CN1067382A - The expression of human papilloma virus's peptide and the application in causing immune composition - Google Patents

Abstract

Comprise and express the papilloma zone or the papilloma protein of having encoded and having brought out, as the peptide of the HPVDNA insert in E6 or E7 zone, antibody and recombinant expression system or cell.Contain these peptides, the compositions of antibody and/or reconstitution cell used as cause immunizing composition and be used in suppress and treatment HPV infects and tumor takes place and the method for making progress in.The specific peptide and the reconstitution cell in the antigen decision zone of expressing HPV16E6 or E7 nucleoprotein have been specifically described, as vaccinia virus and tumor cell.

Description

The expression of human papilloma virus's peptide and the application in causing immune composition

The method that the present invention relates to reconstitution cell, peptide, antibody, compositions and can be used for suppressing and treat human papilloma virus (HPV) infection and cell transformation.

Comprise and express a kind of DNA insert of the albumen zone of HPV of having encoded or a kind of reconstitution cell of the peptide that is brought out by the HPV gene in the mammalian cell is produced out, for example as expressing HPV E ₆Or E ₇An antigen of nucleoprotein gene product determines the recombined vaccinia virus in zone, or comprises and express the E of HPV ₆Or E ₇The fibroblast that rebuilds, epithelial cell, lymph or the tumor cell transfection in a zone of nucleoprotein gene product or recombinant virus infection.With HPV ₁₆E ₆And E ₇The corresponding special peptide in proteic antigen decision zone is produced out, and these peptides have been used to cause in immune composition and the vaccine.And described and suppressed and the intravital HPV infection of the patient of disappearing and the treatment of tumor development and the method for prevention.

To laboratory animal, the particularly rodentine tumors that studies show that great majority are caused by oncogenic virus have been expressed the antigen by the viral gene group coding, and adopt these antigenic immunizations can cause subsequently the rejection that is excited by identical viral-induced tumor cell.Although the major part of these researchs utilizes the zoo virus strain to carry out, for example SV ₄₀, polyoma virus, and Friend, Moloney, or Rauscher murine leukemia virus, but in fact the tumor level and the vertical infection that bring out virus is proved: now can obtain resisting the viral-induced cat leukemia and the commercial vaccine of sarcoma really.

By contrast, the virus causing disease theory of most of human cancers is not proved as yet, and possible exception is hepatitis virus (hepatoma), Epstein Barr virus (nasopharyngeal carcinoma), and human papilloma virus (HPV16) (cervical cancer).Yet, in the past twenty years, verified, some human tumor cell's expressing tumor antigen, the i.e. antigen that tumor cell and its normal cell can be differentiated, that some patient produces cell-conduction for these antigens or humoral immunoresponse(HI) (Hellstrom etc., nineteen sixty-eight, Nature, 220:1352; Morton etc., nineteen sixty-eight, Science, 162:1279-1281; Shiku etc., 1976, J.Exp.Med.144:873-881).These immunoreactive some targets are by the fetus tumor antigen of human gene's group coding or differentiation antigen (Hellstrom etc., 1970, Int.J.Cancer 6:346-351).

Up to this point, the molecular characterization of tumor antigen is still unknown, and the degrees of specificity of this immunological response of tumor is also unclear.And the practice of attempting these information is used in the diagnostic assay of developing cancer or treatment for cancer aspect is also very unsuccessful.Because the spontaneous regression of tumor is almost rare, therefore, people can reach a conclusion, and promptly confirmed immunne response is not effective in vivo in teat glass.For example, can be used to kill tumor cell in the teat glass effectively from the intravital antibody of cancer patient and lymphocyte, and same cancer patient's immunne response is not enough to stop the development of tumor in vivo.

By monoclonal antibody technique that Kohler and Milstein developed (1975, Nature, 256:495-497) guided deep research for human tumor antigen, because not only some antigens provide means (Hellstrom and Brown, 1979, in The Antigens to this technology in order to define like this on molecular level but also aspect specificity, M.Sala, ed., Academic Press, Vol.V:1-66).

In the past few years, a large amount of antigen relevant with tumor has been described, wherein great majority adopted mouse monoclonal antibody carried out defining (Reisfeld and Sell eds. compile and seize, 1985, in Monoclonal Antibodies and Cancer Tllerapy, UCLA Symposia on Molecular and Cellular Biology, New Serres, Vol.27, Alan R.Liss, Inc.New York, pp.1-609).Though in fact all antigens of clearly describing all have been proved to be fetus tumor or differentiation antigen, and they have been found to be quantitative rather than qualitatively for the specificity of tumor, there are several antigens to have enough specificitys (corresponding one 10 to 1000 times the factor usually) for tumor cell and normal cell, so that can be as the potential target of differentiating tumor cell and treatment.Human monoclonal antibody at tumor antigen also obtain (Cote etc., nineteen eighty-three, Proc.Natl.Acad.Sci.80:2026-2030).This has just supported the evidence that quote the front, and promptly some cancer patient has strengthened the immunne response to its tumor.Many these tumor antigens are by the human chromosomal coding, and in some cases, these tumor antigens are results of interior originality or exogenesis viral infection.

The human papilloma virus is well-known epithelial tumor such as wart and the papillomatous infectious agent of producing in its host.Common hands wart and sufficient plantar wart are human modal skin lesions; Yet the squamous cell carcinoma in masculinity and femininity is also common relevant with some HPV infection strain with anogenital cancer.

The papilloma virus gene group comprises a bifilar ring-type super coiled DNA molecule with about 5,000 kilodaltons (KDa) molecular weight.This genome is encoded between 8 and 10 protein, and this number is also uncertain, because a kind of function or protein are not given genomic each open reading framework (ORF ' s) as yet.Be designated as E at first at the ORF ' s that duplicates early stage wart formation, and those ORF ' s that form afterwards were designated as L.But this appointment is no longer valid, and has been found that some E gene outcome is formed at the early stage and late period of infection.

HPV infection and cervical cancer and other human non-anogenital cancer have much relations (ZurHausen etc., 1989, Cancer Res.49:4677-4681; Galloway etc., 1989, Adv.Virus Res.37:125-171).A kind of HPV type, HPV ₁₆, usually with serious hypoplasia relevant (Galloway etc., Supra with cervical cancer; Ikenberg etc., 1983, Int.J.Cancer 32:563-565), in this disease, some viral E gene and protein thereof have demonstrated outstanding effect.Adopt a kind of communication gene, as chloramphenicol acetyltransferase, the trial zone that is carried out shows, E ₆Gene transfer has activated the non-coding section (Phelps etc., 1988.Cell 53:539-547) of HPV DNA.E ₇Nucleoprotein has demonstrated in mammalian cell and to have transformed and keep pernicious phenotypic effect (Tsunokawa etc.,, Proc.Natl.Acad.Sci.USA83:2200-2203 in 1986; Kanada etc., 1988, J.Virol.62:610-613; Crook etc., 1989, EMBOJ.8:513-519).

For laboratory animal, the research that is generally mice shows, adopts the immunity inoculation of cancer cell that live or dead can cause the rejection that is excited by the cancer cell that can survive subsequently.In many cases, be responsible for the target antigen of protective effect and pressed encoding viral, but under its many situations, excite a kind of antigenic characteristic of protective immune response to be still the unknown.

Suggestion on human body, use of cancer vaccine main, theoretic opposing views are, the cancerous cell that " inoculation " for example is killed or not have the people of the preparation of cell may be unresponsive on the immunology.It is believed that this is recurrent, can be used as immunne response target tumor antigen because in some normal cell, exist, though only be micro-, and be considered as " self " by immune system.At this " self " antigenic immunity inoculation,, may cause a kind of autoimmune response if effectively talk about.The antigen relevant with tumor that most (if not all) detected in human tumor by monoclonal antibody also is present in some normal structure, and does not almost have evidence proof cancer patient in vivo these antigens to be had effective response.On the other hand, be external a kind of antigen for the human immunity system, for example a kind of by oncogenic virus, as HPV ₁₆The antigen of coding should bring out a kind of strong immunne response probably.Be included in a kind of appropriate carriers molecular cloning and express the hereditary information of the virus protein of having encoded for prophylaxis of viral infections and cell transformation utilize recombinant DNA technology to produce vaccine, said virus protein can excite immunne response at this protein in host animal.A kind of new preparation for this vaccine may be that useful method is described (Mackett etc., nineteen eighty-two, Proc.Natl.Acad.Sci.79:7415-7419; Mackett etc., 1984, J.Virol.49:857-864; Panicali etc., nineteen eighty-two Proc.Natl.Acad.Sci.79:4927-4931).This method comprises expresses the alien gene that inserts in its genome with vaccinia virus as carrier.Because after being incorporated in the host animal, the vaccinia virus of this reorganization is expressed this alien gene that is inserted into, and therefore excite host immune response to this gene outcome.Because the recombined vaccinia virus of this work can be used as a kind of vaccine, so this method combines the two advantage of subunit and live vaccine.

Expression has been found in the laboratory animal from the antigenic recombined vaccinia virus of adventitious viruses to be brought out, the resistance that adventitious viruses is stimulated.Example comprises expresses a kind of HSV glycoprotein (Cremer etc., 1985, Science 228:1985), a kind of rabies virus surface antigen (Blancou etc., 1986, Nature 322:373) recombinant vaccine virus, non-expression HPV16 expresses bovine papilloma virus albumen (Lathe etc. exactly, 1989, in Vaccines for Sexually Transmitted Disease, A.Meheus and R.E.Spiel, eds, Butterworth Co.pp.166-177) and a kind of recombinant vaccine virus of influenza virus nucleoprotein (Smith etc., 1983, Proc.Natl.Acad.Sci.USA 80:7155; Yewdell etc., 1985, Proc.Natl.Acad.Sci.USA 82:1785).Have and report that the recombined vaccinia virus of expression of influenza virus nucleoprotein has brought out the T cell mesomeric immunity special to influenza virus (Bennink etc.,, Nature 311:578 in 1984) in the mice body of immunity inoculation.In addition, utilization is by the target cell of the recombinant vaccinia virus infection of expression of influenza virus nucleoprotein, confirmed influenza virus nucleoprotein come out by influenza positive serum blood donor's cytotoxin T cell recognition (McMichael etc., 1986, J.Gen.Virol.67:719).Similarly, find recently, discerned by the special people's ctl clone of HSV by a kind of people's of recombinant vaccinia virus infection of the HSV of expression glycoprotein target cell.(Zarling etc., J.Virol.59:506 in 1986).

The present invention relates to reconstitution cell, peptide, antibody, the method for compositions and inhibition and treatment human papilloma virus infection and tumor suppression or degeneration.

Reconstitution cell of the present invention comprises the gene of the peptide of having encoded, and this peptide corresponds essentially to one because human papilloma virus infection and by the amino acid residue sequence of the peptide of being expressed, as a kind of E that corresponds essentially to ₆And/or E ₇The one section zone of gene outcome or the peptide of proteinic one or more snippets the regional chimeric peptide chemical compound of HPV.This peptide can correspond essentially to that a kind of because HPV infects and the HPV albumen of being expressed or corresponding to a kind of because the HPV gene is inserted in the mammalian cell and the cell peptide of being expressed.Reconstitution cell of the present invention comprises that the additional DNA by a protein section that contains the human papilloma virus that encoded is distinguished the eucaryon and the prokaryotic cell of transfection or conversion.Comprise antibacterial, virus as vaccinia virus as the reconstitution cell of illustration, mammalian cell as the epithelium of transfection or fibroblast or lymphocyte with encoded a tumor cell such as the cervical cancer cell with HPV proteins associated matter section.Excite the soluble protein and the peptide of B cell and/or t cell response also to be included among the present invention.

Also be included among the present invention with like this some protein and peptide antibody molecule similar and/or that compete binding site with it.Particularly preferred antibody be at HPV16 E ₆And/or E ₇The antibody of the corresponding peptide in proteic special zone and at the anti-idiotype antibody of these anti-peptide antibodies.

Compositions of the present invention comprises aforesaid reconstitution cell, antibody and/or peptide, and preferably express human papilloma virus's E ₆Or E ₇The reconstitution cell and/or the peptide in the antigen decision zone of nucleoprotein.Compositions of the present invention preferably can excite a kind of immune composition that causes of immune protection response in receptor.

In the method that described reconstitution cell, peptide and compositions are used in inhibition and treatment HPV infects and tumor produces and/or disappear.In the method for the disease that a treatment of the present invention is produced by the human papilloma virus infection, the compositions that contains reconstitution cell of the present invention and/or peptide of treatment effective dose is bestowed the patient to be enough to for one section suppress the time that this disease further develops.

Also conceived a kind of tumorigenic prevention method that after finding the human papilloma virus infection, suppresses.In the method, the present composition of treatment effective dose is bestowed the patient so that excite a kind of protective response in this patient's body, thereby be suppressed at the generation of tumor in the virus infected cell.The invention still further relates to the intravital human papilloma virus infection's of a kind of patient of inhibition method.In the method, q.s causes that immune composition is bestowed the patient so that excite a kind of immunology protection to reply to suppress human papilloma virus's infection effectively in patient's body.

Cause immune composition and can also be formulated into the reconstitution cell that comprises the proteic antigen decision of expression HPV zone.For example, some compositions can comprise the male non-tumorigenic cell of a kind of main histocompatibility complex (MHC) I level like this, in this cell, has inserted a kind of proteic gene that causes the immunity zone of HPV of having encoded.The reconstitution cell of said composition then can be bestowed the patient so that by having excited the immunne response that causes in the peptide zone of being expressed at this, promote the rejection effect to tumor.This peptide zone is also expressed in tumor cell.

This causes immune composition also can be formulated into a kind of vaccine, and in this case, immunogen comprises a kind of reconstitution cell in the antigen decision zone of expressing protein human papilloma virus.Depend on the characteristic that is used as immunogenic recombinant virus, both can make the viral vaccine of deactivation, also can prepare efficient viral vaccine.With this vaccine composition of the present invention or cause immune composition and carry out suitable immunity inoculation and can cause bringing out a kind of immunne response, this immunne response has caused expressing the destruction of the regional tumor cell of HPV antigen decision in by immune object, also suppressed the HPV infection simultaneously.The preferred reconstitution cell of the present invention comprises coding and expresses E ₆Or E ₇The vaccinia virus in the antigen of HPV16 nucleoprotein decision zone, and epithelial cell, fibroblast, lymphocyte, hemocyte and by E ₆Or E ₇The tumor cell of HPV16 gene transfection.

Further superiority of the present invention and helpfulness are by following specific descriptions, and embodiment and claim subsequently will be conspicuous for the person of ordinary skill of the art.

In the accompanying drawings:

Fig. 1 represents the E with HPV16 ₆The open reading framework is cloned into two expression vectors.

A.E ₆Open reading framework (ORF) take from the HPV16 full length DNA and as blunt end Dde I fragment be cloned among the cowpox expression vector pGS62 that has cut with the Sma I.

B.E ₆ORF cloned in pIC20H so that 5 ' and end introduces Hind III site, is used for the pCDM as described below ₈The directed cloning point of the CMV promoter hypomere in the expression plasmid.The E that is not translating ₆5 of ORF ' end has 58 base pairs, at 3 ' end 98 base pairs is arranged.

Fig. 2 represents the E with HPV16 ₇Open reading framework clone becomes two expression vectors.

A.E ₇The open reading framework is used as the Taq I, and Pst I fragment is cloned into from overall length HPV16DNA by among the pIC20R of Cla I and the cutting of Pst I.E ₇ORF is cloned among the cowpox expression vector pGS62 at EcoR I point place.

B.E ₇ORF is cloned in the pIC20H carrier, to introduce a Hind III site and to place pCD M at 5 of this gene ' end ₈Under the control of the CMV promoter in the carrier.For E ₇Have 56 ORF be unconverted sequence 5 ' base pair, and have 24 for E ₇ORF is 3 ' base pair.

Fig. 3 represents by expressing HPV16 E ₆The radioautogram of the radioimmunoprecipitation of the resulting lysate of cell that recombined vaccinia virus infected of proteic two different speckle purification.

The cowpox lysate is according to example 5 described preparations, carries out radioimmunoprecipitation and to the precipitation electrophoresis.Passage 1,3,5 expressions adopt rabbit to E ₆The result of antiserum (providing) gained by D.Lowy; Passage 2,4 and 7 adopts normal rabbit serum, and passage 6 adopts the anti-E of rabbit ₇Serum (α 16E ₇NP).Antigen is explained above channel number.Lysate prepares from be labeled the cell that infects, and running gel is 17.5% polyacrylamide.

Fig. 4 represents to adopt anti-HPV16 E ₆Or E ₇The autoradiography image of the sedimentary recombinant vaccinia lysate of rabbit antiserum of nucleoprotein.

Recombinate infected cell of cowpox is used ³⁵Cys and ³⁵Met labelling one hour.Infective virus is explained above channel number.Apply radioimmunoprecipitation, thereby make passage 1 and the anti-HPV16E of 5 expressions ₆The result of rabbit serum (D.Lowy) gained, the result of normal rabbit serum gained is adopted in passage 2,4 and 7 expressions; And passage 3 and 6 expression α 16E ₇The result that NP obtains, explain on the right side position of the molecular wt labelling that is colored of standard, and E ₆And E ₇The position explain in the left side.

Fig. 5 represents E ₇The pulse tracking research of protein stability.

The cell of vaccinia virus infection is used as after this described ³⁵S-Cys and ³⁵S-Met pulse labeling one hour then, with non-marked medium heat-preserving a period of time, is illustrated in the channel number top during this period of time.Infective virus (vNY or vHPV16/E ₇) be listed in above the time mark.After the indicated time period, on 17.5% acrylamide gel, doing the autoradioimmunography analysis, with cytolysis and remain on 0～4 ℃.Odd chanel is represented α 16E ₇The NP precipitated product, and the passage of even-numbered is represented the sub-seroimmunity precipitated product of normal rabbit.Molecular weight marker is illustrated in right-hand.

Fig. 6 represents E ₇The radioimmunoprecipitation of gene outcome, this product are to use pCDM ₈-E ₇Expressed in the COS cell of the of short duration transfection of mammal expression plasmid.

Passage 1 and 3 has been represented for the anti-Trp E/E of rabbit ₇The resulting combination of sedimentary protein.

Passage 2 and 4 expressions are for the resulting combination of the sedimentary protein of normal rabbit serum.

The N=nuclear consitution

C=cytoplasm component

A. cowpox E ₇The reorganization lysate is used as E ₇Proteinic positive control.

B.pCDM ₈E ₇The COS cell of transfection.

C. the COS cell of untransfected.

Fig. 7 represents HPV16E ₆And E ₇The amino acid residue sequence of nucleoprotein.

Fig. 8 represents at HPV16E ₇The titrimetry of two monoclonal antibodies of proteic peptide 359.Blank square is represented hybrid tumor clone #14, and real square is represented hybrid tumor clone #10.

Fig. 9 represents anti-E ₆The Western trace of the antiserum titrimetry of peptide.Use from E ₆The anti-E that the peptide of ORF is obtained after three days the rabbit immunity inoculation ₆Peptide serum is only at the fused protein (16E of homologous special Trp ₆DS) on Western trace road, do titration.Two different rabbits of immunity inoculation are represented to carry out with identical peptide in numbering (1) and (2).

Figure 10 represents anti-E ₇The titrating Western trace of the antiserum of peptide.After the booster shot three days from using E ₇Obtain anti-E on the animal of the peptide immunity inoculation of ORF ₇Serum is at special Trp fusion rotein (16E ₇NP) titration on the Western trace.Two different rabbits of immunity inoculation are represented to carry out with identical peptide in numbering (1) and (2).

Figure 11 represents anti-E ₆And E ₇Sero-fast two kinds of dilution Western traces of peptide.E uses by oneself ₆Or E ₇The serum of the rabbit of peptide immunity inoculation confirms that with the Western trace reactivity in the serum is specially at HPV16E at fused albumen of special Trp and TrpE vector gene product ₆Or E ₇Proteic.According to aforementioned titrimetry, the highest serum thinner ratio is selected as the terminal point near reactivity.

As former immunogenic specific peptide (with numbering expression) be listed in every group of nitrocellulose passage above; Antigen in each passage is indicated on the top of gel and two kinds of serum dilution being adopted are indicated on the bottom, below the bracket.M represents prestained molecular weight marker.Blood serum sample is drawn out of for these institutes after a week after the first time or the booster shot for the second time and uses.Antiserum α 358, α 360 and α 361 week after the booster shot second time obtains.Antiserum α 359 obtains by force in increase for the first time in a postvaccinal week.

Figure 12 represents anti-E ₇The antiserum of peptide is to E ₇The identification of native protein.It is right to be used to from the antiserum of the anti-peptide 359 that extracts from rabbit in a week after the booster shot first time ³⁵The S-methionine reaches ³⁵The cowpox E of S-cysteine labelling ₇The radioimmunoprecipitation of reorganization infection cell lysate is measured.Contrast α 16E ₇NP(passage 1) 17-18KDa band section is precipitated, α 359 rabbit serum (passage 3 and 4) make the band section of same molecular amount precipitate equally, and α 360 serum (passage 2) fail to make E ₇The section precipitation.Can see molecular weight marker on the right side.

Figure 13 represents from HPV16E ₇The cytoplasm rna of transfection thing and RT-PCR analyze.Passage comprises (from left to right) pCDM ₈/ E ₇Plasmid is as male PCR control; From the cytoplasm rna of N7.4 and N7.2 cell, these cells are the deutero-HPV16E of NCTC2555 respectively ₇The transfection thing; The NCTC2555 cell is as negative control; E ₇C ₃Be M ₂The deutero-HPV16E of melanoma ₇The transfection thing; And M ₂Melanoma cells is as a negative control (par).As seen the alkali of standard to being marked at the left side.

Figure 14 is illustrated in C ₃The intravital E of H/HeN mice ₇C ₃The growth of tumor situation.In square frame A, before tumor cell inoculation, make mouse immune with the NCTC2555 fibroblast, above-mentioned fibroblast is by HPV16E ₇(N7.2 and N7.4 are respectively blank trigonometric sum and are coated with real circle 7 or HPV16E ₆(N6.8, blank triangle) gene transfection.Square frame B is illustrated in E ₇C ₃Before the tumor cell inoculation, and use N _7.2The immunity inoculation of mice is contrasted by giving mouse inoculation phosphate buffer (PBS, blank square) or by human melanoma antigen P97(CL19, being coated with real triangle) research that the NCTC2555 fibroblast of transfection is carried out, square frame C is illustrated in and accepts E ₇NCTC2555 fibroblast (the N of transfection _7.2) the intravital parental generation M of mice of immunity inoculation ₂The tumor growth situation of melanoma cells (par).

Figure 15 represents to resist-CD ₈Antibody is to accepting N _7.2The therapeutic effect of the intravital tumor growth of mice of cellular immunization inoculation.After immunity inoculation, bestowing E ₇C ₃Before the tumor cell, to the anti-CD of injected in mice ₈Antibody (being coated with real circle) or injection resist-CD ₅Antibody (blank triangle).

Figure 16 represents from accepting anti--CD ₈Monoclonal antibody (right group) or anti--CD ₅The CD of the mice of monoclonal antibody (left side group) treatment ₄The positive and CD ₈The dynamic cytometry analysis of male splenocyte.

The present invention relates to reconstitution cell, peptide, antibody, compositions, and be used for suppressing and treatment human papilloma virus infection and tumorigenic method.Reconstitution cell comprises a kind of HPV albumen, preferably E of having encoded ₆And/or E ₇These reconstitution cells of the dna structure of HPV nucleoprotein are expressed the antigen decision zone of a polypeptide, this peptide basically with mammalian cell in since such as infect by HPD or recombination form to insert the peptide of being expressed in this mammalian cell behind the HPV gene corresponding.In a preferred embodiment, peptide of the present invention corresponds essentially to proteic about 8 to 30 the amino acid residue sections of HPV.In a particularly preferred embodiment, peptide of the present invention corresponds essentially to HPV16E ₆Or E ₇A proteic zone, and specifically be can enough antibody when being applied to the host and/or the antigen decision zone of T cell surface molecule challenge corresponding to one.The present invention also comprises the compositions of these reconstitution cells, and can be used as the peptide that causes immune composition, immunization therapy method or be used for the treatment of patient's vaccine.Also considered to compete with these peptides the antibody molecule of binding sites at this.Specifically, produce anti-HPV16E ₆And/or E ₇The antibody molecule of proteic calmodulin binding domain CaM, and the anti-idiotype antibody of these antibody of antagonism, the HPV binding site of they competitions on normal cell protein and tumor cell albumen, as with HPV16E ₇Protein bound retina blastoma gene outcome (RB105).The invention still further relates to and suppress and treatment human papilloma virus infection and neoplastic method.Some concrete methods of the present invention relate to treat by HPV infect the disease cause and find to suppress after HPV infects and the cell that disappears in the prevention method of tumor.Another aspect of the present invention relates to and suppresses the method that HPV infects in patient's body.

In order more clearly to describe the present invention and embodiment, following definition is comprised wherein.

As used herein " transfection " be meant by adding DNA and be incorporated in the eukaryotic cell, obtain new genetic marker.

As used herein " conversion " be meant by adding DNA and be incorporated in the prokaryotic cell, obtain new genetic marker.

" neoplasia " is the tomour specific Phenotype that phalangeal cell obtains to cause uncontrolled cell proliferation as used herein.

" cloning vehicle is meant any plasmid or the virus of foreign DNA to clone of inserting as used herein.

" plasmid " is the extrachromosomal cyclic DNA of a kind of autonomous self-replication as used herein.

" open reading framework (ORF) " is meant that (potentially) can be translated into the protein DNA sequence as used herein.

" helper virus " provides a kind of virus of the function that defective virus lacks as used herein, the latter can be finished in the mixed infection process infect circulation.

" gene (cistron) " is the peptide chain sequence DNA fragment of having encoded as used herein; It can comprise before the coding region and zone (front end and afterbody) afterwards, also has the intervening sequence (introns) between the discrete coding section (exons).

" expression " is to produce the process that a kind of peptide or protein experiences by a structural gene as used herein.It is the combination of transcribing and translating.

What any number with single ancester cell or molecule described in " clone " speech as used herein is equal to cell or molecule.

As used herein " base pair " (bp) speech be adenine in the dna double helical structure (A) and thymus pyrimidine (T), or the combination of cytosine (C) and guanine (G).

" expression vector " speech is any plasmid or the virus that can insert and/or express foreign DNA as used herein.

Be meant in those sequences of expressing the direction front at this employed " hypomere " speech; For example, the peptide-coding region territory of gene is a hypomere or along 3 ' direction away from gene with respect to start codon; Epimere be towards 5 of the sequence of being discussed '.

As used herein " polymerase chain reaction " (PCR) speech be meant that the archaeal dna polymerase repetition DNA chain of a kind of temperature stabilization of use by success amplifies dna molecular, by heating, add introduction and separate its complementary strand, and repeat this process about 30 times to produce about 10 ⁹Individual dna replication dna product.By using round pcr, the DNA of small quantity can be exaggerated and produce the DNA that is enough to be used in the various various process.

" inoculum " speech that occurs with its various different grammatical forms is used herein to the compositions that description includes reconstitution cell of the present invention or peptide, as being used for antibody preparation, or the active component of the T cell activation preparation of anti-human papilloma virus infection/transformation cell.When peptide is used to induce antibody, should be appreciated that this peptide can be used alone occasionally, and be to link to each other with carrier or more frequently, but for ease of expressing, after this these variations will often show in conjunction with other composition.

Inoculum also can comprise adjuvant.Adjuvant is as Freund ' s adjuvant (CFA) completely, and non-complete Freund ' s adjuvant (IFA) and Alumen are the materials of knowing in this area, and can obtain from several sources of goods commercial.

One inoculum is easy to CFA or IFA preparation.For example, a certain amount of reconstitution cell or peptide conjugate are dissolved in about 0.5 milliliter of the PBS(of pH7.2) in, its amount should be enough to each inoculation is provided the reconstitution cell or the peptide combinations of aequum.Then,, wherein comprise this reconstitution cell and/or conjugate with waiting the CFA or the IFA of capacity to mix with solution so that a kind of inoculum to be provided, water, and adjuvant, wherein water and oily ratio are 1: 1, after this, this mixture of homogenize has provided a kind of inoculum.The volume of the inoculum of preparing like this is generally greater than 1 milliliter, and some reconstitution cells and/or peptide combinations, and PBS and adjuvant may be lost in emulsion process.Basically all can be placed in the syringe by sucked back emulsifying agent, then, are injected in the animal body like that as discussed below.Be injected into quantity such as rabbit or the intravital inoculum of this class animal of mice and should be and be at least about 90% of before emulsifying step carries out amount.

In inoculum of the present invention, reconstitution cell and peptide both can be comprised separately, also can be incorporated into a carrier protein, as the key hole

Keyhole limpet hemocyanin (KLH) adds physiologically acceptable diluent, as has the water or the PBS of adjuvant.KLH is the acceptable intravital carrier of animal that is used for, but for the use on the commercial size is very expensive, also consider to use the carrier of replacing, comprising soybean agglutinin, aluminium hydroxide (alum), bovine serum albumin (USA), ovalbumin, peanut agglatinin, tetanus toxoid, and poly-L-Lysine.The glucoside that saponin, a kind of plant produce also is a kind of adjuvant of knowing, can be commercial from the Ohio, and the Berghausen chemical company of Cincinnati obtains with 5% solid solution formation, and can use with aluminium hydroxide at this.

Above-mentioned inoculum original solution is used for illustrating inoculum of the present invention, as confirming that at this they can be used to produce antibody molecule or excite with reconstitution cell of the present invention and/or peptide carry out immunoreactive T cell.

" causing immune composition " speech that occurs with its various grammatical form is used to describe a kind of inoculum type at this, and it comprises reconstitution cell of the present invention or peptide,, is used to bring out an immunocompetent active component in host animal that is.In a preferred embodiment, the immune composition that causes of the present invention can be a kind of vaccine.Because immunocompetence both comprised production of antibodies, also comprise the immunne response that has excited a cell, so vaccine and inoculum can comprise these equal compositions, but its purposes difference.As a rule, the composition that is used for vaccine is different with the composition that is used for inoculum, because many adjuvants that is used for animal cannot be used to the mankind.

The less relatively peptide that in research of the present invention, is adopted be on 403A type applying biological system peptide synthesizer, utilize the solid phase method of Merrifield synthetic (1963, J.Am.Chem.Soc.85:2149-2154), the document is in this application combined.Inserted the cysteine residues of an increase at the C of this sequence end or N end.After splitting off from resin and protecting, this peptide is by the liquid chromatographic purification of reversed phase high efficiency.Before used as immunogen,, adopting the inferior sulfanilamide 4-(N-of bifunctional reagent succinyl maleimide methyl by its cysteine residues) hexamethylene sugar-carboxylic acid is coupled peptide with KLH.

" synthetic " speech is meant by chemical means as used herein, i.e. chemosynthesis, and the peptide molecule of formation, rather than adopt biological means, as gene engineering, preparation.

Vaccine and cause the independent peptide that immune composition comprises said amount, reconstitution cell, conjugate or its combination as used herein.These cause immune composition and also comprise upward permissible diluent of physiology, as water or saline, generally also comprise adjuvant, as complete Freund ' s adjuvant and non-complete Freund ' s adjuvant.

Immunogenic original solution prepares as follows with IFA or CFA: a certain amount of synthetic peptide conjugate and/or the reconstitution cell that are enough to produce required each inoculum concentration are dissolved in the phosphate buffer (PBS).Then, isometric CFA or IFA mixed with this solution to provide comprise this cell and/or peptide, the synthetic of water and adjuvant, wherein water is 1: 1 with the ratio of oil.After this mixture is stirred evenly to obtain original solution.

" antigen decision zone " speech is a range of structures as used herein, as can be in the host exciting a kind of special amino acid residue sequence or peptide section of molecule of specific immune response with antibody molecule or T cell surface molecule.An antigen decision zone can comprise one or more antigenic determinants and/or cause immunologic determinants.

" epitope " speech refers to be responsible for the molecular structure composition that carries out specific reaction with antibody (immunoglobulin) molecule that is excited by antigen identical or that be correlated with or immunogen accordingly or T cell surface molecule as used herein.

The speech that " causes immune determiner " as used herein refers to be responsible for inducing in the host produce and comprises one as antigen the time and the antibody of the bonded antigen binding site of immunogen (genotype) or the molecular structure composition of T cell surface molecule.

This mutually " anti-idiotype " and " anti-idiotype antibody " two speech of using of conversion be meant a kind of antibody, its antigen binding site combines with the idiotype of the original antigen for preparing for anti-specific antigen (as papillomavirus antigen) specifically, and anti-idiotype antibody is combined with antigenic with antigen beginning antibody competition.

" antigen " speech is meant by a kind of owing to antibody or the bonded entity of T cell surface molecule that produces of replying to the constituent that proposed as used herein.

" immunogen " speech as used herein is described in to bring out in the host animal and produces the entity that antibody or specific T-cells are replied.

" unit dose " speech refers to animal is suitable as some physically discrete units of dosage unit, each unit comprises the active substance of scheduled volume, and the active substance of this scheduled volume and required diluent (being a kind of carrier or excipient) combine and can produce desired therapeutic effect.The standard of the unit dose of novelty of the present invention is arranged by following factors, and directly depend on them, the i.e. characteristic of the uniqueness of (a) active substance and the specific therapeutic effect that will obtain and (b) inherent restriction in the technical field of the active substance that synthesizes this treatment usefulness.

" effective dose " speech means as used herein is enough to suppress effectively owing to HPV infects the infection of the cell that causes and/or the generation of tumor.

Effective dose for particular patient can be according to the holistic health situation such as the infection state patient, and application process, the seriousness of side effect etc. be some factors and changing like this.

At this two ends add or deduct maximum 3 amino acid residues about employed " corresponding " speech that occurs with its various different grammatical forms of peptide sequence means described peptide sequence in the arbitrary end or the while of amino and carboxyl terminal at this and in claims, and replacing along only comprising in the specified amino acid residues of this peptide and/or peptide sequence to guard.

Above employed " conservative replace " speech refer to an amino acid residue by another, biology, similar residue substituted.The conservative example of replacing comprises with a hydrophobic residue, as Ile, and Val, Leu, or Met substitutes another or with the alternative another kind of a kind of polar residues is between Arg and Lys, between Glu and the Asp or between Gln and the Asn or the replacement of similar situation.

In some instances, replaced by a kind of ion residue that carries out with opposite electrical ion residue, substitute Asp as Lys, being confirmed as in the present technique field is conservative the replacement, because those ionic groups are considered to seldom solubility be provided assistance.Yet all things considered, since this discuss to substitute with respect to a complete protein be to carry out on relatively short synthetic peptide zone, an ion residue is considered to " group substitutes " by opposite electrical the substituting at this of ion residue of another band, as by non-ionic and ion residue and big residue such as Phe, Tyr and Trp and big slightly residue such as Gly, the alternative of Ile and Val is exactly that group substitutes.

Refer to those respectively under the physiology pH value at this " non-ionic " and " ionic " residue that common implication is used by it, not charged or usually with a kind of amino acid residue of electric charge.The nonionic residue of example comprises Thr and Gln, and the ion residue of example comprises Arg and Asp.

Suppress and the intravital human papilloma virus infection of treatment patient, neoplasia and tumor be purpose of the present invention.Papilloma albumen and the expression of peptide of confirmation in infected cell inspires the present inventor to go to study reconstitution cell described herein, peptide and method.

Reconstitution cell of the present invention comprise one basically to human papilloma virus's the similar gene insert in DNA zone.In one embodiment, the gene insert HPV16E that encoded ₆And/or E ₇The zone of nucleoprotein.In a particularly preferred embodiment, E ₆And/or E ₇Proteic antigen decision zone is expressed.In another embodiment, the gene insert has brought out the expression of cell protein in the recombinant mammalian cells that contains this gene insert.

The present invention has considered to bring out the expression system in HPV albumen zone, and comprise virus, as vaccinia virus and adenovirus, fibroblast, as COS MC and human keratinocyte, tumor cell such as CaSki cervical cancer cell and melanoma cells, and other mammalian cell such as epithelial cell and lymphocyte.In another embodiment, the male and preferably nononcogenic cell of MHC1 level is with the gene transfection in the zone of HPV albumen of having encoded.In a particularly preferred embodiment, HPV16E that encoded ₆And/or E ₇The gene in a proteic zone is inserted in these MHC1 level positive cells or after infecting with the reconstitution cell that contains these genes and is expressed, and as a kind of method of impelling tumour regression, the immune composition that causes that will comprise these cells is applied to and has in the patient of the HPV16 induced tumor body.This tumour regression is along with producing by cause the immunne response that immunocyte brings out in patient's body, and this is replied thereby is directly at tumor.

Reconstitution cell of the present invention is an expression system, or cell, and they include the insertion gene structure in coding HPV albumen zone.Illustrative expression systems more of the present invention or cell comprise such as at a kind of virus such as vaccinia virus that causes in immune composition or a kind of vaccine, retroviral, adenovirus, poliovirus and can bestow other virus of patient in non-pathogenic mode can the infection of enough HPV genes, transfection or cell transformed such as peripheral blood lymphocyte and epithelial cell.Design of the present invention has comprised all cells that can integrate and express a zone of human papilloma virus and gene.By in the amino acid residue of expressing gene product some can very big weakening immunogenicity variation and for E ₆And E ₇The zone has the special peptide section of similar amino acid residue also among design of the present invention.

In one embodiment, gene constructs of the present invention is by cloning ORF preparation, zone of this ORF and human papilloma virus's nucleoprotein such as the E of HPV16 ₆Or E ₇Albumen is corresponding, and it contains the genomic plasmid of more most of HPV16 by restricted enzyme from one and obtains.This restricted fragment passes through then as Auswbel, people such as F.M (1990, " Current Protocols in Molecular Biology ", Greens Publishing Assoc.and Wiley Interscience) and Maniatis, T. wait people (1982, " Molecular Cloning:A Laboratoy Manual " Cold Spring Harbor Laboratory, NY) the standard molecular biology method of Miao Shuing is cloned in the expression vector.

The expression vector that comprises required ORF is further handled then, and be inserted in the genome of host cell with by as homology recombinate or integrate with selection pressure; Or the viral infection by cell is introduced into and produces reconstitution cell of the present invention.

Be selected and be used for will selected ORF fragment being inserted into this cloning vehicle and determined by the known multiple factor of those skilled in the art with the specific site that forms recombinant DNA molecules, these factors are as by peptide or proteic size and the structure expressed, the degraded sensitivity of the gene outcome that is produced by host cell and the position of standard termination codon etc.

In a particularly preferred embodiment, host cell of the present invention is a vaccinia virus.Vaccinia virus comprises a Linear Double helical dna genome of about 1874 base pairs, and duplicates in the Cytoplasm of infected cell.These viruses comprise an infectivity to virus in virus nuclear be the indispensable system of transcriptase completely (comprising the capping enzyme, the sweet enzyme of methylase and polyadenous).Vaccinia virus transcriptional regulatory sequences (promoter) allows by the cowpox RNA polymerase, rather than host cell RNA polymerase transcriptional start.

The expression of the foreign DNA in recombined vaccinia virus need be attached to the cowpox promoter on the protein coding DNA sequences of alien gene.Made up plasmid vector, also claimed to insert carrier, be used for alien gene is inserted vaccinia virus.One class is inserted carrier by constituting down: the vaccinia virus promoter that (a) comprises transcriptional start site; (b) several restricted enzyme cloning sites that are used to insert foreign DNA fragment insert, are positioned at the uniqueness of transcribing beginning site tract; (c) at lateral nonessential vaccinia virus DNA(of promoter such as TK gene etc.) and the guiding of this alien gene insert entered cloning site in this nonessential zone of virus genomic this homology; A bacterial origin that (d) duplicates and the antibiotic resistance label that in escherichia coli (E.Coli), duplicates and select usefulness.The example of some carriers is by people such as Mackett(Mackett like this, and 1984, J.Virol.49:857-864) described.

Recombined vaccinia virus results from the recombinant virus that contains alien gene, and to insert plasmid transfected in the past by after in the cell of vaccinia virus infection.Homology is binned in the interior generation of infected cells and causes alien gene to be inserted in the viral genome.Infected cells can be utilized immunological technique, and the DNA speckle is hybridized soon, or the hereditism of recombinant virus that finally can be separated is selected screened come out.These cowpox recombinants have kept its basic functions and infectivity, and can be built into and comprise the right foreign DNA of about 35 kilobase.The detection that alien gene is expressed can be by utilizing Northem blotting or dot blot and nucleic acid hybridization to detect rna content or by utilizing immunodetection (for example, radioimmunoprecipitation, radioimmunity detection method, or immunoblotting) detect protein level and carry out.

Peptide of the present invention and peptide conjugate have comprised basically and HPV more proteinic regional amino acid sequence corresponding of being expressed.Peptide preferably brings out proteinic those zones corresponding to the HPV that is expressed in reconstitution cell of the present invention.Particularly preferred peptide is corresponding to HPV16E ₆And/or E ₇Proteic antigen decision zone.The solid-phase synthesis of the Merrifield that had been mentioned above peptide both can adopt prepares, and also can adopt the genetic engineering method of standard.

The compositions that comprises reconstitution cell of the present invention and/or peptide is used as and causes immune composition, and vaccine becomes with therapeutic combination.In one embodiment, a kind of expression HPV16E that comprised ₇The compositions of the recombined vaccinia virus in proteantigen decision zone is used as and causes immune composition, and it can be in the patient infects at HPV and/or tumor excites protective response.

Compositions of the present invention except reconstitution cell described here or peptide, also comprises diluent such as the water or the saline of physical compatibility, and generally also comprises adjuvant described above.

The present invention also comprises the reconstitution cell and/or the peptide of preferably expressing HPV antigen decision zone of using effective dose to the patient who suffers from the disease that is caused by the HPV infection.

Generally, reconstitution cell of the present invention and/or peptide are to be applied with pharmaceutical compositions, and compositions comprises reconstitution cell and/or peptide and a kind of pharmaceutical carrier of effective dose.When the parenteral road was used, the present composition was formulated into and the bonded injectable unit dosage form of a kind of pharmaceutical carrier (being generally solution, suspension or emulsion).Some carriers itself are nontoxic and no therapeutical effect like this.The example of this carrier is common saline, RingerShi solution, Glucose Liquid and HankShi solution.Non-aqueous carrier also can be used as not volatile oil and ethyl oleate.Preferred carrier is glucose/saline of 5%.Carrier can comprise the additive of trace as increasing immunogenicity, the material of isotonicity and chemical stability, for example buffer agent and antiseptic.Generally speaking, know in this area for the injection useful carrier.

(bringing out) antibody that produces by reconstitution cell of the present invention and peptide and whole antibody basically, and constituted an alternative embodiment of the invention by the antigen binding site of some Antibody Preparation like this with to the anti-idiotype antibody that these antibody and/or antibody fragment prepare.This antibody is in mammalian hosts, in mice, rat, guinea pig, rabbit, horse and similar animal, by adopt with inoculum described above or be used to form anti-idiotype antibody be attached to that the immunization that monoclonal antibody was produced on the carrier forms.

In a preferred embodiment, antibody molecule of the present invention comprises by carrying out the whole antibody that immunity inoculation forms with the inoculum that comprises above-described reconstitution cell and/or peptide or anti-peptide antibody in mammal.

At E ₆And E ₇The antibody of special peptide preparation will allow E ₆And E ₇The detection of cell homologue.For example, peptide as peptide 359, comprises the part calmodulin binding domain CaM of the retina blastoma gene outcome that is called RB105.Can imitate RB105 and be attached to and E at the antibody of these peptides ₇With having on the cell protein of sequence homologue.This E ₇The identification of cell homologue might be able to identify the cell protein of being responsible for cell proliferation and may be the cell protein of the common ligand of RB105.The anti-idiotype of some antibody can be imitated E like this ₇And identify except that E ₇Ligand or its homologue, the i.e. cellular proliferation protein of the RB105 of itself.What can cause discerning some other and cell proliferation albumino reaction like this raises or the cell protein of downward modulation proliferation function with rising, as tumor suppressor gene albumen or transforming protein.Similarly research can be applicable to E ₆With its ligand p53 and E ₆Or other unknown ligand of p53.

Being used in the reconstitution cell that comprises 50 micrograms to 1.0 milligram in the complete FreundShi adjuvant and/or the inoculum of peptide conjugate is that rabbit carries out immunity inoculation, after two to three weeks, the reconstitution cell or the binding peptide that are used in 10 micrograms to 1.0 milligram in the non-complete FreundShi adjuvant carry out booster shot.The immunization of each rabbit is included in ten the subepidermal injections in back, and wherein twice in subscapular zone, and carries out booster shot in five sites, and one is in subscapular zone.A week (5～8 days) rabbit is being drawn blood after two weeks and the booster injection behind the start injection.

The serum that includes immunoreactivity antibody can adopt the known method in this area to generate from blood then.These antibody for one or more peptides of the present invention be have immunocompetent.Some antibody can be used for producing the anti-idiotype antibody of one or more peptides of the present invention then by similar method like this.

Suitable monoclonal antibody generally is a whole antibody, also can adopt people such as Niman, and 1983.Proc.Natl.Acad.of Sci.USA 802 4949 described hybridoma technologies are prepared, and foregoing description is incorporated among the application by reference.In brief, produce for forming the hybridoma of monoclonal antibody, the lymphocyte that obtains in the cell line of myeloma or other self-perpetuating and the mammiferous spleen with reconstitution cell of the present invention or peptide or antibody hyperimmunize is merged mutually.

Preferably myeloma cell line is from of the same race, and as lymphocyte, but the hybridization that intersection is planted can produce in nude mice.Generally speaking, the mice of Balb/c strain is preferred mammal.The rat bone marrow tumour that is suitable for use among the present invention comprises AG-8 cell and NS-1 cell.

The general Polyethylene Glycol that adopts merges splenocyte and myeloma cell as PEG1500 or PEG6000.Fused hybrid is selected by its sensitivity for HAT medium (hypoxanthine, aminopterin, thymidine).Producing the enzyme relevant with immune absorption measuring method that after this hybrid tumor utilization of antibody molecule of the present invention describe is differentiated.

Monoclonal antibody not only needs to obtain from hybrid tumor supernatant, can also obtain from the mammal ascites fluid of introducing required hybridoma with denseer form usually.Utilizing ascites fluid to prepare monoclonal antibody is prior art, and will no longer discuss at this.

The present invention has also conceived and is used to suppress and treats the method that patient's HPV infects and infected the disease that causes by HPV.

In one embodiment, the treatment method that infects the disease cause by HPV comprises one period that is enough to suppress disease progression of the present composition to patient's administering therapeutic effective dose.The disease of example explanation comprises cervix uteri wart and human cervical cancer, wherein with the treatment prevention of the present composition or delayed further developing of patient disease.

Also conceived another prevention method, according to a kind ofly suppressing tumorigenic protective response and carry out the HPV INFECTION IN DETECTION to suppress the generation of patient's in-vivo tumour for what apply in patient's body that treatment goes up that the synthetic of the present invention of effective dose excites.The using of immune composition that cause that comprises reconstitution cell of the present invention and/or peptide preferably excites the CD8 that causes that inhibition patient in-vivo tumour takes place ⁺Lymphocytic replenishing.

Also conceived the method that suppresses to have the HPV infection that contacts the HPV dangerous patient in the present invention.In the method, the full thing of immune group that causes that comprises reconstitution cell of the present invention and/or peptide of q.s is bestowed the patient to excite a kind of protective immune response to suppress HPV infection subsequently effectively in patient's body.

In a preferred embodiment, causing immune synthetic is to make the vaccine of patient to the HPV infection immunity when using.

Further describe the present invention by the following embodiment that is intended to illustrate rather than limits.

Embodiment 1

E ₆And E ₇The preparation of ORF construction

A.E ₆

E ₆And E ₇ORFS clones from comprise the genomic pBR322 plasmids of whole HPV16 (pBR322/HPV16).The DdeI fragment (bp#25-654) of 630 base pairs has comprised E ₆ORF.The Klenow fragment nose circle of this DdeI fragment and archaeal dna polymerase connects and accept gel electrophoresis in 3% NuSieve genetic technique level (GTG) (FMC Bioproducts Rockland ME) agarose.The DdeI section electrophoretic transfer of 630bp is to NA45 DEAE(Schleicher ﹠amp; Schuell, Keene, N.H) on the paper, and carried and being washed at high-salt buffer (1.0M NaCl, 0.1 mM EDTA, 20 mM Tris pH 8.0) use chloroform extraction then with phenol earlier down at 65 ℃ in, with 100% ethanol precipitation of 2 volumes, then, the DNA precipitate is suspended in Tris-EDTA CTE once more ₇In the buffer (10 mM Tris-Cl, pH 7.5,1 mM EDTA, pH8.0).

The cowpox expression vector that is utilized is pGS62, and it is positioned at the EcoRI site of SmaI site hypomere except the EcoRI site is deleted, only stayed the next one from plasmid, and is identical with pGS20.The SmaI linearisation of this carrier, and handle by the alkali phosphatase (CIAP) that adopts sura and to remove phosphoric acid and purified by gel.By as to obtaining E from pBR322 ₆This fragment is reclaimed in the description that ORF is the same from gel.E ₆ORF adds the sequence of being made up of 58bp5 ' and 98bp3 ' of not translating, and is linked to the hypomere of the 7.5K promoter of pGS62 cowpox expression vector.Recombiant plasmid with structure as shown in Figure 1 is separated by the standard molecular biology method, characterization and breeding, these aspects are as described at following article: people such as Mancatis T., nineteen eighty-two, " MolecularCloning:A Laboratory Manual " (Cold Spring harbour laboratory, New York, 1982) and Ausubel, F.M. wait the people, nineteen ninety, " Current protocols in Molecular Biology " (Green Publishing Assoc. and Wiley Interscience)

B.E ₇

The complete HPV16 genome of being cloned in pBR322 is with TaqI and PstI incision and carry out electrophoresis.Comprise E ₇The 374bp fragment of ORF is as pressing E ₆Description carry out gel like that and purify.PIC20R carrier (Marsh, J.L. etc., 1984, Gene 32:481-485) divides incision and gel-purified with PstI and ClaI.The reorganization pIC20R E of structure shown in having (Fig. 2) ₇Plasmid is separated as described above, characterization and breeding.PIC20R E ₇Handle with EcoRI, and adopt the gel electrophoresis purification to comprise E ₇The fragment of ORF.

Cowpox expression plasmid pGS62 cuts with EcoRI, and purifies with the alkaline phosphatase treatment and the gel of sura.Obtain the E that comprises shown in Figure 2 ₇ORF and E ₇The 56bp of ORF non-translate 5 ', and the recombiant plasmid of 24bp3 ' is to its characterization and numerous.

C.

Two kinds of recombinant vaccinia expression plasmids are extended and by CsCl, and ethidium bromide equilibrium centrifugation partition method is purified.

Embodiment 2

The structure of recombined vaccinia virus

E ₆Or E ₇It is cloning site place in the uniqueness of the hypomere of the transcriptional control element (7.5K starts the factor) of infect early stage and the vaccinia virus of being expressed late period just that clone's step of ORFs is designed at one, insert (Earl in the open reading framework, Deng nineteen ninety, J.Virol.64:2448-2451).ORFs is by the left and right arms side joint of cowpox thymidine kinase (7K) gene so that with the genomic homology reorganization of vaccinia virus.

Employing is by Mackett, people such as M., and 1984, J.Virol.49:857-864, the conventional method of description utilizes the homology reorganization, pGS62/E ₆And pGS62/E ₇Be respectively inserted in the vaccinia virus genome in the thymidine kinase gene.By the parental virus that obtains in the Wyetn antismallpox vaccine, V-NY is bred in the BSC40 cell after three specklees are purified.In brief, before by the parental type vaccinia virus infection, chimeric plasmid is introduced in the cell.The Tk zone of this plasmid is homologue with the Tk zone of virus.The insertion plasmid that alien gene is inserted into the reorganization in the vaccinia virus chromosome set provides recombinant virus Tk ^-Tk ^-Virus is to comprise the Tk that grows in the medium of 5-bromouracil deoxyribose acid in existence ^-Select in the cell.Adopt three-wheel speckle method of purification that recombinant virus is purified, and come out by purifying with viral DNA s and from a kind of bacteria carrier ³²The E of P-labelling ₆Or E ₇DNA is hybridized and is discerned embedded virus.

Embodiment 3

Mammalian cell expression plasmid pCDM ₈/ E ₆And pCDM ₈/ E ₇Structure

E ₆And E ₇HPV16 open reading framework (ORF) send out in early days the Hind III point place of the hypomere of (IE) cytomegalovirus (CMV) promoter (Fig. 1 and Fig. 2) in speed and be cloned into mammals expression vector pCDM respectively ₈(Invitrogen.San Diego, CA) in.By to from pGS621E ₆The BamHI that the vaccinia virus expression plasmid obtains, EcoRI fragment gel purify and it are connected to and use BamH1, the pIC20H plasmid subclones E of EcoRI incision ₆ORF is so that at E ₆ORF5 ' end place obtains a Hind III site that is used for directed cloning to pCDM8.With Hind III and XhoI digested plasmid pIC20H/E ₆, E ₆ORF5 is purified by gel together by the non-sequence of translating that the 98bp of the 58bp and 3 ' of ORF5 ' end end forms, and is linked to the Hind III, in the pCDM8 carrier that xhoI digests.Isolate reorganization pCDM8/E shown in Figure 1 as previously mentioned ₆, to its characterization and breeding.Formed bacterium colony screens with the miniature preparation DNA method of purification of standard, then handle these DNA with restricted enzyme, select these enzymatic compositions, to produce to the diagnosis bands of a spectrum figure of transcriptional orientation with correct orientation (promptly from 5 ' to 3 ') clone's dna fragmentation.Amplify suitable clone, and use CsCl, their DNA of ethidium bromide equilibrium centrifugation partition method purification.

E ₇ORF is by from pIC20R/E ₇In gel purify out as EcoRI, the PstI fragment, and in EcoRI and PstI site by sub-clone in pIC20H (Fig. 2).For at E ₇5 of ORF ' end is introduced Hind III site, utilizes Hind III and PstI with E ₇ORF is from pIC20H/E ₇Take out.Contain E ₇The fragment of ORF by gel-purified and with the non-HPV sequence 5 of translating of ORF56bp ' and E ₇ The non-HPV16 sequence 3 of translating of ORF24bp ' together, be linked to the Hind III, the pCDM that PstI cuts ₈In the expression plasmid, to produce pCDM shown in Figure 2 ₈/ E ₇The screening bacterium colony amplifies and its DNA of purification as described above.

Embodiment 4

With the antigen of the HPV16 nucleoprotein of having encoded decision zone

DNA is inserted in epithelial cell or the fibroblast.

Stay the E of virus corresponding to human mamillary ₆Or E ₇The dna nucleotide sequence at least one antigen decision zone by the above method of in example 3, describing, along with glycerol shock, utilize standard calcium phosphate precipitation method, be inserted in the mammals expression vector also transfection in epithelial cell or fibroblast.Then after transfection, cell is placed in the Dulbecco ' s medium (IMDM) (1 milligram of G418/ milliliter) of the IscoveShi correction that contains G418.When colony growth during, utilize clone's ring that it is taken out, and transfer to and have 24 each ophthalmic of titer plate, and in tissue culture medium (TCM), grow into greater number to visible size.Part is stored in 10%DMSO under liquid nitrogen environment, during 90% cattle fetal blood is clear, and as further research.

Embodiment 5

The E that obtains from recombinant vaccinia virus infection ₆And E ₇The radioimmunoprecipitation of gene outcome

At HPV16E ₇And HPV16E ₆The rabbit antiserum of TrpE fusion rotein is produced and (is respectively α 16E ₇NP and α 16E ₆Ds, and as Jenison etc.,, providing by D.Galloway like that described in the J.Virol.62:2115-2113 in 1988.

At HPV16E ₆Rabbit antiserum such as Androphy etc., 1987, BMBO, described in the 6:989 like that by D.Lowy(National Cancer Institute, Laboratory of Cellular, Bethesda MA) provides.

Infected by cowpox recombinant virus of the present invention or Caski cervical cancer cell (obtaining) in 12 hours the culture dish of a 10cm of BSC40 monkey cell, be supplemented with cattle fetal blood clear (FCS), the 0.25mCi(milli cubic feet per inch of 5% dialysis by ATCC) [ ³⁵S]-methionine, and 0.25mCi(milli cubic feet per inch) [ ³⁵S]-be labeled 60 minutes in the culture medium that does not have methionine of cysteine.Cell is dissolved in (20mM Tris-HCl in molten born of the same parents' buffer of 1 milliliter, pH7.4,50 mM NaCl, 0.5% Nonidet p-40,0.5% deoxycholate, 0.5% sodium lauryl sulphate (SDS), 0.1 aprotinin Tripsin inhibitive factor units per ml and 1mMEDTA) and carried out supersound process in short time.

By being incubated with the normal rabbit serum of 10 microlitres or cowpox immune rabbit serum and a-protein-Sepharose pearl 4 ℃ the time lysate is carried out pre-pure clear processing.After the centrifugalize, discard these globules.Pure clear lysate of handling and rabbit α E ₆(D.Lowy) or α 16E ₇(α 16E ⁷NP) immune serum is incubated together, and this serum is by being incubated with the molten born of the same parents' thing of unlabelled vaccinia virus and being adsorbed in advance.Then, the Sepharose pearl that scribbles protein A is joined immune rabbit antibody and the cell born of the same parents separate in the mixture of thing, and insulation.After the centrifugalize, and globule RIPA buffer (10mM, Tris-HCl(pH7.4), 0.15M MaCl, 1% NP-40,1% dexycholate, 0.1%SDS, 0.1 the aprotinin of trypsin ihhibitor units per ml) clean twice, use high-salt buffer (10mM Tris-HCl(pH7.4) then in succession, 2M NaCl, 1%NP-40,0.5% dexycholate), low salt buffer (0.5%NP-40 0.1% SDS is dissolved among the PBS), 1MMgCl ₂, 1M Tris-Hcl(pH 7.4) and the PIPA buffer solution for cleaning.

By ebuillition of heated in sample buffer 5 minutes, protein is discharged from antibody and globule, and relatively analyze with 17.5% SDS-PAGE and prestained standard molecular weight labelling.

The gel autoradiography photo has confirmed as anti-E ₆Rabbit serum (providing) when being used to precipitate lysate, at E by d.Lowy ₆The bands of a spectrum that have about 17KDa molecular weight in the-cowpox recombinant lysate.(Fig. 3 and 4).And carry out on the sedimentary passage, or with resisting-E with normal rabbit serum ₆Can't see this regional bands of a spectrum on the corresponding position on the passage of rabbit serum precipitation vNY lysate.

Two vaccination E ₆Recombinant speckle 7.1 and 7.8 is extended, analyzes to obtain similar result.

When adopting above-described autoradiography to E ₇When-cowpox recombinant lysate is analyzed, use anti-E ₇Rabbit serum (α 16E ₇NP) post precipitation has been found the 18-20KDa bands of a spectrum.And precipitating with normal rabbit serum or with anti--E ₇Rabbit serum carries out can't see these bands of a spectrum on the relevant position in the sedimentary passage to radiolabeled vNY lysate.Pulse-follow-up study (Fig. 5) shows E ₇Protein was degraded in synthetic back and is carrying (D.Smotkin and F.Wettstein,, E J.Virol.61:1686-1689) in 1987 in the genomic Caski cell of whole HPV16 in 2-6 hour ₇Protein has also demonstrated similar results.E ₇Proteinic migration is identical with observed migration in the Caski cell, and this just illustrates the gene outcome of having made overall length in the recombinant vaccinia cell.

Embodiment 6

The E that obtains from recombinant C OS cell ₇The radioimmunoprecipitation of gene outcome

COS monkey cell is by pCDM8E of the present invention ₇Plasmid transfection, and in culture medium, cultivated 48 hours.According to described like that to the Caski cell in embodiment 5, use then ³⁵S-Met and ³⁵S-Cys carries out labelling to this cell.As at Sato etc., 1989, described in the Virology 170:311-315, cell is separated nucleation, Cytoplasm and cell membrane component.

As described in the example 5,, protein is discharged, and relatively protein is analyzed with 17.5% SDS polypropylene amidine gel and prestained standard molecular weight labelling by ebuillition of heated in sample buffer 5 minutes.Gel autoradiography rings photo and has confirmed using anti-E ₇Rabbit serum carries out the bands of a spectrum that there is about 18KDa in post precipitation.This result is illustrated among Fig. 6.

Embodiment 7

The Western engram analysis

E ₆And E ₇It is as described in the 1988.J.Virol.62:2115-2123 such as Jenison that the Western of peptide immunogenicity inhales trace research, adopts by being attached to HPV16E ₆Or E ₇The fusion rotein that the TrpE of ORF section forms carries out.

Briefly, fusion rotein is utilized its insoluble relatively in nonionic detergent and carries out part and purify.50 milliliters of bacterial culturess that induce are made into piller, are suspended in 10 milliliters of Tris-EDTA buffer (50mM Tris(pH8.0)-5mM EDTA) in and with lysozyme (2 mg/ml) 0～4 ℃ of down digestion 90 minutes.

Add NaCl(5.0M respectively) and 10%ND-40 to the final concentration of 0.3M and 0.7%, and mixture kept 30 minutes down at 0-4 ℃.Allow the solution three times pin by No. 18 standard units reducing its viscosity, and kept other 30 minutes down at 0-4 ℃.

Insoluble component is 16, and 000xg, 4 ℃ were made into the piller sheet in centrifugal 10 minutes down, are suspended in 10 milliliters 10mM Tris(pH8.0)-1.0M NaCl in, and kept 10 minutes down at 0 ° to 4 ℃.Such as mentioned above, insoluble component is made the piller sheet, be suspended in (Leammli in 1.0 milliliters the Laemmli protein example buffer, 1970, Nature 227:680) (the TRIS pH6.8 of 10 milliliters of 0.625M, 20 milliliters of 10%SDS, 20 milliliters of glycerol, 2 milliliters 2 mercapto ethanol, 1 milliliter 1.5% bromophenol orchid (Bromophenol Blue) (in 70% ethanol reagent, preparing), 1 milliliter 1.0% Pyronin Y(Biorad Catalog No.161-0425, or equivalent are at H ₂Prepare among the O) and 36 milliliters deionized water) and be heated to 100 ℃ 5 minutes.

The fusion rotein goods are by separated with 10% the polyacrylamide gel electrophoresis that comprises 0.1% sodium lauryl sulphate (SDS).Fusion rotein utilizes the macroscopy of the blue stained gel of Coomassie to carry out quantitatively.The volume of each quantitative insoluble protein component that every 6mm groove Sheng is carried is adjusted to and provides the blue staining power of the Coomassie that is equal to 5 microgram bovine serum albumin.With protein 100 milliamperes, 16-18 hour, pass in the nitrocellulose in 25mM Tris-195mM glycosides propylhomoserin (pH8.5)-20% ethanol (Western transmits buffer).Trace is soaked in the phosphate buffer that contains 10mM N-ethyl maleimide 10 minutes, degreaser drying Emulsion 5%, 0.9% NaCl, 0.1% Antifoam-A(Sigma chemical company, the St. Louis, Mo.), insulation is 2 hours in 0.1% Hydrazoic acid,sodium salt and the potassium iodide of 1mM (inhale seal agent), and is incubated 1 hour in the trace agent (blotto) that contains 10% hyclone.

The bacterial cultures preparation that is induced by 50ml suppresses reagent, and this culture is made into piller, is suspended in the Tris(pH 8.5 of 3.6 milliliters of 50mM), carry out 20 seconds of supersound process among the 5mM EDTA and with 50 watts of power; Add 20% SDS, with lysate be heated to 100 ℃ 5 minutes.The reagent of this being called as " Blocko " is stored with the aliquot form under (20 ℃).In order to allow the TrpE protein example that the Western trace is played the antigenic effect of negative control, with molten born of the same parents, only comprise carrier (path 10) bring out bacterial cultures as being made into piller to form " Blocko " as described in closing.This cellular lysate is mixed with 1: 1 with 2X Laemmli sample buffer, is heated to boiling 5 minutes, and is added on the gel to check the Trp control protein.

Be that system suppresses reagent, with mixture (" Blocko ") in the trace agent with 1: 20 dilution proportion, add Np40 and NaTDC to respectively doing for oneself 1% ultimate density.With rabbit serum in 25 milliliters inhibition reagent with 1: 100 dilution proportion, and 4 ℃ of following pre-incubations 8 hours.Add the nitrocellulose trace then, be incubated 16-18 hour continuously down at 4 ℃.With trace at 0.5% desalination cholate, 0.1M NaCl, 0.5%Triton X-100, and clean each 20 minutes three times in the 10mM sodium phosphate (pH7.5).(the sub-serum of goat antirabbit IN) joins in the agent of suction seal with 1: 1000 dilution ratio the combined alkali acid phosphate for Boehringer Mannheim Biochemicals, Indianapolis.Under about 27 ℃, be incubated 2 hours after, filtrate is cleaned three times once more, and transfers in the matrix solution (table 1) 10 minutes or till producing color.With filtrate in distilled water, clean finish the reaction.Filtrate is dried and takes a picture.

Table 1

Western Blot Reagents

2 liters of 10 times of transferring buffered doses of taking advantage of

Trometamol alkali (25mM Tris) 60.53 grams

Glycine 288.27 grams

Be diluted to 1 times and add MeOH to 20% of final volume after taking advantage of

4 liters 8 liters of trace agent (bovine lacto transfer technique optimizer)

Defatted milk powder (5%) 200 gram 400 grams

NaCl(0.9%) 36 grams, 72 grams

Antifoaming agent A(0.1%) 12ml 24ml

Hydrazoic acid,sodium salt (0.1%) 4 gram 8 grams

Potassium iodide (1mM) 0.664 gram 1.33 grams

4 liters 8 liters of Western cleanout fluid

Deoxycholic acid (0.5%) 20 gram 40 grams

Triton X-100（0.5%） 20ml 40ml

NaCl(0.1M) 23.4 grams, 46.8 grams

1M sodium phosphate, pH7.4(10mM) 40ml 80ml

(1M sodium phosphate-268 gram Na ₂HPO ₄7H ₂The O[heating for dissolving], about 4ml 85%

H ₃PH4 to pH7.4, capacity to 1 liter)

Alkali phosphatase substrate

Enough 2 traces are used. mix after adding every kind of reagent

Lucifuge; Use in 1 hour

Alkali phosphatase buffer 10ml.

NBT substrate (50 milligrams/ml. is at 70%N, in the dinethylformamide) 66 μ l

BCIP substrate (50 milligrams/ml) 33 μ l at 100%N, in the N-methylformamide

The alkali phosphatase buffer

1M Tris，pH 9.5 10ml（100mM Tris-HCl，pH 9.5）

5M NaCl 2ml （100mM NaCl）

1M MgCl ₂0.5ml（5mM MgCl ₂）

Capacity is to 100ml.

NBT substrate nitroblue tetrazolium (Sigma)

BCIP substrate 3-bromo-4-chloro-3-indyl phosphate (Sigma)

Embodiment 8

The HPV peptide

Synthesized corresponding to HPV16E ₆Or E ₇The synthetic peptide quilt in the specific antigen decision zone of nucleoprotein.This specific peptide is listed in the table 2, and with at E as shown in Figure 7 ₆Or E ₇The amino acid residue that the quilt of reading to the carboxyl terminal from amino terminal in the protein sequence has been indicated is corresponding.

Table 2

Peptide E ₆Or E ₇Amino acid position

359 E ₇29-50

360 E ₇70-81

361 E ₇1-10

358 E ₆148-158

358 E ₆119-134

375 E ₆8-20

376 E ₆1-20

Embodiment 9

Utilize the HPV16 peptide to produce monoclonal gram body as immunogen

20 micrograms with the key hole

The bonded peptide 359 of keyhole limpet hemocyanin (KLH) is emulsified in complete FreundShi adjuvant, and uses to mice at subcutaneous or peritoneal injection.After about 3 and 51/2 week, peritoneal injection is passed through in the booster injection agent that is used in the non-complete FreundShi adjuvant.Take out splenocyte after three days, (, supra) itself and AG8 myeloma system are merged referring to Milstein with standard hybrid tumor technology.Utilization is differentiated the existence of specific antibody with the titer plate of the not binding peptide of every coated 500 nanograms in the 0.1M carbonate buffer solution with healthy clone's supernatant in ELISA detects.Be added into after cleaning with the bonded goat anti-mouse IgG of Radix Raphani peroxidase (HRP) of horse, and adopt the standard ELISA method to carry out substrate reactions through three times.Two high activity clones (clone #10 and #14) are selected as further cloning and in the ELISA of peptide 359 its supernatant being carried out titration, and its result is on Fig. 8.Similarly, at E ₆Peptide 358 and 375(data not shown go out) monoclonal antibody be produced out.

Embodiment 10

The immunogenicity research of peptide-KLH

Carry out its intradermal injection with the 100 microgram peptides of the blended KLH of combining of Freund ' s adjuvant fully rabbit is carried out immunity inoculation by adopting.After three weeks, inject animal is carried out the immune strengthening injection by adopting peptide with 50 micrograms of the blended KLH of combining of non-complete Freund ' s adjuvant to carry out intradermal.After the booster injection three days, rabbit was got blood.

Serum is stored down at (20 ℃).People such as the rabbit antiserum of anti-Trp E-HPV fused protein such as Jenison 1988, J.of Virol.62:2115-2123) describedly is produced like that, and by Dr.Denise Galloway(Fred Hutchinson DKFZ, seattle WA) provides.In order to eliminate antibody at E.Coli-coded protein (the TrpE part that comprises this fusion rotein), utilize the inhibition reagent described in the above embodiment 7, the degeneration lysate of the E.coli of serum his-and-hers watches carrier (pATH) sequence is carried out pre-absorption.

Rabbit serum is done a series of dilution, and at T E-E ₆And E ₇The Western trace of fusion rotein reacts in measuring.All anti-peptide serum and homologous fused protein react, the immunogenicity of peptide conjugate of having demonstrate,proved this.Sero-fast titer is shown in the inverse that still shows reactive high dilution in the Western engram analysis with this specific protein at this, the different sero-fast titer of various differences are illustrated in the table 3, and titrating Western trace is found in Fig. 9 and Figure 10.

Table 3

Rabbit anteserum titer ^*

α357（E ₆） ≥3，200 100 ^＊＊

α358（E ₆） 102，400 ≥6，400

α359（E ₆） ≥109，600 ≥409，000

α360（E ₇） 1，600

α361（E ₇） 800

α16E ₇NP 720，000

α16E ₆DS 40，000-80，000

* show E ₆Or E ₇The inverse of the high dilution of bands of a spectrum positive staining,

Two titer of * represent that two different rabbits are tested.

In these Western traces, anti-peptide 359 antiserums have one 〉=490,600 titer, tool reactivity in measured antiserum.By the diluent of two kinds of serum and the homology Trp fused protein in the Westen trace and Trp contrast antigen are reacted to confirm the specificity of these reactions.This serum be found for homology fused protein (Figure 11) be have specific.In this measurement, sero-fast titer 〉=1000,000 of anti-peptide 359.

At TrpE-E ₆Fused protein, p16E ₆DS-2(Dra III (111)-Sau3a(525)) and E ₇Fusion rotein (p16, E ₇NPI(NsiI(562)-PstI(875) positive control rabbit anteserum (α 16E) ₇NP and α 16E ₆DS) prepared respectively.And by D.Galloway(Fred Hutchinson Cancer Research Center, Seattle WA) provides.

Embodiment 11

Utilize the epitope of ELISA identification B cell

As described in the embodiment 10, in conjunction with E ₆And E ₇Whether the KLH of peptide is used to during KLISA measures, represent by by the HPV16E of bacterial expression to determine them ₆Or HPV16E ₇The antigenic determinant that animal discerned of fused protein immunoprophylaxis.

At three E described in the embodiment 8 ₇Peptide is by anti-Trp-E-E ₇(α 16E ₇NP) the rabbit antiserum of (by D.Galloway, Fred Hutchinson DKFZ, seattle, WA provides) is discerned, as sees Table 4.The HPV16E that identification is bought from Triton Biosciences ₇Monoclonal antibody and aminoterminal peptide 361 mapping one by one.

Table 4

The test antiserum is to E in ELISA ₇The titer of the KLH-binding peptide of open framework ^*

Peptide α 16E ₇NP McAb Triton

（Biascience）

359（aa29-50） 1，600 ＜100

360（aa70-81） 6，400 ＜100

361（aa1-10） 12，800 ≥12，800

* titer is confirmed as demonstrating the dilution inverse of highest serum than the high 4 times of ELISA values of background, and background is to determine at the normal serum that is coated with on the titer plate of same peptide with 1: 100.

E ₆Two kinds of peptides of sequence (357 and 358) also utilize similar approach research.With two kinds of different anti--E ₆Sero-fast result is illustrated in the table 5.Peptide numbering 358 and α HPV-E ₆(Lowy) still responding property under 1: 400 the serum dilution, and α 16E ₆Ds has lower reactivity (1: 100).Peptide 357 is under 1: 100 serum dilution, by α 16E ₆DS identifies reluctantly.

Table 5

The test antiserum is to E in ELISA ₆The titer of the KLH-binding peptide of open framework ^*

Peptide α 16E ₆DS α HPV16-E6(Lowy)

357（aa148-158） 100 ＜100

358（aa119-134） 100 400

* titer is confirmed as showing the inverse of the high dilution of the ELISA value bigger 3 times than background, and background is determined as table 4.

Embodiment 12

Utilize the natural E of anti-peptide antiserum identification ₇Protein

As described in embodiment 8, according to the method for embodiment 10 and as measuring, use E with RIP ₇Peptide carries out immunity to rabbit.The presentation of results that Figure 12 represents utilize anti--E ₇The peptide antiserum is to E ₇The identification of peptide.The antiserum of the rabbit of peptide 389 immunity inoculations of using by oneself is corresponding to E ₇29 to 50-amino acid residues, as radioimmunoprecipitation is determined to the natural E in the cowpox reorganization lysate ₇Protein is discerned.

Embodiment 13

The reverse transcriptase-polymerase chain reaction analysis of RNA

With as pCDM8/E described in the above embodiment 3 ₇The M of transfection ₂Mus melanoma cells and NCTC2555 fibroblast 24 clones separately are utilized RNA point suction seal measurement method and detect.Wherein providing among these clones of positive signal three utilizes reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze further to detect then.

The cytoplasm rna of these transfectional cells according to as at Ausubel etc., 1989, in Current Protocol in Molecular Biology, the described method of Greene Publishing Associates is separated.

Typical primer extension-RT of synthetic first gang of cDNA of cytoplasm rna is used to amplify cDNA with PCR.The cytoplasmic DNA of 1 microgram is used as the template of amplifying reaction.Utilize synthetic first gang of cDNA of reverse transcriptase (Life Science) of murine leukemia virus.

The RT buffer that comprises degeneration RNA sample, random six aggressiveness of 1 microgram degeneration, every kind of Deoxydization nucleotide triphosphate of 1mM (dNTP), the 10mM tetrasodium pyrophosphate, the 5mM dithiothreitol, DTT, the ribonuclease of 10 units (RNasin) (Promega, Madison, WI) and the murine leukemia reverse transcriptase of 18 units under 42 ℃, be held 1 hour, and then 100 ℃ of following degeneration 10 minutes.Supernatant is used to PCR.

The primer that is used for the oligonucleotide of PCR is HPVA22: 5 '-GCATGGAGATACACCTACATTG-3 ' and HPVA20: 5 '-TGGTTTCTGAACAGATGG-3 ' (DNA Factory, San Diego, CA).The cDNA fragment of 292bp is exaggerated.PCR reactant mixture (GeneAmp DNA amplification Reagent Kit, Perkin Elmer Cetus, Norwalk CT) comprises the primer HPVA22 of dNTP1 μ M of 200 μ M and HPVA20 and by the Taq polymerase of the synthetic various cDNA of RT and 25 units.The pCDM8/E of 1 nanogram ₇Plasmid is used in the PCR platform as anti-positive control.Utilize DNA thermocirculator (perkin Elmer Cetus) to carry out PCR(94 ℃ of following degeneration 1 minute with 33 circulations, annealed 2 minutes down and extended 3 minutes down at 50 ℃) at 70 ℃, and with the PCR product of 20 microlitres by at 4% NuSieve agarose gel (FMC with ethidium bromide staining, biological product, Rockland ME) goes up electrophoresis and carries out fractional distillation.

At presentation of results shown in Figure 13 from three kinds of transfection thing (E ₇C3, N7.2 and N7.4) the PCR product shown the dna fragmentation of the 292bp of prediction.Yet parental cell system (melanoma and NCTC2555 fibroblast) does not show these fragments.

When from the isolated mRNA of these transfection things before RT-PCR during with the free RNase pretreatment of DNase-, the PCR product does not demonstrate these fragments by gel electrophoresis therapy determining.

Embodiment 14

Tumor promotion and disappearing

In the 6-10 female C3H/HeN mice (Charles River Breeding Laboratory) in age in week, studied the ability of carrying out the prevention tumor development of immunity with the HPV transfectional cell.

To take from and express HPV-E ₆Or E ₇In antigenic determinant and the antigenic non-oncogenic transfection strain of expression high-caliber main histocompatibility complex (MHC) I level each group mice body by one group of peritoneal injection to 5 mice.Then, mice is by the HPV16E that uses at the hypodermic tumorigenesis dosage in the back, right side of its fine hair of pruning ₇(E ₇C3) M of transfection ₂The melanoma cells deexcitation.Determine tumor mass by measurement to diameter of tumor.Result shown in Figure 14 shows, with expressing E ₇E is passed through in the immunity that the fibroblast of antigenic determinant carries out ₇C3 and cause the of short duration development of tumor, and then disappearing of tumor always.On the other hand, with expressing HPV16E ₆Fibroblastic inoculation of antigenic determinant does not suppress by E ₇The tumor that the C3 cell causes forms, and immune mouse is not being used by E ₇When C3 excites, always generate primary tumo(u)r.

E is promptly used in similar research ₆Or E ₇Transfection strain fibroblast is given injection in the mice abdomen, uses E then ₆The M of transfection ₂Cell activation has produced similar result; Use E ₆Transfection strain fibroblast stops owing to use E ₆The M of transfection ₂Cell forms rather than uses E ₇The M of transfection ₂Plastidogenetic tumor.The result is illustrated in the table 6.

Table 6

Give the inoculum of mice:

The growth of fibroblast melanoma cells tumor progression

No M ₂Be

No E ₇C3 is

No M ₂/ E ₆Be

The nothing of non-transfection is not

The E of non-transfection ₇C3 is

E ₆Do not have not

E ₆M ₂Be

E ₆M ₂/ E ₆Not

E ₆E ₇C3 is

E ₇Do not have not

E ₇M ₂Be

E ₇E ₇C3 is not

E ₇M2/E ₆Be

Use E ₇The C3 cell is that the C3H/HeN injected in mice has produced tumor in the mice body.When using interferon for these mices simultaneously, tumor has taken place disappeared rapidly; Yet, when with the M of non-transfection ₂Cell is given injected in mice together, and interferon does not suppress tumor development.

Studies show that in nude mice for tumor regression takes place, needs functional T cell.Because inoculation M transfection or non-transfection ₂Cell has produced tumor in nude mouse.The result of this research is shown in the table 7, and whether the existence of interferon can not exert an influence to the development of nude mice in-vivo tumour.

Table 7

The growth of inoculum mice tumor progression

M ₂C ₃H/HeN is

M ₂+ interferon C ₃H/HeN is

E ₇C ₃C ₃H/HeN is

E ₇C ₃+ interferon C ₃H/HeN is not

M ₂Nude mice is

E ₇C ₃Nude mice is

E ₇C ₃+ interferon nude mice is

M ₂+ interferon nude mice is

Embodiment 15

CD8 ⁺The T lymphocyte is to the intermediation of tumor regression

Use by HPV16E ₇(N7.2) the deutero-non-tumorigenesis fibroblast of the NCTC2555 of transfection carries out immunity inoculation to the C3H/HeN mice.Then, inject the abdomen liquid of 1.0 milligrams the PBS-dilution that contains 1 milligram of anti--CD8 monoclonal antibody (from the clone 116-13.1 IgG2A of ATCC) separately to eliminate CD8 to these mices ⁺The T lymphocyte.Control group mice accepts to comprise anti--CD5 monoclonal antibody (the hybrid tumor 10.2IgG of homotype coupling _2a, oncogenes) the abdomen liquid of 1 milliliter PBS-dilution.

The result is illustrated among Figure 15.When using E ₇During the C3 cell activation, the mice that anti--CD8 monoclonal antibody that the N7.2 immunity is crossed is handled has produced tumor progression.And using E with the control mice that the anti-CD5 monoclonal antibody that the N7.2 immunity is crossed is handled ₇Resisted the formation of tumor progression during the C3 cell activation.Facs analysis to mouse lymphocyte quantity is by using anti-CD ⁴Or anti--CD ⁸Splenocyte to these mices dyes, monitor by the analysis of fluorescence activated cell analyzer then and carry out.The result is illustrated among Figure 16.CD8 ⁺The elimination of cell is shown it is greater than 90% in the mice that anti--CD8 handles.Anti--the CD4 of combined with fluorescent element measures at the intravital CD4 of those mices ⁺Lymphocyte, and confirm that the CD4 subclass in these mices does not change.

These results show, CD ⁺, HPV16E ₇-special T lymphocyte is exchanged and is saved M in the N7.4 mice immunized body ₂/ E ₇Disappearing of tumor is important effector lymphocyte.

The description of front and the intention of embodiment are to illustrate the present invention, rather than limit.Under the situation that does not depart from connotation of the present invention and scope, can make a large amount of changes and improvement.

Claims (60)

1, a kind of compositions comprises corresponding essentially in mammalian cell because a kind of immunogenic peptide of the amino acid residue sequence of human papilloma virus infection and the peptide of being expressed.

2, compositions according to claim 1 comprises the reconstitution cell of the group that contains the said peptide of having encoded.

3, compositions according to claim 2, wherein said reconstitution cell are virus.

4, compositions according to claim 3, wherein said virus is vaccinia virus.

5, compositions according to claim 2, wherein said reconstitution cell is selected from epithelial cell, fibroblast and MHC I level positive lymphocyte.

6, compositions according to claim 2, wherein said reconstitution cell is a tumor cell.

7, compositions according to claim 2, wherein said gene code correspond essentially to E ₆The peptide in nucleoprotein zone.

8, compositions according to claim 2, wherein said gene code correspond essentially to E ₇The peptide in nucleoprotein zone.

9, compositions according to claim 1, wherein said peptide comprises E ₆Proteinic antigen decision zone.

10, compositions according to claim 1, wherein said peptide comprises E ₇Proteinic antigen decision zone.

11, compositions according to claim 4 comprises a kind of recombined vaccinia virus, wherein contains a kind of coding and expression human papilloma virus's E ₆The antigen decision zone and the gene of nucleoprotein.

12, compositions according to claim 4 comprises a kind of recombined vaccinia virus, wherein contains a kind of coding and expression human papilloma virus's E ₇The gene in the antigen decision zone of nucleoprotein.

13, compositions according to claim 4 comprises a kind of recombined vaccinia virus, wherein contains the human papilloma virus's of the expression of bringing out immunogenic peptide in mammalian cell a gene region.

14, compositions according to claim 6, wherein said tumor cell comprises a kind of recombinant mammalian cells, the human papilloma virus E that wherein contains coding and expressed an antigen determining area of the immunogenic peptide that is produced by said mammalian cell ₆A zone of gene.

15, according to the said compositions of claim 14, wherein said mammalian cell is the human cell.

16, compositions according to claim 6, wherein said mammalian cell comprises a kind of recombined human cell, wherein contains coding and expresses the human papilloma virus E that is determined the zone by an antigen of the immunogenic peptide that said melanoma cells produced ₇A zone of gene.

17, compositions according to claim 16, wherein said mammalian cell is to be selected from hemocyte, a kind of cell in fibroblast and the epithelial cell.

18, a kind of method for the treatment of the disease that is caused by the human papilloma virus infection comprises:

Give a kind of compositions of patient's administering therapeutic effective dose, comprising a kind of peptide, it correspond essentially to since the human papilloma virus infection and in mammalian cell by the amino acid residue sequence in a zone of the peptide of being expressed, treatment continues one period that is enough to suppress described progression of disease.

19, method according to claim 18, wherein said compositions comprises a kind of reconstitution cell, it contains the human papilloma virus's of the expression of bringing out immunogenic peptide in mammalian cell E ₆A zone of gene.

20, method according to claim 19, wherein said cell is a vaccinia virus.

21, method according to claim 18, wherein said compositions comprises a kind of reconstitution cell, it contains the human papilloma virus's of the expression of bringing out immunogenic peptide in mammalian cell E ₇A zone of gene.

22, method according to claim 21, wherein said cell is a vaccinia virus.

23, method according to claim 18, wherein said disease is the cervix uteri wart.

24, method according to claim 18, wherein said disease is a cervical cancer.

25, method according to claim 18, wherein said method suppresses by the propagation of human papilloma virus infection's cell.

26, a kind ofly after detecting the human papilloma virus infection, suppress tumorigenic prevention method in the cell, comprising:

Give a kind of compositions of patient's administering therapeutic effective dose; comprising a kind of peptide, it corresponds essentially to the amino acid residue sequence of the peptide that is brought out by the human papilloma virus infection to excite a kind of tumorigenic protective response that suppresses said virus infected cell in patient's body in mammalian cell.

27, method according to claim 26, wherein said compositions comprise a kind of reconstitution cell that contains the gene of the said peptide of having encoded.

28, method according to claim 27, wherein said reconstitution cell is a vaccinia virus.

29, method according to claim 27, wherein said reconstitution cell is to be selected from epithelial cell, a kind of cell in fibroblast and the MHCI level positive lymphocyte.

30, method according to claim 26, wherein said peptide comprises human papilloma virus E ₆An antigenic determinant of nucleoprotein.

31, method according to claim 26, wherein said peptide comprises human papilloma virus E ₇An antigenic determinant of nucleoprotein

32, method according to claim 26, wherein said peptide comprise a kind of mammiferous peptide that is brought out by a zone of human papilloma virus's gene.

33, a kind of reconstitution cell includes the gene of the peptide of having encoded, and this peptide corresponds essentially to because human papilloma virus infection and cause the amino acid residue sequence that peptide is expressed in mammalian cell.

34, reconstitution cell according to claim 33, wherein said peptide determines area format by said cellular expression with a kind of antigen.

35, reconstitution cell according to claim 33, wherein said cell is a vaccinia virus.

36, reconstitution cell according to claim 33, wherein said cell is an epithelial cell.

37, reconstitution cell according to claim 33, wherein said cell is a tumor cell.

38, a kind of peptide comprises about 8-30 the amino acid residue sequence in a zone that corresponds essentially to protein human papilloma virus.

39, according to the said peptide of claim 38, wherein said zone is said proteinic antigen decision zone.

40, according to the described peptide of claim 38, wherein said protein is the E of HPV16 ₇Protein.

41, according to the described peptide of claim 40, corresponding to E at Fig. 7 ₇Amino acid residue sequence in the aminoacid sequence from about residue 1 to residue 10.

42, according to the described peptide of claim 40, corresponding to E at Fig. 7 ₇Amino acid residue sequence in the aminoacid sequence from about residue 29 to residue 50.

43, according to the described peptide of claim 40, corresponding to E at Fig. 7 ₇Amino acid residue sequence in the aminoacid sequence from about residue 78 to residue 81.

44, according to the described peptide of claim 40, wherein said protein is the E of HPV16 ₆Protein.

45, according to the described peptide of claim 44, corresponding to E at Fig. 7 ₆Amino acid residue sequence in the aminoacid sequence from about residue 1 to residue 20.

46, according to the described peptide of claim 44, corresponding to E at Fig. 7 ₆Amino acid residue sequence in the aminoacid sequence from about residue 8 to residue 20.

47, according to the described peptide of claim 44, corresponding to E at Fig. 7 ₆Amino acid residue sequence in the aminoacid sequence from about residue 119 to residue 134.

48, according to the described peptide of claim 44, corresponding to E at Fig. 7 ₆Amino acid residue sequence in the aminoacid sequence from about residue 148 to residue 158.

49, a kind of method that suppresses the intravital human papilloma virus infection of patient, comprising:

Cause immune composition so that in said patient's body, excite protective immune response effectively to what the patient used q.s at the infection that causes by the human papilloma virus; this causes immune composition and comprises the reconstitution cell that contains the peptide of having encoded, this peptide basically with since the human papilloma virus infection and in mammalian cell by the amino acid residue sequence in a zone of the peptide that brought out, said peptide or both in conjunction with corresponding.

50, according to the said method of claim 49, wherein said reconstitution cell is a kind of virus.

51, according to the said method of claim 50, wherein said virus is a kind of vaccinia virus.

52, according to the said method of claim 50, wherein said reconstitution cell is selected from epithelial cell, a kind of cell in fibroblast and the MHCI level positive lymphocyte.

53, according to the described method of claim 49, wherein said peptide comprises human papilloma virus E ₆An antigen decision zone of nucleoprotein.

54, according to the described method of claim 49, wherein said peptide comprises human papilloma virus E ₇An antigen decision zone of nucleoprotein.

55, a kind of antibody molecule, it can with a kind of receptor of fighting for said peptide of being expressed because HPV infects.

56, according to the described antibody of claim 56, wherein said peptide comprises the E of HPV ₇A proteinic zone.

57, according to the described antibody of claim 56, wherein said antibody molecule is a kind of anti--idiotype antibody at said peptide.

58, according to the described antibody of claim 57, wherein said peptide is corresponding to the E at Fig. 7 ₇Amino acid residue sequence from about residue 29 to about residue 50 in the aminoacid sequence.

59, according to the described antibody of claim 55, wherein said peptide comprises the E of HPV ₆A proteinic zone.

60, according to the described antibody of claim 59, wherein said antibody molecule is a kind of anti--idiotype antibody at said peptide.

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