system commonly used of people.(Invitrogen, San Diego CA) can be used to produce BL1 to for example commercially available plasmid pSE420 in intestinal bacteria.(Invitrogen, SanDiego CA) can be used to production in the S.cerevisiae yeast strain to commercially available plasmid pYES2.Commercially available MAXBAC ^TMFull baculovirus expression system can be used to the production in insect cell.Commercially available plasmid pcDNA I or pcDNA3 (Invitrogen.San Diego.CA) are used in mammalian cell, as the production in the Chinese hamster ovary cell.Persons skilled in the art can be utilized these commercially available expression vectors and system or other, utilize routine techniques and the parent material buied is easily produced hVIP.(referring to, Sambrook etc. for example, Molecular Cloning aLaboratory Mannual, second edition, Cold Spring Harbor Press (1989), this paper with its receive be with reference to).Like this, required albumen promptly can be produced also in prokaryotic system and can produce in eukaryotic system, produces the protein of multiple form processing.

Persons skilled in the art can be utilized other commercially available expression vector or system, or utilize currently known methods and the parent material buied is easily produced carrier.Be applicable to multiple host, the expression system that comprises essential regulating and controlling sequence (for example promotor and polyadenylation signal preferably add hadron in addition) is easy to obtain, and is known in the art.Referring to Sambrook etc., Molecular Cloning a Laboratory Mannual, second edition, ColdSpring Harbor Press (1989).

The expression vector that comprises the DNA of the BL1 that encodes is used to transform the consistency host, cultivates this host then, and holds it under the condition that the foreign DNA expression can take place.Known to those skilled in the art, reclaim the albumen of so producing of the present invention by culture by lysing cell or from nutrient solution.Persons skilled in the art can be separated the BL1 that above-mentioned expression system produces with the technology of knowing.Utilize above-mentioned specificity also can be used for the BL1 that the purification of Recombinant dna technique is produced equally in conjunction with the method for antibody purifying BL1 from natural origin of BL1.

The example of gene constructs comprises the BL1 encoding sequence that is connected with the promotor operability, and described promotor has function as previously mentioned in human body, or has function in construction with cells transfected is.The constitutive promoter example comprises the promotor of cytomegalovirus or SV40.The inducible promoter example comprises mouse mammary gland leukosis virus or metalloprotein promotor.

Except producing the BL1 with recombinant technology, also available automated peptide synthesizer is produced BL1.This technology is known by persons skilled in the art, and can be used for producing the derivative with replacement, and this replacement is can't provide in the dna encoding protein production.

The nucleic acid molecule of coding BL1 can utilize in the multiple transmission composition any one to transmit, for example directly give plasmid, recombinant virus expression vector or other suitable transfer means, thus their introducings in individuality or consistency host cell and expression therein influenced.In general, virus vector can be a dna virus, for example recombinant adenovirus and recombined vaccinia virus, or RNA viruses, for example recombinant retrovirus.Other recombinant vectors comprises can cells infected and the prokaryotic organism of express recombinant gene.Except recombinant vectors, also can consider to use other to transmit composition, the transfection and other the receptor-mediated method that for example be embedded in the liposome, transferrin mediate.The present invention is intended to comprise expression vector and other suitable transfer means of above-mentioned other form, and they have equal effect, and road known in the art successively.

The invention still further relates to the non-human transgenic Mammals, they have the recombinant expression vector that comprises coding BL1 nucleotide sequence.Be used to produce the non-human transgenic animal of recombinant protein, required expression vector and the method for producing transgenic animal, these are all known.In general, transgenic animal comprise recombinant expression vector, contain coding BL1 nucleotide sequence in the carrier, it is connected with a mammalian cell specificity promoter operability, thereby make encoding sequence only in mammalian cell, express, and can from the milk of animal, reclaim the recombinant protein of expressing thus.Persons skilled in the art are according to adopting standard technique can produce the transgenic animal that produce BL1 below with reference to document, described document has: be issued to the United States Patent (USP) 4 of Wagner on October 10th, 1989,873, be issued to the United States Patent (USP) 4 of Leder on April 12nd, 191 and 1988,736,866, more than receive by this paper and to be reference.Better suited animal is goat, sheep or rodent (specifically rat and mouse).

BL1 albumen or the expression vector that is used for its production can be mixed with pharmaceutical composition.

Pharmaceutical composition of the present invention comprises and the transmission component of nucleic acid molecule associating, also comprises pharmaceutically acceptable carrier or media, for example salt solution.Any medium that can successfully transmit nucleic acid all can use.Those skilled in the art are readily appreciated that it is diversified can be used for pharmaceutically acceptable medium of the present invention.Suitable pharmaceutically acceptable vehicle can be referring to Remington ' s Pharmaceutical Sciences, A.Osol, and this is a canonical reference book of this area, this paper receives at this and is reference.Pharmaceutical composition of the present invention can give by variety of way, as long as can make active medicine arrive target cell.Because when oral, peptide is easily digested, and for obtaining good absorption, generally uses administered parenterally, for example vein, subcutaneous, transdermal, intramuscular.Venoclysis can be carried out by means of infusion pump.Pharmaceutical composition of the present invention can be mixed with emulsion.Perhaps, can be mixed with aerosol to be fit to use in the nose or inhale the people.Sometimes, also may need local the use.

Dosage is difference with following factor: pharmacokinetic characteristic; Administering mode and approach; Recipient's age, health and body weight; The nature and extent of symptom; The kind of coupling therapy; The treatment frequency.Usually, peptide dosage is every 50kg body weight 1 to 3000mg: be advisable with every 50kg body weight 10 to 1000mg; Better with every 50kg body weight 25 to 800mg.Generally, divide everyone every day to give 8 to 800mg 1 to 6 time, or utilize slow release formulation, can effectively reach required effect.The local preparation that uses comprises transdermal subsides, ointment, lotion, emulsifiable paste, gel, dropping liquid, suppository, sprays, liquefying agent and powder.May must or should use conventional pharmaceutical carrier, the aqueous solution, powder or oleaginous base, thickening material etc.Suspension or solution, capsule, sachet or tablet that oral compositions comprises powder or pill, is made into water or non-aqueous media.Perhaps, thickening material, seasonings, thinner, emulsifying agent, disperse additive or tackiness agent need.Being used in the parenteral, vein, sheath or the composition of Intraventricular administration can comprise aseptic aqueous solution (wherein containing buffer reagent), thinner and other suitable additive, is good with aseptic and pyrogen-free.According to the present invention, the pharmaceutical composition that is applicable to intravenously administrable is aseptic and pyrogen-free.

With regard to administered parenterally, peptide of the present invention can be mixed with solution, suspension, emulsion or lyophilize powder, and combines with pharmaceutically acceptable parenteral carrier.This class media is water, salt solution, Ringer solution, glucose solution and 5% human serum albumin for example.Also can use liposome and nonaqueous phase media, for example fixed oil.Can contain the additive (for example sodium-chlor, N.F,USP MANNITOL) of keeping osmotic pressure and the additive (for example buffer reagent and preservatives) of keeping chemical stability in media or the lyophilized powder.Preparation is sterilized with routine techniques.For example, be prepared as follows the injection parenteral composition: 1.5% (weight) active ingredient is dissolved in 0.9% sodium chloride solution.

Pharmaceutical composition of the present invention can give by the whole bag of tricks, as long as this method can make active medicine arrive the site of action of animal drug disposition.Pharmaceutical composition of the present invention can be through many administrations, this depend on required treatment be locality or general, also depend on the position of needs treatments.Route of administration can be local (comprise eye with, vagina with, rectum with, nose with, transdermal), oral or parenteral.Parenteral comprises intravenous drip, subcutaneous, intraperitoneal or intramuscularly, lung administration (for example inhale the people or blow the people), perhaps in the sheath or the Intraventricular administration.

Generation is in conjunction with the hybridoma of BL1 antibody, and this antibody itself is used to separate and purifying BL1 and the protein complex that comprises it.In addition, antibody is the active specific inhibitor of BL1.The parent material that adopts known technology and buy easily can be used for this protein of purifying from natural origin in conjunction with the antibody of hVIP with specificity.This antibody-like also can be used to this protein of purifying from product when producing albumen with the DNA recombinant technology.

Term used herein " antibody " refers to complete whole antibody, its Fab fragment and F (ab) ₂Fragment.Complete whole antibody comprises monoclonal antibody, chimeric antibody and the humanized antibody of mouse monoclonal antibody and so on.In conjunction with the antibody of certain epi-position can be used for routine techniques (affinity chromatography for example, promptly antibody not with other albumen generation cross reaction) from natural origin or recombinant expression system, separate and protein purification.It denys described protein that this antibody-like can be used for having in the test sample, and definite cell marking protein whether.

The production method of antibody, complete whole antibody, its Fab fragment and F (ab) ₂Segmental protein structure, and the composition of the gene order of these molecules of encoding is all known, can be referring to Harlow, E. and D.Lane (1988) ANTBODIES:A Laboratory Mannual, Cold Spring Harbor Laboratory, Cold SpringHarbor, NY, this paper receive it and are reference.In brief, give injected in mice BL1 or its immunogenic fragments.Take out the spleen of mouse, separating Morr. cell merges with unlimited breeding mouse cell.Cultivate this hybrid cell or hybridoma, pick out the cell of secretory antibody.Analyze antibody,, then the hybridoma that produces this antibody is cultivated with continuous production antibody is provided if find it specifically in conjunction with BL1.

Following examples have comprised the representative embodiment of content of the present invention.These embodiment are not in order to limit scope of the present invention, but in order to describe.In addition, many contents of the present invention can be summarized with following examples.But more than explanation is not to limit scope of the present invention, but many contents of the present invention are said clearly.Persons skilled in the art are readily appreciated that others of the present invention and embodiment.

Embodiment

Embodiment 1

Below listed the construction that can be used for the inventive method.Carrier pBade.puro produces the multiple construction of listing later as parent material, and it is by Morgenster the earliest, and J.P and H.Land make up and report that referring to 1990, Nucl.Acids Res.18 (12): 3587-3596, this paper receive it and be reference.The pBade.puro plasmid especially is fit to be used for expression alien gene in mammalian cell.The dna sequence dna that needs are expressed inserts cloning site, is under long terminal repeat (LTR) promoter regulation of Moloney murine leukemia virus (Mo MuLV).This plasmid contains anti-tetracycline selective marker.

Plasmid pBa.V α 3-IL-12 contains 2.7kb EcoRI genomic fragment; this fragment coding TXi Baoshouti V α 3 structural domains; this structural domain comprises L, V and J fragment; their cloned people EcoRI sites of pBabe.puro; this plasmid also contains the IL-12 encoding sequence, and this sequence is connected with SV40 polyadenylation series of operations with the CMV promotor.TXi Baoshouti deutero-target protein is used for immunization with opposing and cell-mediated autoimmune disease and clonotypic t cell lymphoma and the leukemia of treatment T.

Plasmid pBa.gagpol-vpr-1L-72 comprises gag/pol and the vif gene among the human cloning pBabe.puro. disappearance vpr gene.This plasmid contains these HIV virogenes, their coding HIV target proteins, also contain the IL-12 encoding sequence that is connected with SV40 polyadenylation series of operations with the CMV promotor, this plasmid can be used for immunization, and HIV infects and the AIDS.HIV dna sequence dna is disclosed in Reiz to resist and to treat, M.S., among the 1992AIDS Res.Human Retro.8:1549, this paper receives it and is reference.This sequence can be obtained to GenbankNo:M17449, and this paper receives it and is reference.

Plasmid pM160 contains the PCR fragment of 2.3kb, this fragment coding HIV-I/3B envelope protein and rev/tat gene, and they all are cloned among the pMAMneoBlue.And nef district disappearance.This plasmid contains these HIV virogenes of coding HIV target protein, and with the IL-12 encoding sequence that is connected with SV40 polyadenylation series of operations with the CMV promotor, this plasmid can be used for immunization to be infected and AIDS with opposing and treatment HIV.The dna sequence dna of HIV-1/3B is disclosed in: Fisher, and A., among the 1985Nature 316:262, this paper receives it and is reference.This sequence can be obtained to Genbank No:K03455, and this paper receives it and is reference.

Plasmid pBa.VL-IL-12 contains the PCR fragment, and the VL district of this fragment coding anti-DNA antibody is cloned on the XbaI and EcoRI site of pBabe.puro, also contains the IL-12 encoding sequence that is connected with SV40 polyadenylation series of operations with the CMV promotor.This antibody deutero-target protein is an example of target protein, can be used for immunization with opposing and cell-mediated autoimmune disease and clonotypic B cell lymphoma and the leukemia of treatment B.The universal method of functional variable region can be referring to Chaudhary on the clonal antibody, and V.K. etc., 1990Proc.Natl.Acad.Sci.USA 87:1066, this paper receive it and are reference.

Plasmid pOspA.B-IL-12 contains coding borrelia burgdorferi Borrelia burgdorferi and causes spirochetal OspA and the antigenic coding region of OspB that lime (Lym) is sick; be cloned on the BamHI and Sall site of pBabe.puro, this plasmid also contains the IL-12 encoding sequence that is connected with SV40 polyadenylation series of operations with the CMV promotor.Referring to: Williams, W.V. etc., 1992DNA and Cell.Biol.11 (3): 207, this paper receives it and is reference.This plasmid contains these pathogenic genes, and their target proteins of encoding can be used for immunization opposing Lyme disease.

Plasmid pBa.Rb-G-IL-12 contains the fragment that PCR produces; its coding for rabies virus G albumen; this fragment cloning is on the BamHI site of pBabe.puro, and this plasmid also contains the IL-12 encoding sequence that is connected with SV40 polyadenylation series of operations with the CMV promotor.The plasmid that contains proteic this pathogenic genes of the mad dog G of coding can be used for immunization with the opposing rabies.Its dna sequence dna is disclosed among the Genebank No:M32751, and this paper receives it and is reference.Also can referring to; Anilionis A. etc., 1981Nature 294:275, this paper receive it and are reference.

Plasmid pBa.HPV-L1 comprises the fragment that PCR produces, the L1 capsid protein of coding human papillomavirus (HPV) (comprising HPV strain 16,18,31 and 33), be cloned in BamHI and the EcoRI site of pBabe.puro, also contain the IL-12 encoding sequence that is connected with SV40 polyadenylation series of operations with the CMV promotor in addition.This plasmid can be used for the cancer of immunization to resist the HPV infection and to be caused by it.Its dna sequence dna is disclosed among the Genebank No:M15781, and this paper receives it and is reference.In addition can be referring to Howley, P., 1990Fields Virology, Vol.2, Channock, editors such as R.M, the 58th chapter, 1625; Shah, K. and P.Howley, 1990Fields Virology, Vol.2, Channock, editors such as R.M, the 59th chapter, more than all received and be reference by this paper.

Plasmid pBa.HPV-L2-IL-12 contains the fragment that PCR produces, the L2 capsid protein of these fragment coding human papillomaviruss (HPV) (comprising HPV strain 16,18,31 and 33), be cloned on the BamHI and EcoRI site of pBabe.puro, also contain the IL-12 encoding sequence that is connected with SV40 polyadenylation series of operations with the CMV promotor in addition.This plasmid can be used for the cancer of immunization to resist the HPV infection and to be caused by it.Its dna sequence dna is disclosed among the GenebankNo:M15781, and this paper receives it and is reference.In addition can be referring to Howley, P., 1990Fields Virology, Vol.2, Channock, editors such as R.M, the 58th chapter, 1625; Shah, K. and P.Howley, 1990Fields Virology, Vol.2, Channock, editors such as R.M, the 59th chapter, more than all received and be reference by this paper.

Plasmid pBa.MNp7-IL-12 comprises the fragment that PCR produces, the coding region of its coding HIV MNgag (core protein) sequence, be cloned on the BamHI site of pBabe.puro, also contain the IL-12 encoding sequence that is connected with SV40 polyadenylation series of operations with the CMV promotor in addition.This plasmid contains these HIV virogenes of coding HIV target protein, can be used for immunization and infects and AIDS with opposing and treatment HIV.Reiz，M.S.，1992AIDS Res。Human Retro。8:1549, this paper receive it and are reference.This sequence can be obtained to Genebank No:M17449, and this paper receives it and is reference.

Plasmid pGA732-2-IL-12 contains the GA733-2 TSA, be cloned into (Seed in the pCDM8 carrier from colon carcinoma cell line SW948, B. and A.Aruffo, 1987, Proc.Natl.Acad.Sci.USA84:3365, this paper with its receive be with reference to) the BstXI site on, other contains the IL-12 encoding sequence that is connected with SV40 polyadenylation series of operations with the CMV promotor.This cancer-related target protein has been represented and can be used for the target protein of immunization with opposing and treatment cancer and so on super proliferative disease.GA733-2 antigen is the useful target antigen of resistive connection intestinal cancer.The antigenic report of relevant GA733-2 is referring to Szala, S. etc., and 1990, Proc.Natl.Acad.Sci.USA 87:3542-3546, this paper receive it and are reference.

Plasmid pT4-pMV7-IL-12 contains the cDNA of coding people CD4 acceptor, is cloned on the EcoRI site of pMV7 carrier, and other contains the IL-12 encoding sequence that is connected with SV40 polyadenylation series of operations with the CMV promotor.The CD4 target protein can be used for immunization with opposing and treatment t cell lymphoma.Plasmid pT4-pMV7 can be to AIDS Repository, and Catalog No.158 obtains.

Plasmid pDJGA733-IL-12 contains the GA733 TSA, is cloned on the BamHI site of pBabe.puro, also contains the IL-12 encoding sequence that is connected with SV40 polyadenylation series of operations with the CMV promotor in addition.This cancer-related target protein has been represented and has been used for the target protein of immunization with opposing and treatment cancer and so on super proliferative disease.GA733 antigen is the useful target antigen of resistive connection intestinal cancer.

Plasmid pBa.RAS-IL-12 contains the Ras coding region, and it is subclone and being cloned on the BamHI site of pBabe.puro from pZIPneoRAS earlier, also contains the IL-12 encoding sequence that is connected with SV40 polyadenylation series of operations with the CMV promotor in addition.This Ras target protein has been represented cytoplasmic signal molecule.The method of clone Ras is referring to Weinberg 1984Mol.Cell.Biol.4:1577, and this paper receives it and is reference.Ras coding plasmid can be used for the super proliferative disease of immunization with opposing and treatment cancer and so on; Especially, Ras dependency cancer, for example cancer of bladder, muscle, lung, brain and bone.

Plasmid pBa.MNp55-IL-12 contains the fragment that PCR produces, its coding p55 coding region, comprising HIV MN gag precursor (core protein) sequence, be cloned on the BamHI site of pBabe.puro, also contain the IL-12 encoding sequence that is connected with SV40 polyadenylation series of operations with the CMV promotor in addition.This plasmid contains these HIV virogenes of coding HIV target protein, can be used for immunization and infects and AIDS with opposing and treatment HIV.Reiz, M.S.1992, AIDS Res.Human Retro.8:1549, this paper receive it and are reference.This sequence can be obtained to Genebank No:M17449, and this paper receives it and is reference.

Plasmid pBa.MNp24-IL-12 contains the fragment that PCR produces from the pMN-SF1 template, coding p24 coding region, comprising the full coding region of HIV MN gag, be cloned in BamHI site and the EcoRI site of pBabe.puro, also contain the IL-12 encoding sequence that is connected with SV40 polyadenylation series of operations with the CMV promotor in addition.This plasmid contains these HIV virogenes of coding HIV target protein, can be used for immunization and infects and AIDS with opposing and treatment HIV.Reiz, M.S., 1992, AIDS Res.Human Retro.8:1549, this paper receive it and are reference.This sequence can be obtained to Genebank No:M17449, and this paper receives it and is reference.

Plasmid pBa.MNp17-IL-12 contains the fragment that PCR produces, its coding p17 coding region, comprising HIV MN gag (core protein) sequence, be cloned in BamHI site and the EcoRI site of pBabe.puro, also contain the IL-12 encoding sequence that is connected with SV40 polyadenylation series of operations with the CMV promotor in addition.This plasmid contains these HIV virogenes of coding HIV target protein, can be used for immunization and infects and AIDS with opposing and treatment HIV.Reiz, M.S., 1992, AIDS Res.Human Retro.8:1549, this paper receive it and are reference.This sequence can be obtained to Genebank No:M17449, and this paper receives it and is reference.

Plasmid pBa.SIVenv-IL-12 contains the fragment of the PCR generation of one section 2.71kb, be that the construction amplification that contains SIV239 from pBR322 obtains, be cloned in BamHI site and the EcoRI site of pBabe.puro, also contain the IL-12 encoding sequence that is connected with SV40 polyadenylation series of operations with the CMV promotor in addition.This plasmid can obtain to AIDS Research and Reference Reagent Program, and catalog number (Cat.No.) is No.210.

Plasmid pcTSP/ATK.env-IL-12 contains the fragment of the PCR generation of the complete HTLV coating of coding coding region, these coding regions are from HTLV-1/TSP and ATK strain isolated, subclone also contains the IL-12 encoding sequence that is connected with SV40 polyadenylation series of operations with the CMV promotor in addition in the pcDNA1/neo carrier.Plasmid pcTSP/ATK.env is reported in 1998, and Proc.Natl.Acad.Sci.USA 85:3599, this paper receive it and be reference.HTLV env target protein can be used for immunization to be infected and t cell lymphoma with opposing and treatment HTLV.

Plasmid pBa.MNgp160-IL-12 contains the PCR product fragment of one section 2.8kb, be to obtain by the construction amplification that contains MNenv among the pSP72, be cloned in point on the BamHI site of pBabe.puro and the EcoRI position, also contain the IL-12 encoding sequence that is connected with SV40 polyadenylation series of operations with the CMV promotor in addition.Reiz, M, S., 1992AIDS Res.Human Retro.8:1549, this paper receive it and are reference.This sequence can be obtained to Genebank No.:M17449, and this paper receives it and is reference.This plasmid contains these HIV virogenes of coding HIV target protein, can be used for immunization and infects and AIDS with opposing and treatment HIV.

Plasmid pC.MNp55-IL-12 contains the fragment that the PCR of one section 1.4kb produces, and is that the gag structural domain amplification by the MN strain isolated obtains, and is cloned in the pCEP4 carrier.These HIV virogenes that this plasmid contains coding HIV target protein and the IL-12 encoding sequence that is connected with SV40 polyadenylation series of operations with the CMV promotor.This plasmid can be used for immunization to be infected and AIDS with opposing and treatment HIV.Reiz, M.S., 1992AIDS Res.Human Retro.8:1549, this paper receive it and are reference.Sequence can be obtained to Genebank No.:M17449, and this paper receives it and is reference.Sequence can be obtained to Genebank No.:M17449, and this paper receives it and is reference.

Plasmid pC.Neu-IL-12 contains the dna fragmentation of one section 3.8kb, this fragment contain cutting from LTR-2/erbB-2 construction and subclone to pCEP ₄The people neu oncogene coding region of carrier, other contains the IL-12 encoding sequence that is connected with SV40 polyadenylation series of operations with the CMV promotor.The pC.Neu plasmid 1987, is reported among the Science 237:178 that by DiFiore this paper receives it and is reference.Neu oncogene target protein has been represented and can be used as the growth factor receptors of immunization with the target protein of opposing and treatment cancer and so on super proliferative disease (the especially cancer of colon, breast, lung and brain).

Plasmid pC.RAS-IL-12 contains the dna fragmentation of one section 1.4kb, the contained ras oncogene encode fragment of this fragment at first is cloned on the BamHI site of pCEP4 from the pZIPneoRAS Central Asia, and other contains the IL-12 encoding sequence that is connected with SV40 polyadenylation series of operations with the CMV promotor.The report of pC.RAS plasmid is referring to Weinberg 1984Mol.Cell.Biol.4:1577, and this paper receives it and is reference.This ras target protein has been represented cytoplasmic signal molecule.Ras coding plasmid can be used for immunization with opposing and treatment cancer and so on super proliferative disease; Especially relevant with ras cancer, for example cancer of bladder, muscle, lung, brain and bony site.

Plasmid pNLpuro-IL-12 contains HIV gag/pol and SV40-puro inserts the personage.This plasmid contains these HIV virogenes of coding HIV target protein, also contains the IL-12 encoding sequence that is connected with SV40 polyadenylation series of operations with the CMV promotor.It can be used for immunization and infects and AIDS with opposing and treatment HIV.

Embodiment 2

Plasmid pCSIL-12 contains the IL-12 encoding sequence that is connected with SV40 polyadenylation series of operations with the CMV promotor.

Embodiment 3

Composition in the certain embodiments of the invention can be by preparing any combination in plasmid pCSIL-12 and the following plasmid.

Plasmid pBa.V α 3 is plasmids of a 7.8kb, contains the EcoRI genomic fragment of one section 2.7kb, and its encode T cell acceptor comprises V α 3 structural domains of L, V and J sections, is cloned in the EcoRI site of pBabe.puro.TXi Baoshouti deutero-target protein can be used for immunization with opposing and cell-mediated autoimmune disease and clonotypic t cell lymphoma and the leukemia of treatment T.

Plasmid pBa.gagpol-vpr is the plasmid of a 9.88kb, contains gag/pol and the vif gene of HIV/MN, is cloned in the pBabe.puro.Disappearance vpr gene.This plasmid contains these HIV virogenes of coding HIV target protein, can be used for immunization and infects and AIDS with opposing and treatment HIV.The HIV dna sequence dna is disclosed in Reiz.M.S., and 1992, AIDS Res.Human Retro.8:1549, this paper receive it and are reference.Sequence can be obtained to Genebank No.:M17449, and this paper receives it and is reference.

Plasmid pM160 is the plasmid of a 11.0kb, contains the PCR fragment of coding HIV-I/3B envelope protein and the rev/tat gene of 2.3kb, is cloned among the pMAMneoBlue.Disappearance nef gene.This plasmid contains these HIV virogenes of coding HIV target protein, can be used for immunization with opposing and treatment HIV and AIDS.The dna sequence dna of HIV-1/3B is disclosed in Fisher, A., and among the 1995Nature 316:262, this paper receives it and is reference.Sequence can be obtained to Genebank No.:K03455, and this paper receives it and is reference.

Plasmid pBa.VL is the plasmid of a 5.4kb, contains the PCR fragment in the VL district of the anti-DNA antibody of encoding, and is cloned on the XbaI and EcoRI site of pBabe.puro.Antibody deutero-target protein has been represented and can be used for immunization with opposing and cell-mediated autoimmune disease and clonotypic B cell lymphoma and the leukemic target protein of treatment B.The universal method of the functional variable region of clonal antibody can be referring to Chaudhary, and V.K. etc., 1990Proc.Natl.Acad.Sci.USA 87:1066, this paper receive it and are reference.

Plasmid pOspA.B is the plasmid of a 6.84kb, contains the OspA and the antigenic coding region of OspB of coding borrelia burgdorferi (causing the spirochete of Lyme disease), is cloned on the BamHI and SalI site of pBabe.puro.Williams, W.V. etc., 1992, DNA and Cell.Biol.11 (3): 207, this paper receives it and is reference.This plasmid comprises the pathogenic genes of the target protein of encoding, and can be used for immunization opposing Lyme disease.

Plasmid pBa.Rb-G is the plasmid of a 7.10kb, contains the proteic PCR product of coding for rabies virus G fragment, is cloned on the BamHI site of pBabe.puro.This plasmid that contains the mad dog G albumen pathogenic genes of encoding can be used for immunization opposing rabies.This dna sequence dna is disclosed among the Genebank No.:M32751, and this paper receives it and is reference.Also can be referring to Anilionis, A. etc., 1981, Nature 294:275, this paper receive it and be reference.

Plasmid pBa.HPV-L1 is the plasmid of a 6.80kb, contains the PCR fragment of the L1 capsid protein of coding human papillomavirus (HPV) (comprising HPV strain 16,18,31 and 33), is cloned on the BamHI and EcoRI site of pBabe.puro.This plasmid can be used for immunization with opposing HPV infection with by its cancer that causes.This dna sequence dna is disclosed among the Genebank No.:M15781, and this paper receives it and is reference.Also can be referring to Howley, P., 1990, Feilds Virology, Vol.2, Channock, editors such as R.M, the 58th chapter: 1625 and Shah, K. and P.Howley, 1990, Feilds Virology, Vol.2, Channock, editors such as R.M, the 59th chapter is all received by this paper and is reference.

Plasmid Ba.HPV-L2 is the plasmid of a 6.80bk, contains the PCR fragment of coding human papillomavirus (HPV) (comprising HPV strain 16,18,31 and 33) L2 capsid protein, is cloned on the BamHI and EcoRI site of pBabe.puro.This plasmid can be used for the cancer of immunization to resist the HPV infection and to be caused by it.This dna sequence dna is disclosed among the Genebank No.:M15781, and this paper receives it and is reference.Also can be referring to Howley, P., 1990, Feilds Virology, Vol.2, Channock, editors such as R.M, the 58th chapter: 1625 and Shah, K. and P.Howley, 1990, Feilds Virology, Vol.2, Channock, editors such as R.M, the 59th chapter is all received by this paper and is reference.

Plasmid pBa.MNP7 is the plasmid of a 5.24kb, contains the PCR fragment of the p7 coding region that comprises HIV MN gag (core protein) sequence, is cloned on the BamHI site of pBabe.puro.This plasmid contains these HIV virogenes of coding HIV target protein, can be used for immunization and infects and AIDS with opposing and treatment HIV.Reiz, M.S., 1992AIDS Res.Human Retro.8:1549, this paper receive it and are reference.This sequence can be obtained to Genbank No:M17449, and this paper receives it and is reference.

Plasmid pGA733-2 is the plasmid of a 6.3kb, contain the GA-733-2 TSA, from colon carcinoma cell line SW948, be cloned into the pCDM8 carrier (referring to Seed B. and A.Aruffo, 1987Proc.Natl.Acad.Sci.USA 84:3365, this paper with its receive be with reference to) the BstXI site on.This cancer-related target protein has been represented and has been used for the target protein of immunization with opposing and treatment cancer and so on super proliferative disease.GA733-2 antigen is useful resistive connection intestinal cancer target antigen.The antigenic report of GA733 is referring to Szala.S etc., and 1990, Proc.Natl.Acad.Sci.USA 87:3542-3546, this paper receive it and are reference.

Plasmid pT4-pMN7 is the plasmid of a 11.15kb, contains the cDNA of coding people CD4 acceptor, is cloned on the EcoRI site of pMV7 carrier.The CD4 target protein can be used for immunization with opposing and treatment t cell lymphoma.Plasmid pT4-pMN7 can obtain to AIDS Repository, catalog number (Cat.No.) No.158.

Plasmid pDJGA733 is the plasmid of a 5.1kb, contains the GA733 TSA, is cloned in the BamHI site of pBabe.puro.This cancer-related albumen has been represented and has been used for the target protein of immunization with opposing and treatment cancer and so on super proliferative disease.GA733 antigen is useful resistive connection intestinal cancer target antigen.

Plasmid pBa.RAS is the plasmid of a 6.8kb, contains the Ras coding region, at first subclone and being cloned on the BamHI site of pBabe.puro from pZIPneoRAS.This ras target protein has been represented cytoplasmic signal molecule.The method of clone ras can be referring to Weinberg 1984Mol.Cell.Biol.4:1577, and this paper receives it and is reference.Ras coding plasmid can be used for immunization with opposing and treatment cancer and so on super proliferative disease; The cancer of especially relevant bladder, muscle, lung, brain and bone with ras.

Plasmid pBa.MNp55 is the plasmid of a 6.38kb, the PCR fragment that contains coding p55 coding region, this coding region comprises HIV NM gag precursor (core protein) sequence, being cloned on the BamHI site of pBabe.puro. this plasmid contains these HIV virogenes of coding HIV target protein, can be used for immunization and infects and AIDS with opposing and treatment HIV.Reiz, M.S., 1992AIDS Res.Human Retro.8:1549, this paper receive it and are reference.This sequence can be obtained to Genbank No:M17449, and this paper receives it and is reference.

Plasmid pBa.MNp24 is the plasmid of a 5.78kb, and it contains the fragment that PCR produces from the pMN-SF-1 template, and coding p24 coding region comprising the full coding region of HIV MNgeg, is cloned on the BamHI and EcoRI site of pBabepuro.This plasmid contains these HIV virogenes of coding HIV target protein, can be used for immunization and infects and AIDA with opposing and treatment HIV.Reiz, M.S., 1992AIDS ResHuman Retro.8:1549, the people this paper that receives is for referencial use.Sequence can be obtained to Genebark No:17449, and it is for referencial use also to include this paper in.

Plasmid pBa.MNp17 is the plasmid of a 5.5kb, contains the PCR fragment that one section coding comprises the p17 coding region of HIV gag (core protein) sequence, is cloned in BamHI and the EcoRI site of pBabe.puro.This plasmid contains these HIV virogenes of coding HIV target protein, can be used for immunization and infects and AIDS with opposing and treatment HIV.Reiz, M.S., 1992AIDS Res.Human Retro.8:1549, this paper receive it and are reference.This sequence can be obtained to Genbank No:M17449, and this paper receives it and is reference.

Plasmid pBa.SIVenv is the plasmid of a 7.8kb, contains one section and includes the 2.71PCR fragment that the construction amplification of SIV239 obtains from pBR32, is cloned on the BamHI and EcoRI site of pBabe.puro.This plasmid can obtain to AIDS Research and Reference Reagent Program; Catalog number (Cat.No.): 210.

Plasmid pcTSP/ATK.env is the plasmid of a 8.92kb, the PCR fragment of the HTLV-1/TSP that the one section coding that contains is complete and the HTLV coating coding region of ATK strain isolated, and subclone is in the pcDNA1/neo carrier.The report of related plasmid pcTSP/ATK.env is referring to 1988, and Proc.Natl.Acad.Sci.USA85:3599, this paper receive it and be reference.HTLV env target protein can be used for immunization to be infected and t cell lymphoma with opposing and treatment HTLV.

Plasmid pBa.MNgp160 is the plasmid of a 7.9kb, contains the 2.8kb PCR fragment that one section construction amplification that contains MNenv from pSP72 obtains, and is cloned in BamHI and the EcoRI site of pBabe.puro.Reiz, M.S., 1992AIDS Res.Human Retro.8:1549, this paper receive it and are reference.This sequence can be obtained to Genbank No:M17449, and this paper receives it and is reference.This plasmid contains these HIV virogenes of coding HIV target protein, can be used for immunization opposing and treatment HIV infection and AIDS.

Plasmid pC.MNp55 is the plasmid of a 11.8kb, contains one section 1.4kb PCR fragment that obtains from the amplification of MN strain isolated gag district, is cloned in the pCEP4 carrier.This plasmid contains these HIV virogenes of coding HIV target protein, can be used for immunization opposing and treatment HIV infection and AIDS.Reiz, M.S., 1992AIDS Res.Human Retro.8:1549, this paper receive it and are reference.This sequence can be obtained to GenbankNo:M17449, and this paper receives it and is reference.

Plasmid pC.Neu is the plasmid of a 14.2kb, contains one section 3.8kb dna fragmentation that comprises people neu oncogene coding region, and this coding region cutting is from the LTR-2/erbB-2 construction, and subclone is in the pCEP4 carrier.The report of relevant pC.Neu plasmid is referring to DiFiore 1987Science 237:178, and this paper receives it and is reference.Neu oncogene target protein has been represented a kind of growth factor receptors, and they can be used as the super proliferative disease that target protein comes immunization opposing and treatment cancer and so on; Especially the cancer of colon, breast, lung and brain.

Plasmid pC.RAS is the plasmid of a 11.7kb, contains one section 1.4kbDNA fragment that comprises ras oncogene coding region, and this coding region is cloned into the BamHI site of pCEP4 carrier earlier from the pZIPneoRAS Central Asia.The report of related plasmid pC.RAS is referring to Weinberg 1984Mol.Cell.Biol.4:1577, and this paper receives it and is reference.The ras target protein has been represented cytoplasmic signal molecule.The plasmid of coding ras can be used for immunization opposing and treatment cancer and so on the cancers relevant with ras such as super proliferative disease, especially bladder cancer, muscle cancer, the cancer of the brain and osteocarcinoma.

Plasmid pNLpuro is the plasmid of a 15kb, contains HIV gag/pol and SV40-puro and inserts fragment.This plasmid contains these HIV virogenes of coding HIV target protein, can be used for immunization opposing and treatment HIV infection and AIDS.

Embodiment 4

In order to improve vaccine, it may be crucial guiding immunne response specifically.It is reported that in the multiple disease type that comprises transmissible disease, autoimmune disease and allergy, immunoreactive type (Th1 or Th2) is crucial.For the strong and stable cell-mediated immune response of inducing anti-HIV to infect, coupling immunological adjuvant and immunomodulator when immunity (for example cytokine) can be strengthened cellullar immunologic response, and guiding antigen dependent immune response is changed to the Th1 type by the Th2 type.

For gene improves intravital immunne response, human cell factor gene (IL-12 or GM-CSF gene) and HIV construction are together transmitted.Check transmitting the immunne response that causes, and induce (especially antiviral CTL replys) of pair cell immunity studied because of these genes and HIV-1DNA vaccine are common.

Respectively with IL-12 and GM-CSF gene clone in expression vector, be under the CMV promoter regulation.Then the dna vaccination box of this gene plasmid expression cassette and HIV-1 (referring to preamble, or U.S. Patent application 08/642,045, apply on May 6th, 1996, this paper with its receive be with reference to) be expelled to together in the mouse body.The common immunization of these gene adjuvant boxes is analyzed in the immunology effect aspect the antigen-specific immune response intensity.See to transmit altogether and reduced humoral response, simultaneously, inoculate moderate altogether with IL-12 and strengthened humoral response with IL-12.Inoculate the increase of all having found antigen specific T helper propagation altogether with IL-12 or GM-CSF.Importantly, induced significant CTL with direct CTL experimental observation jointly to the IL-12 gene.On the contrary, in same research, adopt the GM-CSF gene, almost do not observe any effect of CTL inductive.These results prove and use dna vaccination to produce specific immune response.These results show that also this method is in the purposes of illustrating in the molecular specificity mode aspect the basic immunologic function.

Material and method

Mouse

6 to 8 the week ages the female mouse of Balb/c available from Harlan Sprague Dawley, Inc. (indianapolis, Indiana).These mouse are closed in temperature control, and light-operated (light-cycled) is indoor.Governing principle according to National Institute ofHealth and University of Pennsylvania is nursed them.

Reagent

Preparation dna vaccination preparation pCMN160, pCGN160, pCGag/Pol.With IL-12 and GM-CSF gene clone be inserted in the expression vector that has the CMV promotor.Recombinant vaccinia seedling (vMN462, vVK1, VV:gag and vSC8) derives from NIH AIDS Research and Reference Reagent Program.

The DNA inoculation

Use improved DNA inoculation method, it can use plasmid to transmit gene in vivo and improve intravital protein expression level.Specifically, in Balb/c mouse quadriceps muscle of thigh with Bupivacaine-HCl (Sigma, St.Louis) the solution 100 μ l of No. 27 needle injection 0.25%.After 2 days, inject the DNA construction (with the phosphate buffered saline buffer wiring solution-forming) that 50 μ g are studied in same injection site.The range gene expression cassette be included in injection jointly before selected plasmid is mixed.

Facs analysis

Cell (1 * 10 ⁵) with FACS damping fluid (PBS that contains 1%BSA and 0.1% sodium azide) washing 3 times, under saturation conditions, hatched on ice 30 minutes with FITC and/or the crosslinked mAbs of PE.After FACS damping fluid washing 3 times, with FACScan (Becton Dickinson) analysis of cells.

ELISA

As described in the prior art, (Intracel Corp.Cambridge MA) is made into 2 μ g/ml solution, adds in the droplet hole 4 ℃ of placements of absorption and spends the night with gp120 or gp41 with 0.1M carbonate-bicarbonate buffer (pH9.5).With PBS-0.05% tween 20 washing test plate, in 37 ℃ of sealings 1 hour, (MO) diluent was hatched together for Sigma, St.Louis with the crosslinked goat anti-mouse IgG of HRP-of manufacturers suggestion or IgA then with the PBS solution of 3%BSA and 0.05% tween 20.With TM damping fluid (Sigma) washing and flushing (develope) test panel.On Dynatech MR5000 test panel readout instrument, read the OD value at 450nm place.

The T cell proliferation test

The spleen of results mouse therefrom prepares lymphocyte.The isolated cells suspension is resuspended to 1 * 10 ⁶Cells/ml.In the every hole of 96 hole flat-flooreds, add the equal portions of 100 μ l, include 1 * 10 ⁵Cell.It is the protein of 20 μ g/ml that each hole adds 10 μ l concentration, triplicate.These cells are at 5%CO ₂In 37 ℃ cultivated 3 days.Add 1 μ Ci tritiated thymidine in every hole, cell was cultivated 12 to 18 hours for 37 ℃.The results test panel, (Wallac, Turku Finland) go up the amount of measuring the tritiated thymidine that mixes at β test panel readout instrument.In order to ensure cell health, 10 μ g/ml PHA are used as polyclone stimulant positive control.

The cytotoxic T lymphocyte test

Carry out one time 5 hours chromium ⁵¹Discharge the CTL test.From spleen results lymphocyte, remove red corpuscle, and with fresh substratum washing for several times, be prepared into the effector cell.With 3 * 10 ⁶Individual p815 cell infected 16 hours for 37 ℃, the target cell that the preparation Smallpox Vaccine infects.Target cell is with the Na of 100 μ Ci/ml ₂ ⁵¹CrO ₄Carry out 90 minutes mark, and be used for cultivating the stimulation splenocyte 37 ℃ 4 to 6 hours.Carry out the CTL test, the effector cell: (E: ratio T) was from 50: 1 to 12.5: 1 for target cell.The results supernatant liquor is counted on LKB Clini Gamma γ-calculating instrument.Calculate specificity cracked per-cent with following formula:

100 * { experiment release-spontaneous release } ÷ { maximum release-spontaneous release }

Cracking target cell in the substratum that contains 1%Triton X-100 is measured in maximum release." if spontaneous release " counting surpass " maximum release " 20%, then this test be considered as invalid.

The result

The phenotype of mouse spleen

After the co-inoculation, the spleen of each test group mouse seems inequality.So, the spleen of all inoculation mouse of collecting is weighed and is estimated.Figure 1A has shown the weight of these animal spleens.Although the control mice of injection unitary agent and the spleen weight close (about 100mg) of non-inoculation control mice, the mouse of having injected the Gag/Pol+IL-12 gene, the weight of its spleen is 3 times of contrast spleen.On the other hand, with the mouse of Gag/Pol+GM-CSF inoculation, its spleen does not increase.It is pointed out that list with Gag/Pol or single mouse with the IL-12 inoculation, does not all cause the obvious increase of spleen.Have only when common injections of antigens and IL-12 box gene, just cause splenomegaly, pointing out this is the combined effect of two kinds of gene products.And, shown in Figure 1B, from the lymphocyte quantity of Gag/Pol+IL-12 spleen 3 times of contrast spleen.Equally, compare with contrast spleen cell number, Gag/Pol and IL-12 mice immunized spleen all do not have significantly to increase on lymphocyte quantity.The photo of representative spleen is seen Fig. 2.Corresponding with weight separately, antigen+IL-12 spleen is than the big several times of other spleen.According to existing report, the p35 chain of IL-12 all has constitutive expression in many cell types.So, may have only former this effect that caused of a p40 chain and dna immunization.In order to detect this point, one of IL-12 heterodimer gene (p35 and p40) is given jointly with Gag/Pol respectively.As shown in Figure 3, the both does not cause the spleen enlargement.Above data show, p35 and the p40 gene of IL-12 are injected jointly with dna vaccination together, cause that spleen enlargement and corresponding splenocyte quantity increase.Above data are supported following viewpoint, and these plasmids have advanced the people in vivo same cell has been coordinated all three kinds of compositions: p35 chain, p40 chain and unique antigenic transcribing, thus induced being seen biology to change.

Facs analysis

To carry out signature analysis and carried out facs analysis in order further the cell of enlargement spleen to be formed.As shown in Table 3, use at the antibody of CD3 with at the antibody of B220, CD4 and CD8 and carry out the amphophilic FACS of splenocyte.Show according to the result, compare, the positive B cell of the B220 of coating+IL-12 or Gag/Pol+IL-12 construction immunization group per-cent slightly descend (being respectively 17.46% and 21.62%) with the B cell per-cent (25.43%) in the non-immunity contrast spleen.In addition, compare the CD8+T cell per-cent of coating+IL-12 or Gag/Pol+IL-12 construction immune group slightly raise (being respectively 21.72% and 16.88%) with the per-cent (13.69%) in the non-immunity contrast spleen.

Humoral response

Collect the antiserum(antisera) of immune mouse, with the antigenic specific antibody reaction of elisa assay anti-HIV-1.That Fig. 4 shows is the ELISA result of the 28th day collected sample after the immunization.When extent of dilution was 1: 100, the proteic antibody response of the anti-HIV-1 gp120 that the antiserum(antisera) of pCEnv+pCGM-CSF immune group demonstrates was bigger than single group with the pCEnv immunity.On the other hand, through the same long time, the humoral response of pCEnv+pCIL-12 immune group is obviously less.In repeated experiments, IL-12 generally suppresses the specific antibody reaction and reaches 10-20%, and GM-CSF then shows positive interaction.Such humoral response may be relevant with the being seen variation of B cell quantity in the determined splenocyte of facs analysis.

T cell proliferation

Auxiliary lymphocytic activation of T and propagation are playing an important role by the B cell induction humoral immunoresponse(HI) of expansion antigen activates with in by expansion CD8+ cytotoxic T lymphocyte inducing cell immunne response.In 2 weeks behind the dna immunization, the spleen of collection immune mouse is isolated lymphocyte.Test the T cell proliferation of these cells then as previously mentioned.Fig. 5 shows with the dna vaccination mice immunized of coding HIV-1gag/pol (pCGag/Pol) with the proliferation test result of pCGag/Pol and IL-12 or the common mice immunized of GM-CSF.Reorganization p55 albumen and 20 μ g/ml lectin PHA are used as polyclone stimulant positive control.As shown, inmature mouse spleen control group has the proliferative response of low background level, and extent of dilution is that 1: 2 o'clock stimulation index is 1.2, singly can be observed the proliferative response of medium level with the pCGag/Pol immune group, and 1: 2 o'clock stimulation index of extent of dilution is 9.2.With the group of pCGag/Pol and the common immunity of IL-12 gene with pCGag/Pol and the common immune group of GM-CFS, can be observed the rapid increase of proliferative response, stimulation index is respectively 17.1 and 15.6.

There is not the CTL test of stimulated in vitro

Carried out direct CTL test in order further to study the enhancing of cytoactive.Immune mouse is as previously mentioned collected spleen cell, induces and carries out the CTL test but splenocyte is not carried out stimulated in vitro.Carried out this test on the same day of results spleen, the chromium of measuring specificity bovine vaccine infection and non-specific bovine vaccine target cell infection discharges.The splenocyte of pCGag+IL-12 immunity is observed active significantly increase (Fig. 6) of specific CTL.Single control group with the immunity of IL-12 box gene target cell SL as a result is no more than background level.In addition, single with 50: 1 effects of Gag/Pol immunity: the target ratio can be observed low-level SL (3%).On the contrary, the sample that Gag/Pol+IL-12 gives is jointly being imitated: the target ratio is 50: 1 o'clock, and specificity is cracked into 62%, and at 12.5: 1 o'clock, titre was 9%.On the other hand, there is not detectable CTL activity with Gag/Pol plasmid and GM-CSF plasmid immune result.From also can be observed similar result (Fig. 7) with the common mice immunized of cytokine gene with HIV-1 coating construction.Single have only low-level specific CTL with coating with the group of coating+GM-CSF immunity, is respectively 4% and 1%.On the other hand, the CTL of coating+IL-12 immune group is active significantly to be strengthened, and dissolution rate reaches 59%.In Gag/Pol and the common immune group of coating (Fig. 6 and Fig. 7), the target cell with irrelevant antigen presentation bovine vaccine preparation is carried out same CTL test, the result does not have tangible CTL dissolving.So active significantly reinforcement of the CTL that the common immunity of antigen and IL-12DNA causes not is to be caused by the NK activity, because above result is an antigen-specific.

Discuss

It is reported that with different treatment targets and transmission technology, the inoculation dna vaccination has all produced immunne response in the body.

The key character of many vaccines may be the immunizing power of inducing cell mediation.For example, in the natural infection process, the ctl response of anti-HIV-1 occurs quite early, and temporarily presents the being associated property of foundation with viral settlement.CTL T cell is removed in the virus with destroying virus infected cell at target-seeking and is played an important role.To make and induce immunne response to become possibility by advancing specific CTL to react the immune response that guides antiviral protein at the wide spectrum more of multiple target antigen in the virus.Compared with AIDS patient, easilier in the infected patient of health record antiviral CTL activity, and it is reported that specific CTL reduces along with the progress of the course of disease, this clearly links up ctl response and clinical state preferably.In this respect; a kind of have that high specific CTL replys and the macaca cynomulgus that hangs down antibody response can be protected the attack of avoiding mosaic SIV/HIV (SHIV); though and the monkey with low CTL and high antibody response can be controlled duplicating of virus, but can not be subjected to protecting fully.As if the specific CTL reaction contribute in the asymptomatic stage to some extent to keeping the HIV-1 infection.So, induce in the intensive body HIV-1 specific CTL to reply and may in the development that final protection host avoids HIV to infect, play an important role by dna immunization inoculation.

By can enhancing immunity replying with IL-12 and the common transmission of GM-CSF as gene adjuvant, especially CTL replys, and this is studied with the dna vaccination of HIV-1.With IL-12 gene and GM-CSF gene clone in expression vector and the dna vaccination box of HIV-1 inject to mouse together.The plasmid of coding IL-12 and the common immunization of dna vaccination of HIV-1 cause the remarkable enhancing that antigen-specific CTL replys.Show as the enhancing humoral immunization with the common transmission of GM-CSF, then suppress humoral response about 20% with the common transmission of IL-12.

The dna vaccination of cytokine gene adjuvant and HIV-1 transmitted altogether have many significant immunological effects.At first, the size of its spleen of mouse of injection dna vaccination and IL-12 gene and weight almost are 3 times of contrast spleen.In addition, the white corpuscle in these spleens is more than 2 times of quantity of leucocyte in the contrast spleen.Above result is consistent with former discovery, and the IL-12 that promptly recombinates in the mouse body causes splenomegaly.Discoveries such as Car, in wild-type mice, injection IL-12 causes that spleen weight increases by 500.Above IL-12 induces the variation of generation relevant with significantly increasing of IFN-γ serum level in wild-type mice.But, in IFN-γ acceptor defect type mouse, give IL-12 and also induce the splenomegaly that produces similar quality (reach normal size 2 times).These early stage researchs report that all injection IL-12 albumen causes the spleen enlargement.It is suitable with the research of having delivered of using reorganization IL-12 albumen to do to transmit the gene induced splenomegaly degree of a small amount of IL-12 in the body.It is reported, to a certain degree toxic action may be arranged for injection reorganization IL-12 in the mouse body, for example lose weight even dead the mouse of accepting injection.Be important to note that, give jointly IL-12 gene induced the spleen enlargement, but the mouse of accepting injection is without any the bad variation of visible.The low-level transmission of processing persistence naturally that this prompting and plasmid inoculation cause may be favourable clinically.But the present invention has showed at this and a kind ofly has been used to induce tangible systemic immunne response does not have the DNA transmission method of toxic action.

Except inducing the spleen enlargement, give to handle specific immune response from Th2 to Th1 jointly by IL-12 and dna vaccination.In this respect, the common transmission of dna vaccination and IL-12 reduces the specific antibody reaction, then strengthens the specific antibody reaction with the common transmission of GM-CSF.Above result is that key cytokines is consistent by the Th2 type in the guiding immunne response in the Th1 type changes with the previous IL-12 that reports.The result of these antibody also conforms to the FACS data of splenocyte, in described data, with its B220+B percentage of cells minimizing of mouse of immunogen (HIV-1 coating or Gag/Pol) and IL-12 genetic immunization.In addition, transmit altogether with cytokine and can be observed the tangible antigen-specific hormesis of T cell.Antigen-specific propagation is that CD4 helper cell inductive is well accused of, and it seems that this be two kinds of characteristics that cytokine has.

In order further to illustrate the direct CTL test of the coimmune t cell response of DNA having been carried out the splenocyte of results not being carried out stimulated in vitro.The reinforcement that CTL replys is to prove that the immunne response that dna immunization can be caused is from being turned to the important evidence of Th1 by Th2.Animal groups with dna vaccination and the common immunity of IL-12 is observed the obvious enhancing that specific CTL is replied.In a word, encode jointly in the body immunogenic gene and the gene that causes the key cytokines IL-12 of Th1 type immunne response through T cell proliferation and CTL test determination, show the cellullar immunologic response of reinforcement.

Type and direction (for example guiding is reacted and is converted into the Th1 type by the Th2 type) that the gene that common premunition goes up important molecule helps to guide and handle immunne response can be used to cause clinically more effective immunne response.IL-12 gene and dna immunization are former gives to have suppressed humoral response jointly moderately, replys and greatly strengthened CTL.In addition, also induced the spleen enlargement, this is to give the proteic feature of reorganization IL-12 in the mouse body.So the present invention shows, transmits DNA in the body and not only can be used for producing more effective vaccine of new generation but also can be used as the analysis tool of immunologic function mechanism being carried out the molecule parsing.

Embodiment 5

In order further to guide immunne response in the body, wide spectrum cytokine gene and HIV-1DNA immunogenicity construction transmitted altogether inducing with regulating effect of immunne response studied.Selecting to transmit altogether with cytokine gene is because cytokine plays important adjusting and signal effect in immunization.Though the many cells except that immune system cell can produce and discharge cytokine, the cytokine that lymphocyte produces has special meaning because of it in the effect of regulating immune system cell.For example, the existence of IL-1, IFN-γ and IL-12 activates the Th0 precursor cell becomes Th1 inflammatory T cell.On the other hand, the release of IL-4, IL-5 or IL-10 makes the Th0 precursor cell be transformed into the Th2 helper of arms.In addition, preceding inflammatory cytokine IL-1, IFN-α and IFN-β play active effect in bringing out Inflammatory response.

Common effect of transmitting is studied to preceding inflammatory cytokine (IL-1 α, TNF-α and IFN-β), Th1 cytokine (IL-2, IL-15 and IL-18) and Th2 cytokine (IL-4, IL-5 and IL-10).Specifically, the gene of these preceding inflammatory cytokines, Th1 and Th2 cytokine is cloned into respectively in the expression vector, is under cytomegalovirus (CMV) promoter regulation.Then such gene plasmid expression cassette is injected in the mouse body as mentioned before with HIV-1D dna vaccination box.Having analyzed these gene adjuvant boxes injects altogether to the direction of antigen-specific immune response and the immunological role of intensity generation.

Cytokine gene and dna immunization protogene box injected altogether can regulate antigen-specific immune response.More broadly, this strategy that vehicular gene with immunology significance is transmitted jointly can produce the dna vaccination of a new generation, has strengthened their potential in clinical efficiency and application.

Material and method

The DNA plasmid

HIV-1 envelope protein and gag/pol proteic dna vaccination construction pCEnv and pCGag/Pol are expressed in the parent material preparation of adopting standard technique and buying easily.With standard technique and the parent material that obtains easily with the gene clone of the IL-4 of people IL-1 α, IL-2, IL-5, IL-10, IL-15, TNF-α, TNF-β and mouse and IL-18 to pCDNA3 expression vector (Invitrogen, Inc., San Diego, CA) in.It is reported that people IL-1 α, IL-2, IL-5, IL-10, IL-15, TNF-α and TNF-β have activity in mouse cell.Use Qiagen Maxi Prep test kit (Qiagen, Santa Clara, CA) production of plasmid DNA and purifying in bacterium.

Reagent and clone

Human rhabdomyosarcoma (RD) and mouse hypertrophy cell knurl p815 clone available from ATCC (Rockville, MD).Express HIV-1 coating, gag/pol and tilactase recombiant vaccine vMN462, vVK1 and vSC8 available from NIH AIDS Research and Reference Reagent Program.HIV-1 env peptide (RIHIGPGRAFYTTKN) is with standard technique and the parent material synthetic that obtains easily.Reorganization Pr55 or gp120 albumen derive from Quality Biological (Gaithersburg, MD).Reorganization p24 albumen available from Intracell (Cambridge, MA).Antibody at IL-1 α, IL-2, IL-5, IL-10, IL-15, TNF-α and TNF-β derives from R﹠amp; D Systems (Minneapolis, MN).

The expression of cytokine construction

After being transfected into the RD cell, adopt immuno-precipitation or cytokine ELISA to confirm the expression of cytokine construction.Cell is through the PBS washed twice, starve in the DMEM of serum-free, no methionine(Met) and halfcystine and supported one hour, use then 200 μ Ci/ml (1,200Ci/mmole) ³⁵S protein labeling mixture (NEN/DuPont) mark.At the RIPA of 0.5ml damping fluid (50mM Tris HCl pH7.6; 150mMNaCl; 0.2%Triton X-100; 0.2% Septochol; 0.1%SDK and 1mM PMSF) in cracking labeled cell on ice, 15000rpm clarified in centrifugal 10 minutes then.Lysate after the clarification and associated antibodies (R﹠amp; D Systems) hatches 90 minutes on ice.Immune complex adds A albumen sepharose, and 40 ℃ of joltings mixed 90 minutes.With behind the damping fluid thorough washing that contains high salt concentration and BSA protein precipitation being resuspended in the 50 μ l 1X sample buffers, 100 ℃ were heated 3 to 5 minutes.Get a protein example and carry out SDS 12%-PAGE.In order to carry out fluorography, gel is immersed in the 1M sodium salicylate that contains 10% glycerine 15 minutes, drying is carried out radioactive automatic developing with Kodak X-omat-AR film.Collect the supernatant liquor of the RD cell of transfection, with cytokine ELISA test kit (Pharmingen, San Diego, CA and R﹠amp; DSystem) test is expressed.

The mouse DNA inoculation

6 to 8 the week age balb/c mouse (Harlan Sprague Dawley, Inc., Indianapolis, IN) the various interested DNA constructions of 50 μ g are injected at quadriceps muscle of thigh position, these constructions are formulated in phosphate buffered saline buffer (PBS) and 0.25% Bupivacaine-HCl (Sigma, St.Louis, MO) in.Comprising jointly of range gene expression cassette mixes selected various plasmids, injection then, each 100 μ g.

ELISA

P24 or gp120 albumen to be to be diluted to 2 μ g/ml in 0.1 carbonate-bicarbonate buffer (pH9.5), get 50 μ l4 ℃ and spend the night and be adsorbed onto on the hole of droplet plate.Test panel sealed 1 hour at 37 ℃ of PBS solution with 3%BSA 0.05% tween 20 with PBS-0.05% tween 20 washed twice.With 0.05% tween 20 dilution mouse resisting anteserum, and 37 ℃ hatched 1 hour, and (Sigma, St.Louis MO) are hatched together with the crosslinked goat anti-mouse IgG of HRP-then.With washing of 3 ' 3 ' 5 ' 5 ' TMB (Sigma) damping fluid and flushing.The read test plate is in the optical density value at 450nm place on Dynatech MR5000 test panel readout instrument.

The t helper cell proliferation test

From spleen results lymphocyte, remove red corpuscle and prepare the effector cell for several times with the washing of fresh culture liquid medium.With the resuspended one-tenth 5 * 10 of isolated cell ⁶Cells/ml.Get and contain 5 * 10 ⁵100 μ l equal portions of cell add in each hole of the flat droplet plate in people 96 holes immediately.Add reorganization Pr55 or gp120 albumen in test hole, ultimate density is respectively 5 μ g/ml and 1 μ g/ml, and is triplicate.These cells are at 5%CO ₂In 37 ℃ cultivated 3 days.Add people's 1 μ Ci tritiated thymidine in each hole, cell was cultivated 12 to 18 hours for 37 ℃.The results test panel, (Wallac, Turku Finland) go up the tritiated thymidine amount of mixing the people of measuring at β test panel readout instrument.Calculate stimulation index with following formula:

Stimulation index (SI)=(trial value/spontaneous value)

Comprise 10% foetal calf serum in the test hole of spontaneous value as irrelevant albumen contrast.In addition, the animal with pCEnv or contrast immunity is 1 to the proteic SI of Pr55 generally.Similar, pCGag/pol or contrast are 1 to the proteic SI of gp120 generally.In order to ensure cell health, PHA or con A (Sigma) are used as polyclone stimulator positive control.The SI of PHA or con A control sample is 20 to 40.

The cytotoxic T cell test

Target cell that target cell that infects with bovine vaccine or peptide were handled carried out 5 hours ⁵¹Chromium discharges the CTL test, has carried out external this test that has effector to stimulate and do not have the effector stimulation respectively.In the test of stimulated in vitro, the cytositimulation effector cell who infects with relevant bovine vaccine (vMN462 of coating and the vVK1 of gag/pol), and with the coating specific peptide (RIHIGPGRAFYTTKN) of 0.1% glutaraldehyde or 1M, with 5 * 10 ⁶Cells/ml is fixed 4 to 5 days in the CTL substratum.The CTL substratum is by the improved Dulbecco substratum of 1: 1 Iscove (Gibco-BRL, Grand Island, NY) and Hanks balanced salt solution (Gibco-BRL) (contain 10% foetal calf serum (Gibco-BRL) and 10% and do not contain Con A (Becton Dickinson Labware, Bedford, RAT-T-STIM MA)) form.37 ℃ with 10 to 20 infection multiplicity (MOI) to 3 * 10 ⁶Individual p815 cell carries out 5 to 12 hours infection, prepares the target cell that bovine vaccine is infected thus.The p815 cell is hatched the target cell that the preparation peptide is handled with the peptide of 1 μ M concentration.Carry out the standard chlorine release test, promptly use 100 μ Ci/mlNa ₂ ⁵¹CrO ₄Target cell is carried out 60 to 120 minutes mark, this cell was cultivated 4 to 6 hours for 37 ℃ with irriate effect splenocyte.Measure the CTL dissolving, the effector cell: (E: ratio T) was from 50: 1 to 12.5: 1 for target cell.The results supernatant liquor is counted on LKB CliniGamma γ calculating instrument.Measure SL per-cent with following formula:

100 * { test burst size-spontaneous burst size } ÷ { maximum burst size-spontaneous burst size }

Maximum burst size is measured with the nutrient solution dissolving target cell that contains 1%Triton X-100." if spontaneous release " surpass " maximum discharge " 20%, then this test be considered as invalid.

The complement dissolving of CD8+T cell

(Pharmingen, San Diego CA) handle, and hatch 45 minutes with 37 ℃ of rabbit complements (Sigma) then, and the CD8+T cell is removed from splenocyte with anti-CD8 monoclonal antibody.

The result

The expression of cytokine gene box

Cytokine gene is cloned into (Figure 10) in the pCDNA3 plasmid expression vector respectively.By these cytokine-expressing boxes are confirmed in the sequential analysis of whole insertion fragment (at 5 ' terminal and 3 ' end).In addition, these cytokine in-vitro transfections are in the RD cell, according to the materials and methods part of preamble, by immunoprecipitation that uses associated antibodies or the expression that confirms these constructions by cytokine ELISA.

Humoral immunoresponse(HI) after the common injection of cytokine gene

Collect antiserum(antisera), reply with the antigenic specific antibody of elisa assay anti-HIV-1 with the pCGag/pol mice immunized.

In these experiments, the same day and the 14th day, intramuscular injection gave the various DNA of 50 μ g jointly.Before the injection and injected back 28 days, serum is collected in mouse (4 every group) bloodletting.Did serial dilution 1: 100,1: 200,1: 400,1: 800,1: 1600 and 1: 3200.The background indensity of ELISA is lower than 0.015.It is similar to repeat these experimental results.As shown in data, measured the terminal point of the antibody of inoculation group with the proteic ELISA of anti-p24gag and tired, also the antibody endpoint of having measured inoculation group with the ELISA of anti-gpl20 envelope protein is tired.Following data are the gag/pol-specific antibody titres that produce in the serum that the 28th day collects behind dna immunization:

Anti-p24 antibody titer

Single with pCGag/pol 400

pCGag/pol+IL-1α 1600

pCGag/pol+TNF-α 1600

pCGag/pol+TNF-β 1600

pCGag/pol+IL-2 3200

pCGag/pol+IL-15 800

pCGag/pol+IL-18 3200

pCGag/pol+IL-4 3200

pCGag/pol+IL-5 3200

pCGag/pol+IL-10 1600

The group of injecting altogether with IL-2, IL-4, IL-5 and IL-18 has the highest terminal point level of tiring.Compare with the group of pCGag/pol immunity with single, inject the remarkable humoral immunoresponse(HI) of having strengthened altogether with IL-1 α, TNF-α, TNF-β and IL-10.In the group with the pCEnv immunity, also observed similar result.

Anti-gpl20 antibody titer

Single with pCEnv 200

pCEnv+IL-1α 800

pCEnv+TNF-α 800

pCEnv+TNF-β 400

pCEnv+IL-2 800

pCEnv+IL-15 400

pCEnv+IL-18 800

pCEnv+IL-41 600

pCEnv+IL-51 600

pCEnv+IL-101 600

Equally, the serum of the group of injecting altogether with IL-4, IL-5 and IL-10 is seen the highest terminal point level of tiring.

The generation of t helper cell

Auxiliary lymphocytic activation of T and propagation play an important role in inducing humoral immunoresponse(HI) that produces by the B cell and the immunne response that produces by CD8+ toxicity T cell.Mouse has been accepted two kinds of dna immunizations (being 50 μ g), at interval 2 weeks.In first week after the booster immunization injection, mouse is killed results spleen, isolated lymphocytes, the propagation of test t helper cell.

Preceding inflammatory cytokine is injected altogether

Carry out proliferation test with the spleen of injecting pCEnv or pCGag/pol and preceding inflammatory cytokine IL-1 α, TNF-α and TNF-β mouse altogether.Behind preceding inflammatory cytokine IL-12, TNF-α of injection and the TNF-β, estimate the proliferative response of t helper cell altogether, this experimental technique is as described below.In the pCEnv DNA first time (being 50 μ g) immunity two weeks of back, mouse (4 every group) is carried out booster immunization with identical dosage.After another week, gather the immune mouse spleen, isolated lymphocytes resists the test of reorganization gpl20 albumen (ultimate density is 5 and 1 μ g/ml).Inject two weeks of back altogether at the first time pCGag/pol and DNA (being 50 μ g), mouse (4 every group) is carried out booster immunization with identical dosage.After another week, gather the immune mouse spleen, isolated lymphocytes is carried out the test of anti-Pr55 albumen (ultimate density is 5 and 1 μ g/ml).These experiments have repeated 2 times, and the result is similar.Draw following data:

The T cells with antigenic specificity proliferative response

	Gp120 albumen

5 μ g/ml	1μg/ml
5 μ g/ml	1μg/ml	Single with pCEnv pCEnv+IL-1 α pCEnv+TNF-α pCEnv+TNF-β contrast	2.2 2.3 6.1 3.1 0.5	1.3 0.1 2.7 1.8 0.2

	Gp24 albumen 5 μ g/ml	1μg/ml
	Gp24 albumen 5 μ g/ml	1μg/ml	Single with pCGag/pol pCGag/pol+IL-1 α pCGag/pol+TNF-α pCGag/pol+TNF-β contrast	2.4 2.8 12.4 4.0 0.6	0.8 1.4 2.2 1.9 0.8

Above data presentation, the proliferation function of the level of having powerful connections in control group is in single proliferative response that medium level is arranged in the group with the pCGag/pol immunity with pCEnv or list.Even the group of having injected IL-1 α jointly with pCEnv or pCGag/pol, do not cause the increase of t helper cell proliferative response yet, but the group that pCEnv+TNF-β injects altogether causes the obvious enhancing of t helper cell proliferative response, and stimulation index is 3.1 when the gpl20 protein concentration is 5 μ g/ml.Similarly, when the Pr55 protein concentration is 5 μ g/ml, injects pCGag/pol+TNF-β altogether and produce 4.0 stimulation index.PCEnv+TNF-α and pCGag/pol+TNF-α inject altogether and observe even higher t helper cell propagation level, and stimulation index is respectively 6.1 and 12.4 (protein concn is 5 μ g/ml).

The Th1 cytokine is injected altogether

Study transmitting Th1 cytokine IL-2, IL-15 and IL-18 altogether.The stimulation index of pCEnv+IL-18 and pCGag/pol+IL-18 injection group altogether is respectively 4.4 and 10.0 (protein concentration respectively is 5 μ g/ml).To the proliferative response that mouse is injected IFN-γ inducibility Th1 cytokine IL-12 and IL-18 postevaluation t helper cell altogether, this experimental technique is as follows.In two weeks of the first time pCEnv and DNA (being 50 μ g) immunity back, with identical dosage to mouse (4 every group) booster immunization.After another week, gather the immune mouse spleen, isolated lymphocytes resists the test of reorganization gpl20 albumen (ultimate density is 5 and 1 μ g/ml).In two weeks of the first time pCGag/pol and DNA (being 50 μ g) immunity back, with identical dosage to mouse (4 every group) booster immunization.After another week, gather the immune mouse spleen, isolated lymphocytes is carried out the test of anti-Pr55 albumen (ultimate density is 5 and 1 μ g/ml).These experiments have repeated 2 times, and the result is similar.Draw following data:

	Gp120 albumen 5 μ g/ml	1μg/ml
	Gp120 albumen 5 μ g/ml	1μg/ml	Single with pCEnv pCEnv+IL-18 pCEnv+IL-12 contrast	2.2 4.4 7.8 0.5	1.3 0.8 3.8 0.2

	Gp24 albumen 5 μ g/ml	1μg/ml
	Gp24 albumen 5 μ g/ml	1μg/ml	Single with pCGag/pol pCGag/pol+IL-18 pCGag/pol+IL-12 contrast	2.4 10.0 12.0 0.6	0.8 2.1 3.8 0.8

In addition, pCEnv+IL-2 and the pCGag/pol+IL-2 stimulation index of injecting altogether is respectively 6.0 and 12.0 (protein concn is 5 μ g/ml).But, transmit IL-15 altogether and cause that comparatively demulcent t helper cell proliferative response strengthens.Inject IL-2 acceptor dependent T h1 cytokine IL-2 and IL-15 altogether to mouse, estimate the proliferative response of t helper cell then, this experimental technique is as follows.In the first time pCEnv and DNA (being 50 μ g) immunity two weeks of back, give mouse (4 every group) booster immunization with identical dosage.After another week, gather the immune mouse spleen, isolated lymphocytes resists the test of reorganization gpl20 albumen (ultimate density is 5 and 1 μ g/ml).Inject two weeks of back altogether at the first time pCGag/pol and DNA (being 50 μ g), give mouse (4 every group) booster immunization with identical dosage.After another week, gather the immune mouse spleen, isolated lymphocytes is carried out the test of anti-Pr55 albumen (ultimate density is 5 and 1 μ g/ml).These experiments have repeated 2 times, and the result is similar.Draw following data:

	Gp120 albumen 5 μ g/ml	1μg/ml
	Gp120 albumen 5 μ g/ml	1μg/ml	Single with pCEnv pCEnv+IL-2 pCEnv+IL-15 contrast	2.2 6.0 2.3 0.5	1.3 2.1 2.0 0.2

	Gp24 albumen 5 μ g/ml	1μg/ml
	Gp24 albumen 5 μ g/ml	1μg/ml	Single with pCGag/pol pCGag/pol+IL-2 pCGag/pol+IL-15 contrast	2.4 12.0 2.6 0.6	0.8 2.5 0.6 0.8

The Th2 cytokine is injected altogether

Except that preceding inflammatory cytokine and Th1 cytokine are injected altogether detect, also studied the effect that Th2 cytokine IL-4, IL-5 and IL-10 and pCEnv and pCGag/pol transmit altogether.Compare with the group of pCEnv or pCGag/pol immunity with single, two groups that inject altogether with IL-4 or IL-5 all show moderate t helper cell proliferative response and strengthen.Injection is with Th2 cytokine IL-5 and the proliferative response of IL-10 postevaluation t helper cell altogether, and this experimental technique is as follows.In two weeks of the first time pCEnv and DNA (being 50 μ g) immunity back, with identical dosage to mouse (4 every group) booster immunization.After another week, gather the immune mouse spleen, isolated lymphocytes resists the test of reorganization gp120 albumen (ultimate density is 5 and 1 μ g/ml).In two weeks of the first time pCGag/pol and DNA (being 50 μ g) immunity back, with identical dosage to mouse (4 every group) booster immunization.After another week, gather the immune mouse spleen, isolated lymphocytes is carried out the test of anti-Pr55 albumen (ultimate density is 5 and 1 μ g/ml).These experiments have repeated 2 times, and the result is similar.Draw following data:

	Gp120 albumen 5 μ g/ml	1μg/ml
	Gp120 albumen 5 μ g/ml	1μg/ml	Single with pCEnv pCEnv+IL-4 pCEnv+IL-5 pCEnv+IL-10 contrast	2.2 3.8 2.4 4.5 0.5	1.3 2.9 1.8 2.3 0.2

	Gp24 albumen 5 μ g/ml	1μg/ml
	Gp24 albumen 5 μ g/ml	1μg/ml	Single with pCGag/pol pCGag/pol+IL-4 pCGag/pol+IL-5 pCGag/pol+IL-10 contrast	2.4 5.0 3.1 8.0 0.6	0.8 3.1 2.4 2.4 0.8

It is more obvious to the reinforcement of t helper cell propagation to inject IL-10 altogether, and its stimulation index is respectively 4.5 and 8.0 (protein concn is 5 μ g/ml).

The generation of cytotoxic T lymphocyte

Carried out cytotoxic T lymphocyte (CTL) test for the reinforcement of further research immunization of cell, use be splenocyte with pCEnv and pCGag/pol mice immunized.Mouse is accepted twice dna immunization (each 50 μ g), is spaced apart for 2 weeks.In a week behind booster immunization, mouse is put to death the taking-up spleen, isolated lymphocytes, test ctl response.Carried out two kinds of tests respectively, i.e. the chromium release that specificity and non-specific bovine vaccine infection or peptide are handled target cell is tested in stimulation and not stimulatory effect lymphocyte earlier then.In order to calculate the SL rate of target cell, specificity target cell (having infected vMN462 or vVK1) dissolving per-cent is deducted non-specific target cell (having infected vSC8) dissolving percentage.

The ctl response of effector cell's stimulated in vitro

In order to estimate the cytotoxic T lymphocyte reaction, after injecting various cytokines altogether, draw following data:

The antigen-specific ctl response

	50∶1	25∶1	12.5∶1
	50∶1	25∶1	12.5∶1	PCEnv pCEnv+IL-1 α pCEnv+TNF-α pCEnv+TNF-β pCEnv+IL-2 pCEnv+IL-15 pCEnv+IL-12 pCEnv+IL-18 pCEnv+IL-4 pCEnv+IL-5 pCEnv+IL-10 contrast	10％ 14％ 30％ 20％ 22％ 46％ 35％ 22％ 4％ 13％ 13％ 3％	4％ 10％ 23％ 16％ 16％ 28％ 24％ 16％ 4％ 11％ 8％ 2.0％	3％ 6％ 18％ 13％ 4％ 10％ 19％ 13％ 7％ 9％ 3％ 3％

	50∶1	25∶1	12.5∶1
	50∶1	25∶1	12.5∶1	PCGag/pol pCGag/pol+IL-1 α pCGag/pol+TNF-α pCGag/pol+TNF-β pCGag/pol+IL-2 pCGag/pol+IL-15 pCGag/pol+IL-12 pCGag/pol+IL-18 pCGag/pol+IL-4 pCGag/pol+IL-5 pCGag/pol+IL-10 contrast	12％ 16％ 29％ 12％ 14％ 30％ 38％ 19％ 2％ 0％ 6％ 3.3％	11％ 8％ 20％ 13％ 11％ 21％ 24％ 15％ 2％ 3％ 6％ 2.8％	7％ 1％ 7％ 3％ 10％ 7％ 15％ 4％ 2％ 2％ 0％ 4.5％

Inflammatory cytokine IL-1 α, TNF-α and the reaction of TNF-β postevaluation cytotoxic T lymphocyte before the injection altogether, in this experiment, in the first time DNA and pCGag/pol (being 50 μ g) injection two weeks of back altogether, with identical dosage to mouse (4 every group) booster immunization.After another week, gather the immune mouse spleen, isolated lymphocytes is tested ctl response with the target cell that specificity bovine vaccine (vVK1) and non-specific bovine vaccine (vSC8) are infected.In the first time pCEnv and DNA (being 50 μ g) injection two weeks of back altogether, with identical dosage to mouse (4 every group) booster immunization.After another week, gather the immune mouse spleen, isolated lymphocytes is tested ctl response with the target cell that specificity bovine vaccine (vNM462) and non-specific bovine vaccine (vSC8) are infected.These experiments have repeated 2 times, and the result is similar.

Inject the reaction of IFN-γ inductive Th1 cytokine IL-12 and IL-18 postevaluation cytotoxic T lymphocyte altogether, in this experiment, in the first time DNA and pCGag/pol (being 50 μ g) injection two weeks of back altogether, with identical dosage to mouse (4 every group) booster immunization.After another week, gather the immune mouse spleen, isolated lymphocytes is tested ctl response with the target cell that specificity bovine vaccine (vVK1) and non-specific bovine vaccine (vSC8) are infected.In the first time pCEnv and DNA (being 50 μ g) injection two weeks of back altogether, with identical dosage to mouse (4 every group) booster immunization.After another week, gather the immune mouse spleen, isolated lymphocytes is tested ctl response with the target cell that specificity bovine vaccine (vNM462) and non-specific bovine vaccine (vSC8) are infected.These experiments have repeated 2 times, and the result is similar.

Inject the reaction of IL-2 acceptor dependent T h1 cytokine IL-2 and IL-15 postevaluation cytotoxic T lymphocyte altogether, in this experiment, in the first time DNA and pCGag/pol (being 50 μ g) injection two weeks of back altogether, with identical dosage to mouse (4 every group) booster immunization.After another week, gather the immune mouse spleen, isolated lymphocytes is tested ctl response with the target cell that specificity bovine vaccine (vVK1) and non-specific bovine vaccine (vSC8) are infected.In the first time pCEnv and DNA (being 50 μ g) injection two weeks of back altogether, with identical dosage to mouse (4 every group) booster immunization.After another week, gather the immune mouse spleen, isolate lymphocyte, test ctl response with the target cell that specificity bovine vaccine (vNM462) and non-specific bovine vaccine (vSC8) are infected.These experiments have repeated 2 times, and the result is similar.

Altogether injection is with Th2 cytokine IL-5 and the reaction of IL-10 postevaluation cytotoxic T lymphocyte, in this experiment, in the first time DNA and pCGag/pol (being 50 μ g) two weeks after the injection altogether, with identical dosage to mouse (4 every group) booster immunization.After another week, gather the immune mouse spleen, isolated lymphocytes is tested ctl response with the target cell that specificity bovine vaccine (vVK1) and non-specific bovine vaccine (vSC8) are infected.In the first time pCEnv and DNA (being 50 μ g) injection two weeks of back altogether, with identical dosage to mouse (4 every group) booster immunization.After another week, gather the immune mouse spleen, isolated lymphocytes is tested ctl response with the target cell that specificity bovine vaccine (vNM462) and non-specific bovine vaccine (vSC8) are infected.These experiments have repeated 2 times, and the result is similar.Inflammatory cytokine before injecting altogether

PCEnv or pCGag/pol and preceding inflammatory cytokine IL-1 α, TNF-α and TNF-β are injected mouse jointly, obtain the data of CTL test-results, from control animal observe specific killing to background level, single animal with the pCEnv immunity shows low-level ctl response.Inject altogether with pCEnv+IL-1 α or pCEnv+TNF-β, the CTL activity is had moderate raising.On the other hand, after injecting altogether with pCEnv+TNF-α, the specific killing that can be observed expressing the target cell that HIV coating bovine vaccine (vNM462) infects has more obvious reinforcement.When imitate target than (E: when T) being 50: 1, inject pCEnv+TNF-α altogether after, the specificity target cell dissolution rate surpasses 30%.Similarly, after mouse is used the pCGag/pol+TNF-alpha immunization, (cleavage rate when E: T is 50: 1 is 29%) significantly strengthened in the antigen-specific CTL dissolving of expressing the target cell that HIV-1gag/pol bovine vaccine (vVK1) infects, and injected the slight increase that only causes ctl response altogether with pCGag/pol+IL-1 α or pCGag/pol+TNF-β.

The Th1 cytokine is injected altogether

Studied the common effect of transmitting of Th1 cytokine and dna vaccination.

Obtain single with pCEnv immunity and the CTL test-results of having injected Th1 cytokine IL-12 and IL-18 mouse altogether.Give the IL-12 difference together, inject IL-18 altogether the enhancing of ctl response is more relaxed.Give the moderate reinforcement that IL-2 also causes ctl response altogether.On the other hand, after the common immunity of pCEnv+IL-15, observed the more significant reinforcement of ctl response, the SL rate is 46%.Similar, also significantly strengthen (30%) through the mouse antigen specific CTL dissolving that pCGag/pol++IL-15 injects altogether.

The Th2 cytokine is injected altogether

Outside the effect that inflammatory cytokine and Th1 cytokine are transmitted altogether before research, also studied common injection Th2 cytokine IL-4, IL-5 and IL-10 effect to the ctl response level.Though strengthened the t helper cell proliferative response with the common injection of these cytokines, IL-4, IL-5 or IL-10 and pCEnv or pCGag/pol injected any specificity booster action that does not cause ctl response altogether.

Measure the I class MHC restriction in the ctl response

Whether strengthen because of the restricted stimulation of CD8+I class MHC in order to determine to inject altogether the ctl response that TNF-α and IL-15 cause, carry out the CTL test with HIV-1 env peptide (RIHIGPGRAFYTTNK), this peptide has been proved to be a unique epi-position of balb/c mouse I class MHC Restricted CTL.It respectively is the DNA construction immunity of 50 μ g that mouse is accepted twice, 2 weeks of interval, and in 1 week of back of immunity for the second time, the results spleen.With carrying out the CTL test behind the coating specific peptide stimulated in vitro splenocyte.After injecting altogether with IL-15 and TNF-α, see ctl response and all significantly strengthen, E: T is that 50: 1 o'clock specific killing is respectively 25% and 32% (11A-11E).After from effector cell group, removing the CD8+T cell, measure the CTL activity and confirmed this observation by the complement dissolving.With each plasmid immune mouse twice of 50 μ g, pitch time is the same.Carry out CTL test, one group of Treatment Effects cell as previously mentioned, another group is removed the CD8+T cell.Shown in Figure 11 F-11O, remove the reinforcement that the CD8+T cell has suppressed antigen-specific CTL behind common injection IL-15 and the TNF-α.Above result shows that the reinforcement of cytotoxic activity is an antigen-specific, and I class MHC is restrictive and the CD8+T cell is dependent.

Direct ctl response (not stimulated in vitro effector cell)

Study injecting the direct ctl response level of IL-15 and TNF-α inductive altogether, observed constant high level ctl response (having carried out stimulated in vitro) because organize altogether from these two.Carried out chromium-release test on the same day of separating Morr. cell.Inject the being seen direct CTL difference of IL-12 together, do not observe after pCEnv and TNF-α or IL-15 inject altogether and induce direct CTL activity (Figure 12).

Discuss

The general objective of any immunization strategy all is to induce strong and persistent cause of disease specific immune response with minimum immunization.But the relation between a kind of cause of disease and the provide protection may change with the variation of the cause of disease, and, can reach the improvement of final result by the guiding immunne response.For example, think that high-caliber specific antibody replys avoiding hepatitis b virus infected provide protection very importantly, the provide protection of avoiding lymphocytic choriomeningitis virus to infect is then mainly mediated by the T cell-mediated response.The design that direction that can be by regulating institute's induce immune response and intensity are improved the dna vaccination strategy is so that the relation between provide protection and the target cause of disease is more suitable.

As a kind of new methods of vaccination, nucleic acid immunization is proved to be in several animal models, in vivo inducing antigen-specific humoral immunization and cellullar immunologic response.The direction of control immunne response and the strategy of intensity can be produced more effective clinically vaccine.Better control can be by realizing with common transmission of cytokine and the gene that is total to molecule significant on pungency molecule and so on the immunology by the particular type and the direction of the immunne response of vaccine or immunotherapy generation.

As the releaser and the attemperator of immunological network, cytokine plays an important role in immune response and Inflammatory response.In immune unique function, these cytokines can be further divided into preceding inflammatory cytokine, Th1 cytokine and Th2 cytokine according to them.Preceding inflammatory cytokine IL-1, TNF-α and TNF-β play an important role to the starting person of damage and infection reaction as the host.IL-1 produces IL-2 by inducing T cell and raises IL-2R indirect activation T cell.IL-1 also synthesizes by differentiation, growth and the IgG that induces the B cell influences the B cell.At least the IL-1 that has two kinds of forms is called IL-1 α and IL-1 β, and they show similar activity.TNF-α and TNF-β are closely-related albumen (the amino-acid residue homology are about 30%), and they are in conjunction with identical cell surface receptor.TNF-α is produced by activatory scavenger cell, monocyte and neutrophil(e) cell, activated lymphocytes and NK cell, and TNF-β is then produced by lymphocyte.TNF-α and TNF-β also involve gram-negative bacteria and infect septic shock and the rheumatoid arthritis that causes.And TNF-α is considered to play keying action in regulating other preceding inflammatory cytokine.

The Th1 cytokine is regulated cell or T cell-mediated immune responses.IFN-γ, to be prototype Th1 cytokine produced by Th1, CD8+ and NK cell, and be proved to be and have antivirus action and immunoregulation effect, for example raises I class and II class MHC antigen.Have been found that a kind of new cytokine, the IFN-γ inducibility factor (IGIF) or IL-8 suppress to increase when IL-10 produces the generation of IFN-γ in irriate PBMC.In human peripheral blood single nucleus cell (PBMC), IL-18 also strengthens NK cell (NK) cell activity, and irrelevant cytokine IL-12 is similar on this point and its structure.Mainly activated T cells produces IL-2 by outside stimulus; It expands most important for the clone of the propagation of T cells with antigenic specificity.IL-2 plays a crucial role in the activation of T cell by the reacting to each other of a receptor system with 3 chains (α, β and γ c) formation.IL-15, a kind of IL-12 homologue of new evaluation is a kind of multiple-effect cytokine, has the similar T cell-stimulating activity with IL-2.

The Th2 cytokine is regulated body fluid or antibody-mediated immunne response.IL-5 is a kind of dimer cytokine, and control B cytodifferentiation becomes the plasmocyte that produces antibody.Verified, IL-5 induces mouse or human B cell to produce the IgA of antigen-specific.In addition, IL-5 also promotes growth and the propagation of eosinophil.Produced by the Th2T cell colony though show IL-10 at first, it is also produced by B cell and monocyte.As prototype Th2 cytokine, IL-10 has been proved to be the type cytokines that the monocyte that can suppress mitogen-activated produces IL-1 α, IL-16, IL-18 and TNF-α, and suppresses the macrophage activation effect of IFN-γ.It is reported that IL-10 may work in HIV-1 infects; Compare with the PBMC of non-infected individuals, the IL-10mRNA level raises among the PBMC of asymptomatic HIV positive individuals, and the IL-10 level improves.In addition, prove that IL-10 has reduced virus replication in the vitro human scavenger cell.

Develop preceding inflammatory, Th1 and Th2 cytokine expression box, analyzed they play instrumentality effect in the body in dna vaccination inductive immunne response ability.Cytokine gene is delivered in the mouse body with the former construction intramuscularly of dna immunization, analyzes them the direction of institute's inductive immunne response and the effect of intensity.Inject altogether with IL-2, IL-4, IL-5, IL-10 and IL-18, observe the remarkable reinforcement of antibody response.Inject altogether with TNF-α, TNF-β, IL-12, IL-10 and IL-18, cause the remarkable reinforcement of t helper cell proliferative response, then cause than the proliferative response of demulcent t helper cell with the common injection of IL-5 and IL-15 and strengthen.And, in all common injection combinations, have only TNF-α and IL-15 to inject altogether to cause to be similar to the CTL level reinforcement (the SL rate is higher than 30%) that IL-12 injects altogether.With only compare with the animal groups of the former immunity of dna immunization, inject altogether with TNF-β, IL-12 and IL-18 and cause than the demulcent ctl response and strengthen.As injecting finding altogether, inject being seen ctl response altogether with TNF-α and IL-15 and strengthen the restriction that is subjected to I class MHC and CD8+T cell with IL-12 or CD86.

IL-18 it is said to have the activity similar to IL-12.For example, IL-18 has strengthened natural killer (NK) cell activity in human peripheral blood single nucleus cell (PBMC) cultivation, and this point is similar to incoherent cytokine IL-12 on its structure.The PBMC that IL-18 also strengthens concanavalin A (Con A) to be stimulated produces IFN-γ, suppresses the generation of IL-10 simultaneously.Also observe, IL-18 induces IFN-γ by IL-12 dependent/non-dependent approach.Though transmit the remarkable T cell-mediated response level moderate reduction humoral response simultaneously of having strengthened altogether with IL-12, with IL-18 give but not see similar effect altogether.On the contrary, IL-18 significantly improves antibody titer altogether.In addition, to transmit remarkable reinforcement CTL altogether different with IL-12, and IL-18 injects the CTL that does not induce similar level altogether and strengthens.As if the immunomodulatory properties of IL-18 similar to IL-10.

In the body that IL-12 and IL-18 give altogether effect variant, inject altogether with IL-2 and to inject the direction and the intensity that also cause immunne response altogether different with IL-15.IL-2 and IL-15 once reported to have similar biological activity, comprised having identical IL-2 receptor y chain and identical T cytositimulation signaling mechanism.Cause significantly strengthening antibody response and t helper cell proliferative response altogether with IL-2, IL-15 injects altogether and then causes significantly strengthening ctl response.Perhaps, this species diversity can be explained with the pleiotropy of IL-15.For example, IL-15 once was reported in the generation of significantly inducing TNF-α by activating synovia T cell in the rheumatoid arthritis.On the other hand, IL-2 inductive TNF-alpha levels is much lower.With the Notes of Key Data in the upper body, these two kinds of molecules participate in activation signal mechanism by different way.

The Th2 cytokine can be used for improving the Th2 immunne response, and does not influence the level of T cell-mediated response.IL-4, IL-5 and IL-10 it is said it is strong Th2 cytokine.IL-4, IL-5 and IL-10 transmit altogether and cause significantly improving of antibody response level and t helper cell proliferative response level.On the other hand, the level of ctl response does not improve.Above result shows, utilizes IL-4, IL-5 and IL-10 to give artificially to transform the Th2 type altogether and reply.

The multifunction immunity modulator effect that important discovery is TNF-α and IL-15.

Studied the upward significant cytokine of panimmunity and DNA has been inoculated the effect of inducing host immune response.Generalized as Figure 13 institute, by giving cytokine gene altogether, can the people be the immunne response of inducing unique types.This cytokine gene adjuvant system is emphasized to regulate and control inducing of specific immune response on new level, so that the provide protection that immunologic process produces has relation preferably with the disease difference.This accuracy controlling to vaccine and immunotherapy is unprecedented.As a result, the direction and the degree of control immunne response will help various immunization strategies.For example, based on the T cell-mediated response, and humoral response does not need in addition deleterious situation under, can select for use the IL-12 gene to transmit altogether as immunomodulator and specific DNA immunogen.On the contrary, in order to build the vaccine of target, can inject altogether with for example IL-4, IL-5 or IL-10 gene to the outer bacterium of born of the same parents.And, if CD4+T helper and antibody all play an important role, can transmit GM-CSF and IL-2 altogether in provide protection.At last,, can inject TNF-α altogether, comprehensively add powerful antibody, t helper cell and CTL and reply if this immunne response of three types all is critical.

Embodiment 6

By PCR reaction, utilize suitable Restriction Enzyme, with the insertion fragment BL1 clone of Figure 14 with to be connected to the PCR3 carrier for expression of eukaryon be in the carrier pBBKan (as Figure 15).The BL1 construction is given jointly with different HIV-1 antigen, measure it at the intravital immunoregulation effect of mouse.Result among Figure 16 and 17A, B, C and the D shows that the dna fragmentation in the BL1 has been strengthened immunne response when being total to immunity with HIV-1 antigen.Viewed is to reply different-effect aspect the reinforcement at spleen enlargement and antibody and CTL.

Table 1

Picornaviridae

Rhinovirus: (medical) causes 50% common cold.

Etheroviruses:(is medical) comprise poliovirus, Coxsackie virus, dust can (ECHO) virus, and human enteric virus is hepatitis A virus (HAV) for example.

Apthoviruses:(animal doctor with) these are sufficient oral cavity virus.

Target antigen: VP1, VP2, VP3, VP4, VPG

Calcivirus section

The Norwalk papova belongs to: (medical) these viruses are important virulence factors of epidemic gastroenteritis.

Togaviridae

Alphavirus: (medical and animal doctor with) example comprises Senilis virus, RossRiver virus and east and western equine encephalitis virus.

Reovirus belongs to: (medical) rubella virus.

Flaviviridae

For example: (medical) dengue fever virus, yellow fever virus, japanese encephalitis virus, St. Louis meningitis virus and lice pass encephalitis.

Hepatitis C virus: (medical) these viruses do not belong to any section, but are considered to or togavirus or flavivirus.Major part is similar to Togaviridae.

Coronaviridae

(medical and animal doctor uses)

Infectious bronchitis virus (fowl)

Pig can be transmitted marcy agent (pig)

Pig blood clotting encephalomyelitis virus (pig)

Feline infectious peritonitis virus (cat)

Cat small intestine coronavirus (cat)

Dog coronavirus (dog)

The human respiratory coronavirus causes about 40% common cold.EX.224E，0C43。Attention: coronavirus may cause the hepatitis of non-first, non-second and non-third liver.

Target antigen:

E1-claims M albumen or stromatin again

E2-claims S albumen or spike protein again

E3-claims HE or hemagglutination elterose glycoprotein (not being present in all coronavirus) again

The N-nucleocapsid

Rhabdoviridae

Vesiliovirus belongs to

Rabies virus: (medical and animal doctor uses) rabies

Target antigen: G albumen, N albumen

Filovirus section: (medical)

Hemorrhagic fever virus, for example Marburg virus and Ebola virus

Paramyxoviridae:

Paramyxovirus genus: (medical and animal doctor uses) mumps virus, new city eqpidemic disease virus (important chicken is caused a disease former)

Morbillivirus: (medical and animal doctor uses) Measles virus, dog pest heat

Pneumovirinae: (medical or animal doctor uses) respiratory syncytial virus

Orthomyxovirus section (medical)

Influenza virus

Anilius Viraceae (Bungavirus)

Krait Tobamovirus: (medical) galifornia encephalitis, LA Crosse

Phlebotomus fever virus belongs to: (medical) Rift valley heat

Hantavirus: Puremala is a kind of hemahagin fever virus

Nairovirus: (animal doctor uses) Nairobi sheep disease

Also have many unclassified Anilius Tobamovirus

Arenaviridae (medical)

LCM, Lassa fever virus

Reoviridae

Reovirus belongs to: may be a kind of people's cause of disease

Rotavirus: children acute gi tract inflammation

Orbivirus: (medical and animal doctor uses) Colorado lice heat transfer virus, Lebombo (people), equine encephalitis virus, blue tongue rims

Retroviridae

Oncovirus subfamily: (animal doctor uses and be medical) feline leukaemia virus, HTLVI and HTLVII

Lentiviridae: (medical and animal doctor uses) HIV, feline immunodeficiency virus, horse-sickness virus, anemia virus

Spumavirinae

Papovaviridae

Polyomavirus subfamily: (medical) BKU and JCU virus

Papilloma virus subfamily: the viral adenovirus of many kinds (medical) that (medical) is relevant with cancer or papillomatous malignant development

EX AD7, ARD., O.B.-causes respiratory tract disease

The part adenovirus, for example 275, cause enteritis

Parvoviridae (animal doctor uses)

Feline panleucopenia virus: cause feline enteritis

Feline infectious enteritis virus

Canine parvovius

Pig parvoviral

Herpetoviridae

Alphaherpesvirinae

Simplexvirus: (medical) HSVI, HSVII

Varicellavirus: (medical and animal doctor uses) pseudorabies-varicella zoster virus

Betaherperesvirinae

Cytomegalovirus belongs to: (medical) HCMV, Muromegalovirus

Gammaherpesvirinae

Lymphocryptovirus belongs to: (medical) EBV-(Burkitts lymphocyte), Rhadinovirus

Poxviridae

Banded poxvirus subfamily (medical-animal doctor uses)

Variola virus belongs to

Cowpox belongs to

Parapoxvirus belongs to-(animal doctor uses)

Capripoxvirus-animal doctor uses

Rabbitpox virus belongs to

Suipoxvirus

The Entemopoxviridue subfamily

Hepadnaviridae

Hepatitis B virus

Non-classified

Hepatitis

Table 2

Bacteria pathogeny

Coccus comprises the gram-positive cause of disease: streptococcus pneumoniae; Staphylococcus; And suis.

Coccus comprises the Grain-negative cause of disease: meningococcus and gonococcus.

Intestinal bacilli comprises the pathogenicity bo Grain-negative: enterobacteria; Pseudomonas, acinetobacter calcoaceticus and eikenilla; Pseudoglanders; Salmonella; Shigella; Influenzae; Venereal ulcer; Brucellosis; Soil draws bacterium heat; Pasteurella; Streptobacillus; Candidiasis and spirillum; Listeria monocytogenes; The single aeontium bacterium of pig erythema; Diphtheria corynebacterium; Cholera bacilli; Anthrax bacillus; The Du Nuofan bacterium; And bartonia bodies.

Anerobe comprises pathogenicity bo: tetanus bacillus; Botullnus and other carboxylic bacterium; Tubercule bacillus; Leprosy bacillus and other mycobacterium.The pathogenicity bo spirochetosis comprises: syphilis; Treponematosis; Yaw; Its sick and endemicity syphilis of product; And leptospirosis.Other infection that is caused by more senior pathogenic bacteria and pathogenic epiphyte comprises: actinomycosis; Nocardiosis; Torulosis, blastomycosis, histoplasmosis and coccidioidomycosis; Moniliosis, aspergillosis, and mucormycosis; Sporotrichosis, Brazilian blastomycosis, petriellidosis, Torulopsis, mycetoma and chromoblastomycosis; And tinea.

Rickettsial infection comprises rickettsial and rickettsiosis.

The case of mycoplasma and choamydiae infection comprises: mycoplasma pneumonia; Lymphogranuloma venereum; Psittacosis; With the choamydiae infection in term.

The eukaryotic cell cause of disease

The infection that is caused by pathogenic protozoon and worm comprises: loeschiasis; Malaria; Leishmaniasis; Trypanosomiasis; Toxoplasmosis; The sub-parasitosis of alveolar; Knuckle; Babesiosis; Giardiasis; Trichonematosis; Filariasis malayi; Schistosomicide; Nematode; Fluke; And cestode infection.

Claims

1. plasmid comprises:

A) nucleotide sequence of coding immune modulator, be selected from IL-12, GM-CSF, IL-1, TNF-α, TNF-β, IL-2, IL-4, IL-5, IL-10, IL-15, IL-18 and the BL-1 that forms by sequence shown in Figure 14, this sequence is connected with the controlling element operability; With

B) the immunogenic nucleotide sequence of encoding;

Condition is that when described immune modulator was IL-12 or GM-CSF, what be connected with effective controlling element operability in the mammalian cell was the nucleotide sequence of coding IL-12 or GM-CSF only, and only comprises single immunogenic nucleotide sequence.

2. plasmid according to claim 1, wherein said immunogen are the target proteins that is connected with the controlling element operability, and described target protein is pathogenicity bo antigen, cancer associated antigen or the antigen that is connected with the autoimmune disease relevant cell.

3. plasmid according to claim 1, wherein said immunogen are HIV-1 antigen.

4. plasmid according to claim 1, wherein said immune modulator are strand IL-12.

5. plasmid according to claim 1, it comprises the coding panimmunity and regulates proteinic a plurality of nucleotide sequence, described immune modulator is selected from IL-12, GM-CSF, IL-1, TNF-α, TNF-β, IL-2, IL-4, IL-5, IL-10, IL-15, IL-18 and the BL-1 that is made up of sequence shown in Figure 14, the nucleotide sequence of each coding immune modulator all is connected with the controlling element operability.

6. pharmaceutical composition, it comprises the described plasmid of claim 1.

7. the purposes of the described plasmid of claim 1 in the immunological reagent of preparation opposing cause of disease.

8. composition, it comprises multiple plasmid, comprising:

First plasmid, it comprises the nucleotide sequence of the coding immune modulator that is connected with the controlling element operability, described immune modulator is selected from IL-12, GM-CSF, IL-1, TNF-α, TNF-β, IL-2, IL-4, IL-5, IL-10, IL-15, IL-18 and the BL-1 that forms by sequence shown in Figure 14 and

Second plasmid, it comprises the immunogenic nucleotide sequence of coding;

Condition is, when described immune modulator is IL-12 or GM-CSF, what be connected with effective controlling element operability in the mammalian cell in first plasmid is the nucleotide sequence of coding IL-12 or GM-CSF only, and second plasmid only comprises single immunogenic nucleotide sequence.

9. composition according to claim 8, the antigen that the described second plasmid-encoded immunogen is selected from pathogenicity bo antigen, cancer associated antigen or is connected with the autoimmune disease relevant cell.

10. composition according to claim 8, wherein said immunogen are HIV-1 antigen.

11. composition according to claim 8, wherein said immune modulator matter is strand IL-12.

12. composition according to claim 8, wherein said first plasmid comprises a plurality of nucleotide sequences of multiple certain immune modulator of encoding, described immune modulator is selected from IL-12, GM-CSF, IL-1, TNF-α, TNF-β, IL-2, IL-4, IL-5, IL-10, IL-15, IL-18 and the BL-1 that is made up of sequence shown in Figure 14, the nucleotide sequence of each coding immune modulator all is connected with the controlling element operability.

13. composition according to claim 8, it also comprises the 3rd plasmid, described the 3rd plasmid comprises the nucleotide sequence of the coding immune modulator that is connected with the controlling element operability, described immune modulator is selected from IL-12, GM-CSF, IL-1, TNF-α, TNF-β, IL-2, IL-4, IL-5, IL-10, IL-15, IL-18 and the BL-1 that is made up of sequence shown in Figure 14.

14. the purposes of the described composition of claim 8 in the immunological reagent of preparation opposing cause of disease.

15. recombiant vaccine, it comprises the nucleotide sequence of the coding immune modulator that is connected with the controlling element operability, these immune modulators are selected from IL-12, GM-CSF, IL-1, TNF-α, TNF-β, IL-2, IL-4, IL-5, IL-10, IL-15, IL-18 and the BL-1 that is made up of sequence shown in Figure 14, this recombiant vaccine also comprise the immunogenic nucleotide sequence of coding.

16. the antigen that recombiant vaccine according to claim 15, wherein said immunogen are selected from pathogenicity bo antigen, cancer associated antigen or are connected with the autoimmune disease relevant cell.

17. recombiant vaccine according to claim 15, wherein said vaccine are the varicella vaccines of reorganization.

18. recombiant vaccine according to claim 15, wherein said immune modulator are strand IL-12.

19. recombiant vaccine according to claim 15, wherein said immunogen are pathogenicity bo antigen.

20. recombiant vaccine according to claim 15, it also comprises the coding panimmunity and regulates proteic a plurality of nucleotide sequence, described immune modulator is selected from IL-12, GM-CSF, IL-1, TNF-α, TNF-β, IL-2, IL-4, IL-5, IL-10, IL-15, IL-18 and the BL-1 that is made up of sequence shown in Figure 14, the nucleotide sequence of each coding immune modulator all is connected with the controlling element operability.

21. the purposes of the described recombiant vaccine of claim 15 in the immunological reagent of preparation opposing cause of disease.

22. recombiant vaccine according to claim 15, described recombiant vaccine are a kind of attenuated live cause of diseases.

23. a plasmid comprises:

A) nucleotide sequence of the p35 subunit of coding IL-12, this sequence is connected with effective controlling element operability in the mammalian cell;

B) in the nucleotide sequence of p40 subunit of coding IL-12, this sequence and mammalian cell effectively another controlling element operability be connected with

C) the immunogenic nucleotide sequence of encoding.