CN1054613A - A kind of in prevention and treatment human body retrovirus as antiviral agent and immunogenic nonreplicative recombinant retrovirus particle - Google Patents

Abstract

The inventive method comprises the recombinant vaccine virus and the recombinant vaccine virus co-infected mammalian host cell that has the HIV-2env gene with band HIV (human immunodeficiency virus) I type (HIV-1) gag and proteinase gene, prepares the HIV-1 particle of non-replicability reorganization preparation.The HIV-1 particle that these and natural HIV-1 have the non-replicability reorganization preparation of very approaching immunology and morphological feature can have the immunogenicity of height in vivo in the infection of external prevention HIV alive.The HIV-1 particle of reorganization preparation can be used as the immunogen in antiviral agent and the vaccine product, suppresses effectively or the infection of prevention HIV and/or the development of acquired immune deficiency syndrome (AIDS).

Description

A kind of in prevention and treatment human body retrovirus as antiviral agent and immunogenic nonreplicative recombinant retrovirus particle

1. preamble

The present invention relates to the reversal of viral particle of non-infectious reorganization preparation, with produce this particulate vitro system, and in the prevention of anti-human body retrovirus such as HIV (human immunodeficiency virus) (HIV) and therapeutic process as antiviral agent and immunogenic purposes.The HIV particle of reorganization of the present invention preparation is combined with the HIV core and the glutelin of correct processing, and on the morphology and closely similar with natural HIV on the immunology.And because Recombinant HIV particle of the present invention does not contain the genomic whole compositions of the necessary HIV of virus replication, so these virions are noninfectious.

2. background of invention

Identified two types human body retrovirus, leukosis virus and acquired immune deficiency syndrome (AIDS) (AIDS) virus or acquired immune deficiency syndrome (AIDS) dependency virus.The retroviral main target of human body is T lymphocyte and central nervous system cell.All human body retrovirus all are to be undertaken infectious by the infection of breast-feeding after close contact, blood contamination and intrauterine infection or the birth.Seeming whole human retrovirus originates from Africa, and might by kind between infect, may meet with the mankind by the green ape in Africa or relevant kind.The human retrovirus of Fa Xianing first, T lymphocytes in human body affinity I C-type virus C (HTLV-I) and T lymphocytes in human body affinity II C-type virus C (HTLV-II), T4 cell and some T8 cell had a kind of deflection affinity, and it is have tangible sequence homology, and main relevant with T chronic myeloid leukemia and lymphoma.To another human body retrovirus, generally be referred to as HIV (human immunodeficiency virus) (HIV) and carry out more detailed discussion below.Two main differences are arranged between this human body retrovirus of two types: (1) has very big genome mutation in various HIV choristas, and the genome of HTLV-I and HTLV-II virus is stable; (2) HTV that enters recently among the crowd is more much more than HTLV-I and HTLV-II virus.

2.1 human immunodeficiency virus and acquired immune deficiency syndrome (AIDS)

Human immunodeficiency viruses (HIV) is a kind of retrovirus that causes cytopathy, and is the paathogenic factor of acquired immune deficiency syndrome (AIDS) (AIDS).Identified two types HIV.Prototype virus HIV-I was called as the paathogenic factor of most of acquired immune deficiency syndrome (AIDS) (AIDS) case that Lymphadenopathy-associated virus (LAV) and T lymphocytes in human body affinity III C-type virus C (HTLV-III) be in the world to be reported in the past.Another kind of retrovirus HIV-2 is initial to be isolated from the aids patient in western Africa, and has pathogenic dependency with HIV-1.On the genetics level, HIV-2 in fact with simian immunodeficiency defective virus (SIV), a kind of retrovirus that infects monkey, more closely related.

To on May 31st, 1989, only 97,000 many cases AIDS patients have been reported, the death over half among these patients in the U.S..It may be the asymptomatic carrier of HIV virus that 3,000,000 more than number is arranged in this country, and can propagate this virus.Estimate that the U.S. in 1991 has 270,000 routine acquired immune deficiency syndrome (AIDS) patients and (U.S. public health service portion, 1986, Public Health Rep.101:341) occur.The mortality ratio of acquired immune deficiency syndrome (AIDS) is alarming height, and it is dead within back 3 years of diagnosis surpassing 80%, and may reach 100% in during more longer.

Worldwide, the popular of acquired immune deficiency syndrome (AIDS) may comprise about five to 10,000,000 present infected patients, and especially alarming is resulting statistical information from the African continent, and wherein millions of people is considered to infect HIV, dead scope is at hundreds of thousands of, and heterosexual transmission is preponderated.Up to the present, both there be not a kind of known treating AIDS method, the vaccine that does not yet have a kind of anti-effectively HIV to infect.

2.2 the pathogenesis of HIV virus infection

HIV virus is retroviral non-conversion, a member of cytopathy slow virus (Lentivi-rus) section.HIV can cause a kind of disease that typically causes, and its feature shows as serious immune deficiency or neurodegenerative disease, or the two all has.The immunosuppressant main basis of HIV inductive is to express CD4 molecule (T4 or CD4 ⁺Cell) the lymphocytic assistant agent of T/inductor subunit exhausts, and it is the high-affinity cell surface receptor of virus.The T4 lymphocyte is each immunologic function almost in the inductor directly or indirectly, and its disappearance can cause the pathogenic infection of extensive chance and the susceptibility of tumour.

Except the T4 lymphocyte, some other cell of expressing the CD4 molecule also is target, the especially monocyte-scavenger cell of HIV infection and some neurone and the neurogliocyte of brain.The HIV virus infection also can cause serious B cellular abnormality, comprises that many cells system activates, hypergammaglobulinemia, and the circulating immune complex level raises, and autoantibody.Quantity by discovery feature natural killer cell (NK) in the acquired immune deficiency syndrome (AIDS) patient body reduces.

CD4 ⁺The infection of cell is caused by the interaction of CD4 molecule with a large amount of HIV chitose albumen gp120, then then take place virus particle internalization and shell, ThermoScript II by encoding viral is transcribed into DNA with geneome RNA, and the proviral DNA that obtains is incorporated among the host cell chromosome DNA, and the proviral DNA of Zheng Heing can not put aside in the cell that infects in a large number, may be obvious paathogenic factor (the Shaw et al. of HIV cytopathy, 1984, Seience 226:1165).In reproduction process, the mRNA transcript of the proviral DNA of integration is translated into the HIV viral protein.These albumen are processed then, make it to assemble with the HIV geneome RNA, and the mature virion that grows in the T lymphocytic cell surface that infects and the scavenger cell combines with the host cell membrane lipoid and forms viral shell.

Though HIV can keep dormancy at certain hour after infecting,, when the active replication of virus occurring, host CD4 ⁺Cell generally can be killed.Although proposed several mechanism (as, do not integrate in a large number the savings of viral DNA in cells infected; When a large amount of viruses permeability of cell membrane when cell surface grows increases; Infer that HIV can cause the end differentiation of the T4 cell of infection, causes the lost of life), but producing its accurate mechanism that causes cytopathic effect by HIV does not still know.Show that gradually CD4 molecule and viral shell causing in the HIV cells infected all plays certain effect in the cytopathic effect.Outstanding feature is exactly forming of the multinuclear syncytium that seemingly caused by the gp120/gp41 glutelin in the cytopathology that HIV infects.On the contrary, the scavenger cell of HIV-infection can continue to prepare HIV virus and do not have cytopathic effect in long-time.

Have to think that monocyte and scavenger cell play main effect in the pathogenesis that HIV infects.Except by phagolysis with the virus of eating, the CD4 surface antigen can be expressed by some subunit of monocyte-scavenger cell, thereby and can be attached on the HIV shell.Monocyte-scavenger cell is the main cell type that infects in the brain, and relevant with the neuropsychiatry phenomenon that the HIV infection takes place.And, in the patient who infects, generally can observe the functional defect of monocyte-scavenger cell.These defectives can produce the distinctive infection chance of acquired immune deficiency syndrome (AIDS) patient.

It is significant that HIV can survive with dormant state in monocyte-scavenger cell.Perhaps be that the monocyte of infection does not show the cytolysis effect that HIV is had the T4 cell because the density of cd4 cell surface receptor is lower.Thereby monocyte to can be used as HIV storage main, as much as possible virus is transferred to brain, each organ in central nervous system and the body.The virus that is present in the monocyte may be passed through hemato encephalic barrier, influences monkines, and the release of enzyme and CF causes neuronic destruction or damage and brain tissue inflammation (Ho et al., 1987, N.Engl.J.Med.311:278; Fauci, 1988, Science 239:617).In the various neurological syndromess that directly originate from the HIV infection, the most subacute encephalitis or aids dementia (almost 90% AIDS patient), its Clinical symptoms comprises dementia, psychomotor retardation, and behavior change.

2.3 the morphology of HIV and genome difference

Fine structure (Gelderblom et al., 1988, the Micron and Microscopia 19:41 of HIV virus have been determined by immunological method and electron microscopic analysis technology; Gelderblom et al., 1987, Virology 156:171).Between HIV-1 and HIV-2 strain, do not identify morphology difference (Gelderblom et al., 1988, Micron and Microscopia 19:41).The HIV virus particle is the spheroidal particle of a kind of diameter about 100 to 120nm, and contain an electron density, P24, the tube core that gag albumen is formed, matrix under a kind of film of forming by gag P17, and a kind of by being dispersed in the shell that glutelin gp120 between the lipoids two membranes and gp41 form.The HIV geneome RNA is present in the part of core as nucleoglucoprotein (RNP) mixture, and this mixture is combined with ThermoScript II molecule (this enzyme catalysis rna transcription becomes proviral DNA) and nucleoprotein.The capsid protein gp120 and the gp41 that obtain by proteolysis cutting from precursor gp160 are embedded in the middle of the film in conjunction with mixture as non-covalent.As if observe these glutelin mixtures, be revealed as and have the about 14nm of maximum diameter, the sphere of height 9-10nm is outstanding, and arrange with the symmetric icosahedral structure of virus form in T=7 left side.Should outstanding contain gp120, loosely be connected on its transmembrane gp41 ancora.Shell gp120 automatically comes off from virus surface to a great extent, and this phenomenon can influence the pathogenesis of HIV disease.Virus maturation can take place after germination process neutralization has just finished.After the surface from cells infected grows bud, from precursor cutting HIV nucleoprotein, become the mature structure albumen that tissue forms this nuclear structure by a kind of HIV-encoded protein enzyme.

The HIV viral genome contains the gene of three these virus particle primary structure compositions of coding: the env(capsid protein of encode), the gag(nucleoprotein of encoding) and the pol(ThermoScript II of encoding, proteolytic enzyme, and endonuclease).These three gene both sides are the Nucleotide extensions that are known as the long end section of duplicating (LTR), and these LTR contain the sequence that has certain effect in the control process of viral gene expression.But, not resembling other retroviruss, the genome of HIV comprises at least 6 other genes, wherein three have known regulatory function.The expression of these regulatory gene is considered to that the HIV pathogenesis is had certain influence.The tat genes encoding a kind of protein, this protein has strong activator function for the HIV expression of gene, thereby plays an important role in the amplification of virus replication.This rev gene product is regulated the connection of HIV mRNA and is passed on.On the contrary, the nef gene is then down regulated the expression of virus.The vif gene forms not absolute demand for virus particle, but for producing infective particle effectively and being crucial at the external viral communication that influences.The Vpr genes encoding a kind of immunogene albumen of unknown function.At last, the nearest Vpu open reading frame of reporting a kind of protein that participates in regulating virus maturation and cause cytopathic effect of having encoded.

Obtained the many different isolates of HIV, and determined their nucleotide sequence, having shown has genome difference significantly in the env gene.Env gene region point with obvious branching characteristic stops and is stored in the central zone of various different isolates.Estimation is as whole HIV isolates are attached on the cd4 cell surface receptor molecule, and a kind of zone of preservation like this is the CD4 land.Isolate relevant still distinguished HIV-1 variant on one's body from the individual AIDS patient who infects for some time, wherein some has the antigen polytropy.These isolates have nothing in common with each other for the tropism of particular cell types.As if about this respect, some isolate inclines each at CD4 ⁺T cell or duplicate in the scavenger cell that brain produces shows that the HIV virus infection produces various clinical manifestation because of pathogenesis optionally.

2.4 vaccine prospect

The tradition research of virus vaccines comprises with the form of attenuation form of living or deactivation preparation uses complete virus particle.These researchs successfully have been used for resisting numerous disease such as smallpox, poliomyelitis, measles, mumps, rubella etc.But have problems for the possibility that these researchs is used for the HIV vaccine production always.Except having the actual danger and active deficiency relevant, owing to these traditional processing comprise whole reversal of viral gene is imported in the healthy individual, thereby generation causes disease, for example theoretic danger of acquired immune deficiency syndrome (AIDS) with answer.Therefore, all concentrate on the recombination method at present, no matter be with subunit's form or with the form of vector-viral vaccine at the most of energy aspect the preparation AIDS vaccine.

Research to the HIV target antigen is confined to capsid glycoprotein aspect to a great extent always, on less degree, is confined to cAg.About the information of the immune pathogenic aspect of capsid sugar-protein compound (also being the gp120-gp41 polycomplex, opposite) or capsid-cAg mixture,, also few of even exist with solubility gp120 or gp160.A series of signs show that it may be important having capsid sugar-protein compound and cAg in ad hoc structure simultaneously.At first, studies show that of hepatitis B surface antigen(HBsAg), as the sort of antigenic existence of part grain pattern than soluble antigen more meaningful (Cabral et al., 1978, J.Gen.Virol.38:339).The second, the epitope that certain is given, the immunogenicity matter of (as basic with the surface antigen decision in the genus specificity of adenovirus six adjacent bodies) has the form dependency.The 3rd, because the cAg of HIV is stored in the various isolates relatively, cAg can be inspired immune response to various HIV-1 isolates widely as immunogen.Therefore, the vaccine that design and evaluation combine traditional method and recombination method superiority is significant, that is to say, preserve the vaccine of reorganization preparation of the immunogenicity matter of natural viral particle, all do not have infection activity and other potential shortcomings of whole virus formulation.

Except its preventive use, vaccine can also be used for metainfective immunotherapy.For example, there is the potential result's of breaking out patient to use effective rabies vaccine to rabies virus.Since infect and morbidity between arranged long latent period, the someone proposes immunization method and prevents AIDS for the people who has infected HIV and also have value (Salk, 1987, Nature 327:473-476)

3. summary of the invention

The present invention relates to the retroviral particle of non-replicability reorganization preparation, the vaccine preparation that contains the retroviral particle of non-replicability reorganization preparation, prepare method and the reorganization of non-replicability that the reorganization of non-replicability prepare retroviral particle and prepare the purposes of retroviral particle as antiviral agent.Reorganization of the present invention prepares retroviral particle and contains retrovirus core and capsid protein, and they are assembled into has the structure that is in close proximity to those natural retroviral virus particle at immunology and morphological feature.Main structure difference between the retroviral particle of reorganization preparation of the present invention and the natural retroviral particle is that the former lacks a complete reverse transcription virus gene group.If the reverse transcription virus gene group that has a kind of can control expression the, particularly retrovirus to infect and duplicate necessary range gene product, the recombinate retroviral particle of preparation of the present invention does not then have infectivity fully and can not breed.But, because the counter-transcription-ing virus particle of reorganization preparation structurally is configured to the infectivity counter-transcription-ing virus particle, therefore, they are high immunogenic, and not only can excite a kind of anti-pre-property immunne response to resist specific meaningful retrovirus, but also can stop the infectivity of retrovirus effectively.

Applicant and be used to prepare the non-replicability recombinant Retroviruses of the present invention particulate method and comprise, co expression retrovirus core and capsid structure albumen in mammalian host cell, this host cell can be controlled the maturation of retrovirus core and capsid protein, and promotes them to combine with the sprout particle of correct assembling.Use the various technology of having set up as infecting by live vector and using the dna vector transfection, can finish that these retrovirus cores of coding and the proteic nucleotide sequence of capsid structure are imported in the mammalian host cell.In addition, during the applicant believes that also the nucleotide sequence of coding retrovirus proteolytic enzyme should be imported to mammalian host cell learns, to guarantee that the retrovirus core protein is carried out suitable processing.

Furtherly, the present invention relates to the HIV virion of non-replicability reorganization preparation, the vaccine of anti-HIV (human immunodeficiency virus), prepare non-replicability reorganization preparation the HIV virion method and use the HIV virion of non-replicability reorganization preparation to suppress the purposes that HIV infects and HIV virus patient has been infected in treatment.In the specific embodiments that this paper describes by way of example, recombinant vaccine virus is used as carrier, gag with HIV (human immunodeficiency virus), proteolytic enzyme and capsid gene import in the middle of the mammalian host cell, have the similar particle of HIV-1-that immunology and morphological feature are in close proximity to those natural HIV-1 with control preparation.The HIV-1 particle of these reorganization preparations can stop HIV virus alive in external infectivity, and has the immunogenicity of height in vivo.

4. the simple description of accompanying drawing

Fig. 1. with the proteic radioimmunoprecipitation assay of HIV-1 of expressing in the recombinant vaccine virus infected B SC-40 cell.The individual layer of BSC-40 cell is grown in containing the improved Eagle's medium of Dulbecco ' s (DMEM) of 10% foetal calf serum (FCS) converge (confluency).With v-env5(A and B group), V-env5+v-gag2(C and D group), v-gag2(E and F group) and, perhaps V-NY parental virus (G and H group) infects these cells with the MOI of each viral 10PFU/ cell.In infection back 12 hours, with [ ³⁵S]-methionine(Met) and [ ³⁵S]-halfcystine (100uCi/ml) pair cell radio-labeled 4 hours.Collect substratum and wash cell, collect, then dissolving in RIP damping fluid (1%NP40,0.5% deoxycholate salt contain the PBS of 0.1%SDS) with PBS.Pass through RIP with human body polyclone anti-HIV-1 serum (following 6.1.2 joint), and then in 11.5% acrylamide matrix, carry out fractional separation by SDS-PAGE, to with substratum (B, D, F and H group) and nuclear back cytolysis thing (A, C, E and G group) carries out the HIV-1 protein determination abreast.By radioautography radiolabeled albumen is observed.The molecular weight marker thing is represented with kilodalton (KD).

Fig. 2 infects the cell surface compartmentization of the cell synthetic HIV capsid protein of BSC-40.With the MOI of every kind of viral 10PFU/ cell, use the v-env5 similar to infect or with v-env5 and these cells of v-gag2 co-infected to parental generation V-NY virus.In the time of back 16 hours, extract substratum and washed cell, in infection then by the catalytic reaction 0.5mCi[of lactic acid peroxidase ¹²⁵I] to this cell carry out radio-labeled (Haffar et al., 1987, Mol.Cell.Biol.7:1508).According to described in figure I and the following 6.1.2 joint, carry out the HIV protein determination to examining back cytolysis thing then by RIP and SDS-PAGE.

Fig. 3 contains HIV-1gag and separates with the proteic Recombinant HIV particulate of env.(1) radioautogram: use in the time of back 5 hours in infection [ ³⁵S]-methionine(Met) and [ ³⁵S] halfcystine (60 μ Ci/ml) carried out radio-labeled 10 hours to infected B SC-40 cell as described in Figure 2.Collect the substratum (14ml) under each infectious condition, and made the cell clarification in centrifugal 10 minutes at 600xg.The gained supernatant liquor of collecting 2 milliliters carries out initiator (TS) mensuration.With remaining 12ml in each sample in the SW55Ti whizzer 120, centrifugal 3 hours of 000xg is separated into supernatant liquor (S) behind a particle piller and the particle.With the washing of this piller, resuspending and is covered on the 15% sucrose pad of a 2ml in the middle of PBS.With 120, the rotating speed of 000xg carries out ultracentrifugation made the particulate matter redeposition in 1.5 hours in a kind of SW55Ti whizzer.With solids precipitation piller (P) resuspending of gained in the RIP damping fluid.As described in the following 6.1.2 joint, measure the HIV albumen of P part (B, E and H group) with RIP, and measure 2ml(abreast from total amount 12ml) S part (C, F and I group) and 2ml TS material (A, D and G group.(2) graphic representation: the super centrifugal gained particle piller first time of the BSC-40 culture supernatants of double infection is covered on the continuous sucrose density gradient (15%-60%), and 120,000xg precipitated in centrifugal 1.5 hours in the SW55Ti whizzer.Collect gradient composition with branch forms such as 200 μ l from the gradient end.The material of collecting is divided into two parts, and measures gag p24 content according to the EIA method described in following 6.1.2 joint.The peak value of EIA is partly represented each nanogram(ng) amount of collecting the p24 that measures in the component (real point line), and relevant with sucrose concentration (circle is represented).Western trace response analysis shows that the component (not expressing) at gradient top does not contain any p24.

Thinly-sliced electron microscope of Fig. 4 and immunoelectron microscope are to the Recombinant HIV-1 particulate analysis of assembling.To use precipitation obtains from culture supernatant particle piller (Fig. 3) with v-env5 and each virus of v-gag2(MOI=10PFU/ cell with paralleling) the complete BSC-40 cell of co-infected fixes 20 minutes in 4% Paraformaldehyde 96.Use 0.8% bovine serum albumin then, 0.1% gelatin and 5% contains the PBS flushing sample and the blocking-up of common sheep blood serum.With 6.1.2 joint below MAbs110-4 or the 41-1() join in the middle of the various samples according to described method as ascites fluid (1: 2000 blocking-up damping fluid).3.5 after hour, wash sample, and cultivate, to be used to carry out the EM analysis of step described in the following 6.1.3 joint with golden bonded goat-anti-mouse IgG with PBS.

Nucleic acid content in the HIV-1 particle of the reorganization preparation that the round spot blot hybridization measuring method described in Fig. 5 such as the following 6.2.4 joint is measured.A group: gag-specific probe.B group: env-specific probe.Concentration with ng p24 determination of equivalent reorganization preparation HIV-1 particle and deactivation HIV virus particle.(inactivation of viruses: 1 swimming lane, 2600ng p24; 2 swimming lanes, 260ng p24; 3 swimming lanes, 26ng p24; 4 swimming lanes, 2.6ng p24; Reorganization preparation HIV particle: 1 swimming lane, 300ng p24).C group: be respectively applied for gag-pol gene (258-3317) in the preparation process of recombinant vaccine virus v-gag2 and v-env5 and the coordination line chart between the env gene (5671-8572).Arrow represents to be positioned at gag-pol gene (Lever et al., 1989, the J.Virol.63:4085-4087) site of the RNA packaging sequence (300-319) of upstream.

The structural representation of Fig. 6 plasmid pv-G2E5.

The Western trace reaction of Fig. 7 recombinant vaccine virus cells infected.Infect the BSC-40 cell with the MOI of 5pfu/ cell, and in infection in the time of back 24 hours, collecting cell carries out PAGE and analyzes.The cytolysis thing that infects is carried out electrophoresis and electrotransfer to soluble cotton on the 7-15% acrylamide gel.React with HIV+ human serum (Trimar) and immunoblotting, and then react with it with peroxidase bonded sheep-Anti-Human IgG.As substrate trace is developed with 2-chloro-naphthols.According to described method filling gel bands of a spectrum.

Recombinant HIV-1 particulate the radioimmunoprecipitation assay of Fig. 8 to preparing with V-G2E5 infected B SC-40 cell.

The diagram of plasmid vector structure described in 8.1 joints below Fig. 9.

The HIV-1 particulate immunoreactivity of the reorganization preparation that Figure 10 produces in transfection CHO cell.From 3010-C ₆Collect the HIV particle of reorganization preparation in the substratum of cell, by high speed centrifugation it is concentrated, and carry out fractional separation by tape transport in the saccharose gradient of a 15-60%.Top: use the Gag protein content of Gag antigen EIA analysis gradient composition, and analyze sucrose density with a kind of light instrument of analysing.The bottom: the aliquots containig of selected component (indicating with * in top group) is carried out electrophoresis on the component polyacrylamide gel of selecting on the 7-15% gradient, and electrotransfer is detected it with the a-protein of a kind of acquired immune deficiency syndrome (AIDS) patients serum and 125-I mark to a kind of nitrocellulose filter.Also load unseparated virus particulate aliquots containig and isolating HIV virus.

Figure 11 is to the HIV-lenv that encoded, the analysis of the HIV-specific antigens of expressing in the HeLa cell of the plamid vector transfection of tat and rev gene.With every bands of a spectrum of CmHiTgfbEnv5() add the common transfection HeLa cell of tat that every bands of a spectrum top is indicated and rev plasmid [" BS " is a kind of control plasmid that contains " blue line adds (Bluescribe plus) " carrier that coded sequence inserts].In substratum, added zinc before 24 hours at the collection sample, represent with "+Zn ".Whole cytolysis things are carried out electrophoretic analysis on 10% polyacrylamide gel, and electrotransfer is to a kind of nitrocellulose filter, detects with the monoclonal antibody 110-4 of 125-I mark.Also load aliquots containig with vaccinia virus v-env5 infected B SC-40 cell.

The Western engram analysis of Figure 12 recombinant vaccinia virus-infected cell.Infect the BSC-40 cell with the MOI of 10pfu/ cell, and infection in the time of back 23 hours collecting cell carry out PAGE and analyze.The dissolved cells infected is carried out electrophoresis and makes it electrotransfer to soluble cotton on a kind of 8.5% acrylamide gel.React with HIV+ human serum (Trimar) and immunoblotting, and then react with it with peroxidase-conjugated sheep-Anti-Human IgG.As substrate trace is developed with 2-chloro-naphthols.The gel bands of a spectrum by shown in load.

The HIV-1 particle that Figure 13 prepares with recombinating is to the analysis of T-lymphocytoblast class (T-Lymphoblastoid) cell infectivity.Described in following 7.1 joints, perhaps cultivate with the HIV-1 particle (corresponding) of reorganization preparation or with HIV virus (corresponding) and lymphocytoblast (CEM) with 5pg p24 gag with 3ng p24gag.In back 5 days collection of cellular samples from each hole of infection, and according to following 7.1 the joint described in method analyze by HIV antigen in the indirect immunofluorescence pair cell.Coming easily with Evans Blue dyeing (red colouring agent), pair cell positions.A group: the cem cell that the HIV-1 particle for preparing with recombinating is cultivated.B group:, show the fluorescence positive with the cem cell of HIV virus culture.C group: the syncytium of using the cem cell of HIV virus culture.

Figure 14, from (A) reorganization preparation HIV-1 particle, the relative antibody titers of measuring by ELISA with the serum sample of gathering in the New Zealand white rabbit of the HIV-1 immunity of (B) psoralene deactivation.The details of being measured is 14.1 joints of face as follows.

Figure 15, the humoral immunoresponse(HI) in the immune animal.HIV-1 particle (R#238 and R#241) and non-activity HIV-1 virus (R#239 and R#243) with the reorganization preparation are carried out immunity to New Zealand white rabbit.Different time after immune response begins is collected serum sample at interval, and by whole virus (A and B group) or purifying gp120(C and the D group of ELISA in fragmentation) go up and measure the HIV-1 specific antibody.Data (ordinate) be with 2 times cause HIV-1 that the immune serum titre calculates terminal point titre with heterogenetic antibody.The initial immune response of value representative of abscissa begins serum sample is gathered in the back in week time.Arrow is represented (4 all R#241 and 243,5 all R#238 and 239) and (18 all R#241,35 all R#238) immunoreactive time for the second time for the third time.

Figure 16 carries out neutralization reaction with the HIV-1 infectivity of rabbit anteserum and cem cell.In the HIV-1 specificity in the serum sample that mensuration is selected from immune rabbit (A group R#238 and R#241, B group R#239 and R#243) and active.Cultivated 45 minutes down at 37 ℃ with suitable serum and homology virus (BRU isolate) earlier before in joining cell.Under 37 ℃, from cell, remove virus and serum after 1 hour, and replace it with the blood serum medium of suitable dilution.Neutralizing effect is determined in the minimizing of using a kind of EIA to measure cell release p24gag protein content.The p24 level reduces 75% in being indicated and in titre (ordinate) the expression substratum.Abscissa value such as Figure 15 describe.

Figure 17, the antibody reactivity of the individual virus protein of measuring by the Western engram analysis.For used experimental technique with to the detailed description of the discussion of results 14.2.4 joint of face as follows.

Figure 18, with the HeLa cell of CD4 gene transfection earlier with Recombinant HIV-1 particle, and then the same focal argon laser scnning micrograph after cultivating with the HIV specific antibody.To the detailed description of method 15.1 joints of face as follows.

5. detailed description of the present invention

The present invention relates to counter-transcription-ing virus particle in the non-replicability reorganization preparation that is in close proximity to retrovirus virus particle alive aspect immunology, structure and the morphological specificity.The inventive method be applicable to member be similar to any artificial transcribe virus (as: the HTLV-I, the HILV-II, HIV-1, reorganization HIV-2) prepares particle.

The invention particularly relates to the HIV particle of reorganization preparation, as natural HIV, the HIV particle of reorganization preparation contains HIV core and the capsid protein of still preserving anti-HIV seroimmunity reactive behavior through correct processing and assembling.But the HIV particle of reorganization preparation does not contain the HIV genome, thereby is reproducible not.Method of the present invention is described in detail by embodiment, in these embodiments, has used a kind of new vitro system preparation surface to have Recombinant HIV-1 particle of gp120/gp41 capsid protein mixture.Those skilled in the art should be understood that and present invention includes many specific embodiments.And this paper do not describe particularly and illustrate anyly drop on technology within the claim scope of the present invention all within the scope of the invention.

Following some feature proof the present invention of the present invention is a kind of novel method for AIDS vaccine.At first, no matter the HIV particle of reorganization preparation is all to be in close proximity to real HIV virus particle aspect morphology or aspect antigenic characteristic.When using as a kind of immunogen, these particles demonstrations are similar to the antigenicity of antigen manifestation mode in the HIV course of infection, thereby inspire immune response relevant for the natural infection height and that have potential protective capability.Disclosed any recombinant subunit vaccine can not reach this feature of the present invention at present.The second, the inventive method provides a kind of antigen bonded handiness that obtains from the different isolates of HIV-1 and HIV-2 that makes according to recombinant DNA technology, thereby produces definition reaction immunne response the most basic for the effective vaccine of anti-AIDS.Can also use recombinant DNA method to remove or modify and make viral infection or the deleterious surface antigen decision of pathogenic enhanced potential base.The 3rd, Recombinant HIV particle described herein does not have infectivity and does not contain complete HIV genome.Therefore, the immune response of carrying out with these particles can not bring the potential infection risk that interrelates with deactivation or attenuation intact virus vaccine.Below chapters and sections in also can be further described these and other feature of the present invention.

The HIV particle of reorganization preparation of the present invention also can be used as the specific immunity stiffeners.Generation by acquired immune deficiency syndrome (AIDS) in the human body that excites anti-this viral immune response to prevent to infect HIV virus.This concrete feature of the present invention has instructed reinforcement and preserved the immunoprotection factor that has induced in the middle of the seropositivity patient.Dr.Jonas Salk uses deactivation, removes the HIV preparation of capsid protein a kind of similar method is estimated.

Recombinate other desired use of HIV particulate of preparation of the present invention comprise the application of the antiviral agent that they are infected as a kind of HIV of interference, produce the application of the monoclonal antibody of HIV core and capsid protein antigen, the application of preparation anti-idiotypic antibody and the application of illustrating HIV virus involucrum (encapsidation) process.Recombinant HIV of the present invention-1 virion has antiviral effect (as follows 12 of face and 13 joint), and can excite HIV-specificity humoral and cellullar immunologic response (seeing 14 and 16 joints respectively) in the rabbit of immunity and maeaque monkey body with this particle.

5.1. as anti-HIV (human immunodeficiency virus) vaccine

The HIV particulate preparation of non-replicability recombinant chou preparation

The preparation Recombinant HIV particulate method that the applicant provides is included in the coding of co expression HIV env in the mammalian cell and the structural protein gag coding.Selected cultivation host cell must be able to synthesize and correct processing HIV protein.Can use the method that has been established well known in the prior art, comprise, infect and use the method for dna vector transfection, env and gag gene are imported in the middle of the host cell as vaccine virus and retroviral vector by live vector.In host cell, successfully give expression to after the HIV albumen, can use method for normalizing in the prior art from substratum, to separate the HIV particle of recombinant chou preparation.

In the specific embodiments that describes in further detail among the embodiment of the 6th part below, with two kinds of recombinant vaccine virus (the complete gag gene of a kind of HIV-1 of having and the another kind of complete env gene that has HIV-1) co-infected African green monkey kidney embryo (BSC-40) cell, prepare the HIV particle of reorganization.Recombinant HIV-1 particle that this double infection causes being assembled grows sprout from the BSC-40 cell surface.Biochemical analysis shows that this particle combines sophisticated immunoreactivity gag and env albumen.By electron microscope observation, it is identical with the HIV reality of living that this reorganization prepares the particulate form.

In a relevant embodiment, introduce a kind of utilization virus vector and produced the another kind of system of recombinant retrovirus particulate of the present invention, in this embodiment, the single recombinant vaccine virus that use contains the env of HIV-1 and gag gene is used for transfection mammalian cell, produces the HIV-1 particle (the 7th of face the joint as follows) of reorganization preparation then

In another embodiment, use this system of DNA transfection mammalian cell of these retrovirus structural protein of having encoded to produce the retroviral particle of reorganization.As giving an example of this specific embodiments, the 8th part has below been done detailed description further.With two kinds of plasmid carrier transfection CHO cells of encoded respectively HIV-1 gag and HIV-lenv group, this then cell controls then that to be mounted to Recombinant HIV-1gag and env antigenic synthetic.Other schemes relevant with present embodiment comprise, but be not limited to the compound plasmid vector that contains a plurality of HIV genes and carry out transfection, use basic and adjustable enhancers/promoters original paper control HIV expression of gene, combine with the HIV expression of structural gene and its HIV that controls is regulated protein expression, the use that the use of various clone and coding are modified the proteic plasmid vector of HIV.

For example, described during 8.3 joints below, 9 joints and 10 save and be used to express the HIV structural protein and produce other schemes of Recombinant HIV particulate.

Use system of the present invention can obtain some selection schemes and control gained particulate character.Can use these selections at virus structure and period of infection.

5.1.1. the preparation of recombinant DNA and virus vector

According to the pendent together U.S. Patent application 779 of the application, 907(applying date 1985.9.25); The application in 9,842,984 and 1986 on September of on March 27th, 1986 application 905,217 described in method can make up recombinant DNA carrier and virus vector such as vaccine virus, by reference, the present invention combines the full content of these inventions.

In one particular embodiment of the present invention, structure carries the recombinant vaccine virus of HIV env and gag sequence and it is used as carrier.In brief, be structured in the vaccine promotor transcribe control under the plasmid vector that contains HIV core and capsid protein coded sequence, influence HIV gene order and the genomic integration of vaccine virus by the vitro recombination method with it.According to the method described in the patent documentation of above-cited co-applications, identification, purification of Recombinant vaccine virus are also assessed them to HIV albumen synthetic controllability in the cells infected.

In another embodiment, the various bonded recombinant plasmid vectors of member a kind of encoded HIV structure and/or regulatory gene, and come transfection can produce reorganization preparation HIV particulate cell as carrier with it.Next the 8th part is described the typical arrangement structure of these carriers.

In the building process of recombinant DNA carrier, recombinant vaccine virus etc., can utilize recombinant DNA technology to modify the recombinate exact nature of preparation particulate each protein ingredient of the present invention.In this way, also can determine the existence and the structure composition thereof of reverse transcription virus antigen determinant on this particle.For example, these antigenic determinants can comprise that the different antigenic determinant of the HIV gp120 that obtains is to produce the immunne response of cross reaction from different HIV isolates.Equally, various HIV gag gene order can be attached in the middle of the recombinant vectors, produce and to improve the Recombinant HIV particle that causes immunological competence and antiviral effect to change reorganization preparation HIV particulate immunogenicity, also can use the encoded carrier of sudden change HIV gene order.The applicant thinks, the retroviral particle that is combined with the reorganization preparation of the core of this modification and/or capsid protein belongs within the scope of the present invention and back claims.

5.1.2. with recombinant vectors infection/transfection host cell

To produce the reverse transcription disease toxalbumin of reorganization preparation

The composition that not only depends on used recombinant vectors to a great extent that is controlled to the retroviral particle character of reorganization preparation, and depend on infect or transfection process in the combination of used carrier, this being chosen in the entire method of the present invention is a main variable factor.As briefly bright, the applicant finds can form the HIV-1 core protein that is mounted in the virion with the single recombiant vaccine strain infection BSC-40 host cell that carries HIV-1 gag gene (v-gag2).Yet, when with v-gag2 with when carrying these same cells of recombiant vaccine co-infected of HIV-1 env gene (v-env5), cause these assembling particles to be combined with and present HIV-1 capsid protein (back the 6th is partly) in its surface.Therefore, form the particle that only contains core protein, v-env5 is joined to produce the more complicated particle of structure that is combined with capsid protein in this infection system though infect the back with v-gag2.A step is goed deep in above explanation again, expect to use v-gag2, v-env5 and the third recombiant vaccine co-infected host cell that carries the env gene that obtains from HIV-2 can be formed with the white xenogenesis particle of HIV-1 core and HIV-1 and HIV-2 capsid dawn.Select as another kind, also single kind of recombinant vaccine virus of a plurality of HIV genes of having encoded can be used as infection carrier.As described by back the 7th part example, the host cell that infects with this vaccine virus carrier also produces the HIV-1 particle of immunocompetent reorganization preparation.

Same principle also is applicable to other carrier systems, comes transfection host cell to produce the reorganization particle as plasmid vector.For example, can be with the plasmid vector of independent one encoded HIV env and gag or a coding HIV env, the carrier transfection host cell of gag and other HIV genes.Also can come transfectional cell with a plurality of different HIV genes or HIV gene bonded plasmid vectors of encoding respectively.And, can also carry out transfection once more to this cells transfected system with the carrier that is designed to this particle generation system is increased other HIV genetic expressions.Can use has encoded can regulate the carrier of promotor, and (as the 8.3.2 joint of back) regulated in proteic expression to HIV in the transfection host cell.Can use the carrier of other HIV genes of having encoded to influence in transfectional cell, they with HIV structure gene together with expression, this HIV can be regulated and/or a kind of method that changes particle characteristic and/or their production level of the expressional function of accessory protein in this system.

Be important to note that be to form particulate under the situation that does not have gag albumen to exist, therefore, in this recombinant vectors, must include basic HIV core protein gene sequence.Although do not need hiv protease when this particle of assembling, it is (the Peng et al.1989.Virology 63:2550) with effect in forming infectious virus particle process.Therefore, in the HIV granules preparation process of reorganization preparation, preferably include the function of hiv protease, so that these particles and natural viral particle are very approaching.In addition, can use from HIV-1, the gag gene that obtains in HIV-2 or their the different isolates makes up recombinant vectors.As this area professional can know clearly, above-mentioned multiple infection method and use the similar approach of other recombinant vectorss can be used to prepare to have the Recombinant HIV particle on surface and cAg feature widely.The number of various various combinations is hard-core basically.

Selected particular host cell also can influence with the prepared particulate character of the inventive method.Selected cell is wanted to express and the ripe HIV albumen of processing treatment correctly.Because HIV capsid protein gp120 and gp41 be by glycosylation, and be to derive from by bigger gp160 precursor, therefore will select the processing modification process after a kind of cell can be controlled these translations by the proteolytic ferment cutting.Certainly, if use recombinant viral vector, host cell must have susceptibility to the infection of recombinant virus.In a preferred embodiment of the invention, used and derived from the mankind, ape or rodentine host cell.

Can be with recombinant vaccine virus according to the condition infection host cell described in following the 6th part, perhaps use recombinant plasmid vector according to transfection host cell described in following the 8th part, when using the described recombinant vaccine vector of following the 6th part system, can infect with infection repeat number (MOI) pair cell of every kind of about 10PFU/ cell of recombinant vaccine virus.But the Technology professional understands, can increase or reduce the MOI of one or more recombiant vaccines, with the influence particulate character that produced.That have a liking in design or this will be an important factor during xenogeneic particle more.For example, can obtain the required ratio of HIV-1 and HIV-2 capsid antigen on the xenogenesis particle surface by infection with a suitable corresponding MOI ratio.

In the middle of the specific embodiment of in following the 6th part, describing in detail, use kidney (BSC-40) cell of the cercopithecus aethiops of recombinant vaccine virus v-env5 and v-gag2 co-infected cultivation.Infected BSC-40 cell synthesizes HIV-1 capsid protein gp120, gp41 and gp160 precursor, and HIV-1 gag albumen p24, p17, p15, p55, p45, and p39.And p24 at least, p17, gp120 and gp41 assembling particle be to polyclone anti-HIV-1 antiserum(antisera) and to p17, p24, gp120 and gp41 have specific monoclonal antibody be have immunoreactive.HIV-1 particle by thin layer tangent plane electron microscope and the preparation of immunity gold (immunogold) pairs of markings group carries out analysis of Ultrastructure, shows that it and natural HIV are consistent basically on morphology.About this point, HIV-1 particle that observing reorganization preparation be have diameter 100 and 120nm between spherical object, and contain a cylindricality or a thick kernel of spheric electronics that depends on the tangent plane angle.By capturing enzyme immunoassay and using the monoclonal antibody of anti-p24 and p17 to carry out the immunoelectron microscope analysis, confirmed the consistence of HIV core texture respectively.Use is carried out the immunoelectron microscope analysis revealed to HIV-1gp120 and the special monoclonal antibody of gp41 and have the gp120/gp41 title complex on the HIV-1 particle surface of reorganization preparation.Also observe and the corresponding to various forms of prematurity particles of the morphology generating process of HIV-1 virus particle.These features are actual with observed feature from the similar analysis of HIV-1 virus particle to be consistent (with people such as electron micrograph shown in Fig. 4 and Gelderblom 1988, people such as Gelderblom among the Micron and Microscopia 19:41 are 1987, and those electron micrographs that provide among the J.Virol.156:171 compare).The HIV-1 particulate ultrastructure that applicant's result shows these reorganization preparations and the ultrastructure difference of the HIV-1 that live only are that they do not contain viral genome or ThermoScript II.

Can also be with the HIV gag that encoded, the recombinant plasmid vector transfectional cell of env and other HIV gene produces Recombinant HIV particle of the present invention.The various specific embodiments of this respect of the present invention have been described in 8.2 joints below.In a particular embodiment, with the HIV-1 gag that encoded, env, the plamid vector transfection of tat and rev gene China hamster ovary (CHO) cell.The processed env and the proteic stable Chinese hamster ovary celI of gag that have obtained expression and secretion Recombinant HIV-1 particle form are.Use by various plasmids or its Hela in conjunction with transfection, BSC-40 and Vero cell can obtain similar result (face as follows; For example 8.3.1 saves, 8.3.2 joint and 8.3.3 joint).

5.2 identification with separate contain the immunoreactivity core and

The non-replicability reorganization particle of capsid protein

Can develop by various immuno-chemical methods and/or by electron microscope (EM) and discern the particle of reorganization preparation.Can use immunochemistry detection method such as radioimmunoprecipitation (RIP), capture enzyme immunoassay (EIA), Western engram analysis and similar approach thereof.The 6th following part mode has by way of example described how to use some specific embodiments of these methods identification reorganization preparations HIV particulate.Can use various EM methods, thin section EM and immune electron microscopy described in following 6.1.3 joint, discern the HIV particle of reorganization preparation and describe its characteristic.Operable other EM technology comprises scanning electronic microscope (SEM), the copying surface electron microscope, with the freezing ultra micro otomy of immunity (immunocryoultramicrotomy), verified all these methods all can be used for illustrating fine structure (the Gelderblom et al of HIV, 1988, Micron and Microscopia, 19:41).

Can from the substratum of host cell, isolate the particle of reorganization preparation with the standard method of knowing in this technology.But importantly, employed separation method will make the level of coming off of gp120 reduce to minimum, so that make the HIV particulate immunogenicity of reorganization preparation increase to maximum.

5.3 measure non-replicability reorganization preparation particulate immunogenicity

Can measure reorganization preparation particulate immunogenicity by the immunne response of monitoring immunity back laboratory animal.Laboratory animal can comprise mouse, rabbit, chimpanzee, even the people.Admissible several immunization route comprises oral cavity, intradermal, intramuscular, approach such as in intraperitoneal, intravenously, subcutaneous, the nose.Can analyze from three aspects to the HIV particle immunogen institute inductive immunne response of reorganization preparation: (a) known technology of utilization such as ELISA assay method (ELISA), immunoblotting, the immune serums that mensuration produced such as radioimmunoprecipitation are to the antigenic reactivity of real HIV, (b) immune serum is in external and the infective ability of HIV (Robert Guroff, 1985, Nature 316:72), (c) prevent that immune animal HIV from infecting and/or minimizing infection symptoms (Francis, 1984, Lancet 2:1276; Gujdusek, 1985, Lancet 1:55).

In a particular embodiment, measure isolating reorganization and prepare HIV-1 particle (the 6th following part) the intravital immunogenicity of rabbit (below the 14th part etc.).The result of this analysis shows that the HIV-1 particle of reorganization preparation has the immunogenicity of height, because this particle can excite HIV capsid and core texture albumen is had specific tangible body fluid and cellullar immunologic response.And the rabbit of the HIV-1 particle immunity for preparing with recombinating produces the neutralizing antibody of HIV-1.

In a relevant embodiment, to having been carried out estimating (the 16th part of face as follows) by immunity in the inhuman primate of the HIV-1 granulating of reorganization preparation.Specifically, with the HIV-1 particle and the antigen combined use of other HIV-1 of reorganization preparation the Macaque monkey is carried out immunity.Can pass through various measuring methods, comprise the ELISA of whole viruses, gp120 ELISA, focus immunoassay (focal immunoassay) and lymphadenosis are reacted and are measured the intravital immunne response of immune animal.Described as the embodiment in following the 16th part etc., as unique immunogen elementary and the second immunisation reaction, the HIV-1 particle of reorganization preparation can excite HIV-specificity humoral and cellullar immunologic response.When being used for immunity when using a kind of animal of encoded HIV-1 env and the antigenic recombinant vaccine virus of gag in advance, the HIV-1 particle of reorganization preparation is especially effective.

5.4 vaccine preparation

One particular embodiment of the present invention are exactly to prepare to excite to help to prevent retroviral infection or the vaccine of the immunne response of take a turn for the worse viral relative disease of record such as acquired immune deficiency syndrome (AIDS).As immunogen, this immunogen is combined with some important retrovirus core and capsid proteins to this vaccine ingredients with the retroviral particle of reorganization of the present invention preparation, so be polyvalent in nature.In some relevant embodiment, the retroviral particle that can prepare with recombinating is as the specificity immunology stiffeners, and being used for making it, the intraindividual reverse transcription relative disease of retroviral infection develops toward the good aspect.

5.4.1 the vaccine of anti-HIV (human immunodeficiency virus)

Another specific embodiment of the present invention comprises can excite the vaccine preparation that helps to prevent the HIV infection or the immunne response of acquired immune deficiency syndrome (AIDS) takes place.This vaccine ingredients uses the HIV particle of reorganization preparation as immunogen.

According to the conventional method, virus vaccines is to be prepared by intact virus attenuation or inactivation.Never have the vaccine that a kind of good method can be used for designing a kind of main anti-HIV-1, this mainly is because a large amount of preparation virus, the potential incomplete virus is active and the HIV genome is input to the danger (Minor that may bring in the healthy acceptor, 1989, J.Antimic-robial.Chemotherapy 23, Supp.A:55).Therefore the development of HIV vaccine has focused on above the subunit vaccine.Though tend to use the subunit's preparation that contains reorganization gp120 capsid protein at first, clear at present, gp120 and gp41 all be the generation neutralizing antibody (Chahn et al., 1986, EMBO is J.5:3065; Ho et al, 1987, J.Virol.61:2024; Skinner et al., 1988, J.Virol.62:4195), transfer cell toxicity (Tyler et al., 1989, the five acquired immune deficiency syndrome (AIDS) international symposiums, Abstract T.C.O.33:521), with bring cytotoxic T lymphocyte to kill and wound susceptibility (Zarling et al., 1987, target antigen J.Immunol.139:988).These results advocate will include gp120 and gp41 when design HIV-1 vaccine.

Because gp41 and cytolemma combine, and it and gp120 have more weak non-covalent interaction, makes the solubility gp120/gp41 title complex of purifying intact become unrealistic.What is more important, other virus antigen such as hepatitis B surface antigen(HBsAg) (Cabral et al. that occurs with the membrane structure composition of picture, 1978, J.Gen.Virol.38:339) and herpes simplex virus glycoprotein (Ho et al., 1989, J.Virol.63:2951) the same, membrane-bound gp120/gp41 title complex has more immunogenicity than the homologue of solubility probably.

Discussed in the background of invention part as the application, the several characteristic of HIV makes that a kind of effective HIV vaccine of preparation is complicated.The present invention can avoid many problems, if not the words of having avoided most problems.Except in they contain gag and the env albumen in the structure naturally, can also prepare the HIV particle to reorganization of the present invention and design, keep needed antigenic determinant, and remove or modify unwanted antigenic determinant.And, the proteic several different variable antigenic determinants of same HIV can be attached in the particle that is used for the vaccine ingredients by method provided by the invention.The present invention also provides a kind of HIV particulate method that produces xenogeneic reorganization preparation, uses this particle can prepare a kind of vaccine that can prevent that HIV-1 and HIV-2 from infecting.

One of new feature that utilizes the inoculation method that the present invention carries out is one group of these and other this antigenic determinant can be merged in the middle of the immunogenicity particle.And, these antigenic determinants and they the same being brought in this immunity system on natural HIV, thus induce the immunne response of resisting natural viral infection effectively.

5.4.1.1 HIV-1 vaccine

Protection immunity for anti-HIV does not still get across fully.Because HIV can infect with the form irrelevant or relevant with cell, it is generally acknowledged the immunizing power that may need neutralizing antibody and cell to transmit.In the individuality that HIV-1 infects, identified the neutralizing antibody that to discern a plurality of different HIV antigenic determinants, comprised that those can be identified in the height reserve area of capsid protein gp120 and the antibody of the antigenic determinant on the Variable Area.Equally, identified the antigenic determinant of anti-gp41 and p17 neutralizing antibody (Papsidero et al., 1989, J.Virol.63:267-272).Detected other antibody comprises that those have specific antibody to the gp120 zone that is attached on the cd4 cell surface receptor in the individuality that HIV infects.Also have other antibody on some hypervariable regions that are attached to gp120 can suppress cytogamy that HIV infects become syncytium (Rusche et al., 1988, Proc.Natl.Acad.Sci.O.S.A.85:3198).In the middle of the human body that HIV infects, can also see cellullar immunologic response, specifically, identify anti-env, the cytotoxic t-lymphocyte of gag and pol gene product (Walker et al., 1987, Nature 328:345; Nixon et al., 1988, Nature 336:484; Riviere et al., 1989, J.Virol.63:2270-2277).

One embodiment of the present of invention are vaccines of the HIV-1 virus of the current popular form of a kind of anti-HIV, this vaccine has used the vaccine of the HIV-1 particle virus of reorganization preparation, the HIV-1 particle of the reorganization preparation described in following the 6th part, this particle contains the ripe core and the capsid protein of I C-type virus C, be assembled in one with the similar structure of the morphology of the HIV that lives and antigenicity in.This embodiment comprises the HIV-1 particulate vaccine preparation of the various reorganization preparations of utilization.For example, can use the particle that has the gp120/gp41 title complex on two or more HIV isolates simultaneously to induce the protection antibody that produces anti-a plurality of variable regions antigenic determinant.Can prepare some viruses like this with a plurality of different recombinant vaccine virus co-infected host cell that has the env gene that from different HIV-1 isolates, obtains respectively.Can produce the immunne response of anti-multiple different HIV-1 strain with the individuality of this particle immunity.

Equally, many preferendums particulate structure also can increase the usefulness of this vaccine preparation.In this embodiment, designed particle is combined with the antigenic determinant with different preferendums that obtains from the HIV-1 strain.For example, utilize the multiple infection method can prepare the HIV-1 particle of demonstration to the reorganization preparation relevant HIV-1 strain uniqueness of monocyte and that combine with the total determinant of T4 CAV strain.Specifically, can use three kinds of recombiant vaccines, a kind of gag gene that from a kind of bacterial strain, obtains that has, a kind of env gene that from this same bacterial strain, obtains that has, and another kind has the env gene that obtains from the different bacterial strain of a kind of preferendum, and the co-infected host cell prepares this particle.

The vaccine of effective anti-HIV-1 comprises HIV-1 particle and other immunogen of reorganization preparation of the present invention is united use.About this point, in the non-human primate,, the HIV-1 particle of reorganization preparation as second immunisation when former, is presented and excites body fluid and cellular immunization most effectively with behind the reorganization gp160 initial immunity.

5.4.1.2. HIV-2 vaccine and heterovaccine

Other embodiment of the present invention relates to vaccine and the heterovaccine of anti-HIV-2, can comprise the independent a kind of vaccine that for example is used for HIV-1 and HIV-2.The reorganization that is used for this heterovaccine prepares particle can comprise for example core protein of HIV-1 and the capsid protein of HIV-and HIV-2.Select as another kind, also can wish to prepare a kind of vaccine of the HIV granulometric composition by two or more different reorganization preparations.Especially all the more so when a kind of vaccine of design can prevent the many different isolate of HIV-1 and HIV-2.

Can contain independent a kind of Recombinant HIV grain type or the dissimilar vaccine that combines with a kind of suitable assistant agent preparation, to strengthen to they antigenic immunological responses.Suitable assistant agent is including, but not limited to mineral rubber, and surfactant is as lysolecithin, the polyion polyvalent alcohol, polyanion, peptide, oiliness emulsion and the human assistant agent that comes in handy such as BCG(bacille Calmette-Guerin vaccine bacille Calmette-Guerin) and CBP.

Can use the above-mentioned several different methods of in this technology, knowing to inoculate this vaccine preparation, comprise intradermal cut, intravenous injection, modes such as subcutaneous injection, intramuscular injection, intranasal administration, oral administration.

6. embodiment: the HIV-1 that non-replicability recombinant chou is made

Particulate produces and separates

Described herein is a kind of HIV-1 particulate vitro system that recombinant chou is made that produces, and this particle contains the core and the capsid protein matter of assembling, and presents envgp120 and gp41 antigen in its surface.Briefly, with carrying the complete capsid gene (v-env5) of HIV-1 or the recombined vaccinia virus coinfection BSC-41 cell of complete HIV-1 gag and proteinase gene (v-gag2).HIV-1 albumen is expressed in the BSC-40 cell, and is assembled into by in the blastogenic HIV-1 particle on the cytolemma.The particle that is produced is non-replicability, its can with HIV-1 capsid and core protein are had specific monoclonal antibody reactive, and on morphology similar in appearance to the HIV-1 virus particle.

6.1. general method

6.1.1. cell and virus

EagleShi substratum (DMEM in the DulbeccoShi improvement of adding 10% foetal calf serum and each 100 unit/ml penicillin and Streptomycin sulphate, Gibco, Grand Ishand, NY) propagation African green monkey kidney cell (BSC-40 cell strain in, it is for being derived from the BSC-1 cell, the continuous cell line of the cercopithecus aethiops cell of ATCC No.CCL26).

Carry the recombined vaccinia virus of HIV-1 env and gag gene order according to the preparation of method described in 905, No. 217 U.S. Patent applications that await the reply jointly (September 9 1986 applying date), and it is assessed.Recombined vaccinia virus v-env5 carries the complete env gene of HIV-1.Recombined vaccinia virus v-gag2 then carries complete HIV-1 gag and Prt gene and part pol gene.

From available from Wyeth Laboratories(Marietta, the New York City Board of Health virus strain of purifying vaccinia virus in commodity variola virus PA) (the Dryvax Lot 321501G) goods.See below with PBSAM() dilute variola virus and on the BSC-40 cell, carry out continuous three plaque purifications.Produce reserve (V-NY hereinafter referred to as) in the isolate by this plaque purification on the BSC-40 cell, and in order to make up recombinant virus.

6.1.2.HIV the calibrating of capsid and cAg

Use four kinds of technical test HIV-1 env and gag albumen: radioimmunoprecipitation (RIP), capture enzyme immunoassay (EIA), Western engram analysis method and immuno-electron microscope microscopy method.

Radioimmunoprecipitation be basically by state method (Haffar et al., 1988, J.Cell.Biol., 107:1677) (Trimar Inc.) carries out end user's polyclone anti-HIV-1 serum.Briefly, promptly infect the BSC-40 cell with the superinfection number (MOI) of each each cell 10PFU of virus.Infected back 12 hours, with [ ³⁵S] methionine(Met) and [ ³⁵S] halfcystine (100 μ Ci/ml) pair cell radio-labeled 4 hours.Collect substratum and wash cell with PBS, harvested cell, and dissolve it at RIP damping fluid (adding 1%NP40,0.5% deoxycholate salt, 0.1%SDS among the PBS).Make nuclear back cellular lysate or substratum and polyclonal antiserum reaction 30 minutes then.The insulation of antibody-antigenic compound and 10% glutaraldehyde fixed streptococcus aureus (Staph A) is 30 minutes under the room temperature.Centrifugation Staph A-antibody-antigenic compound, and wash twice with RIP lavation buffer solution (being added with 1%NP40,0.1%SDS among the PBS).Resulting agglomerate with SDS-PAGE sample buffer dissolving (Laemmli, 1970, Nature 227:680), in 11.5% polyacrylamide gel fractional separation, and manifest the protein of immunoprecipitation with radioautography.

Can use anti-P24 monoclonal antibody by stating method (Hu et al., 1987, Nature 328:721), to capture enzyme immunoassay and the Western blotting detects HIV-1 p24 specifically.Be used in the reorganization fusogenic peptide immune mouse that limits the crossover sequence in the p24 albumen, be used to capture the monoclonal antibody of enzyme immunoassay and Western blotting with generation.At Genetic Systems(Seattle, WA) go up generation monoclonal antibody 25-2 and 25-3, these antibody are addressed in U.S. Patent application 054, No. 026 (April 30 1987 applying date) and 105, No. 761 (on October 7th, 1987).

The MAb110-4(Thomas et al. that use is special to gp120,1988, AIDS Res.Hum.Retroviruses 2:25; Linsley et al., 1988, J.Virol., 62:3695) and the MAB41-1(Gosting et al. special to gp40,1987, J.Clin.Microbiol., 25:845), gp120 and gp41 capsid protein on the HIV-1 particle surface made from immuno-electron microscope detection method calibrating recombinant chou, and illustrate the HIV particulate morphology of ripe recombinant chou manufacturings, hereinafter 6.1.3. describes technique for electron microscopy in saving.

6.1.3. electron microscopic microscopy art

Separate and prepare the HIV-1 particle of the recombinant chou manufacturing that is used for the EM developing as follows: collect substratum and merge it by the cell culture that infected 15 hours.In refrigerated flat-top centrifuge tube, centrifugal with 600xg, by clarifying out substratum in the contamination of cells.With 120, the 000xg ultracentrifugation is by isolating particulate fraction in the clarifying supernatant liquor then.(S) collects resulting supernatant liquor as material behind the microparticle.Particulate fraction is suspended in the pH7.4 phosphate buffered saline (PBS) (PBS) again, and by 15% sucrose solution layer, with 120, the 000xg ultracentrifugation precipitates it again.Twice sedimentary material is particulate fraction (P).

With sucking-off behind the topped gently precipitation agglomerate of PBS, so sedimentary solids precipitation thing (P) is washed several times repeatedly.To precipitate agglomerate with 4% Paraformaldehyde 96 then and fix 20 minutes, and wash with PBS once more.With similarity method, with the little individual layer BSC40 of suitable recombinant virus infection cell (10 ⁴-10 ⁵Individual cell).Discard growth medium after 15 hours, and with PBS cell monolayer is washed several times again, fix 20 minutes with 4% Paraformaldehyde 96 down in 22 ℃ then.

With PBS fixed cell and fixed solids precipitation thing are washed 5 times, then with PBS solution (sealing damping fluid) sealing that contains 0.8% bovine serum albumin (BSA), 0.1% gelatin and 5% normal sheep serum 30 minutes.Deblocking solution and add monoclonal antibody (as the ascites fluid of dilution in 1: 2000 in lock solution) and be incubated 2 to 3 hours in cell inclines.Wash cell with PBS, then with 1: 5 the dilution golden bonded goat-anti mouse second antibody (IgG is attached on the 15nm colloidal gold; Janssen, Piscataway NJ) continues insulation 2 hours, washes and fixing in 2% glutaraldehyde with PBS.Sample in 22 ℃ down with 1% perosmic anhydride (OsO in 0.1M Cacadylate damping fluid ₄) be incubated 30 minutes.With PBS thorough washing sample, and continuous insulation made it dehydration in 3 minutes in 35%, 50% and 75% ethanol, and the uranyl acetate (uranyl acetate) that then sample is used under 22 ℃ in 70% ethanol dyeed 30 minutes.As stated above, once more in 80%, 90%, 95% and last 100% ethanol (3 times) in 22 ℃ of insulations continuously, further dehydrations.

As follows sample is embedded in the methacrylate resin then: use dehydrated alcohol/plastics (2: 1) with sample preparation 1 hour respectively, spend the night with 100% process in plastics.Under 60 ℃, make resin polymerization 2 days then.Go up machinery by the garden dish and remove the plastics of the sample of embedding, and thinly slice (100nm), be collected in then on the grid of formvar coating.Grid with saturated uranyl acetate/Tiorco 677 (dyeing of Millonig ' s) 10 minutes, and thorough washing it.Grid with 60kv, is observed with the JEOL100B transmission electron microscope for 100,000 times in dry back.

6.2. contain the non-replicability recombinant chou of ripe gag and env albumen

The HIV-1 particulate of manufacturing produces

6.2.1. the BSC-40 that analyzes at recombinant vaccinia virus infection

The HIV-1 protein of expressing in the cell

By above the 6.2.1 joint is described, end user's polyclone anti-HIV-1 serum, in the generation of being infected by v-env5, v-gag2, two kinds of recombinant chous or parental generation vaccinia virus V-NY with the RIP methods analyst, penetrated the lysate and the growth medium of the BSC-40 cell of radio-labeled, with detection HIV-1 protein.As shown in fig. 1, in the lysate of the cell of v-env5 infection, detect gp160env precursor protein matter, and proteolysis processed products gp120 and gp41(swimming lane A).In addition, detect the gp120(swimming lane B of the emiocytosis of v-env5 infection).Synthetic p55gag precursor also is subject to processing in the cell that v-gag2 infects, and produces precursor p45 and p39(swimming lane E in the middle of sophisticated gag protein p24, p17, p15 and two kinds) (Gowda et al., 1989, citation).Interesting is also to have detected gag protein (swimming lane F) in the culture supernatant.

Expressed person identical (swimming lane C, with swimming lane A and E relatively) in env that processes that is produced by the BSC-40 cell of v-env5 and v-gag2 coinfection and gag protein and the indivedual cells that infect.Ripe env that cell produced and gag protein by these double infections concentrate on (swimming lane D compares with swimming lane B and F) in the culture supernatants equally, and they have identical kinetics.In addition, the iodization of the enzymatic plasmalemma related protein of lactoperoxidase is shown, in the cell that v-env5 infects with the cell of double infection in the same, gp160, gp120 and gp41 all are transported to (Fig. 2, swimming lane A and B) on the cell surface.

6.2.2. separate the HIV-1 particle that non-replicability recombinant chou is made

The composition that can be used as soluble protein or budding particle for extracellular gp120 and the gp41 that whether detects by infected B SC40 cell expressing is released, culture supernatants is separated into behind particle (p) and the particle (S) partly (6.1.3 that sees above joint), and carry out parallel RIP with unsegregated culture supernatants (TS) and test, to analyze the HIV protein content of each several part.Shown in Fig. 3 (1) (swimming lane A, B and C), predict by the cell-derived extracellular gp120 of v-env5 infection mainly in the S part, this points out this protein is not the composition of budding particle.Yet, record by the cell-derived gp120 of V-env5 and v-gag2 coinfection in P part and S part (seeing Fig. 3 (1) respectively, swimming lane E and F), very little although particle forms relevant gp120 to the contribution of the extracellular gp120 aggregate level that can detect.

Opposite with gp120, only in the P part supernatant of the cell of double infection, detect gp41(Fig. 3 (1), swimming lane E is to D and F), this shows that gp41 only combines with budding particle.What is interesting is,, in the P of the cell conditioned medium of double infection part, detected gp160env precursor (Fig. 3 (1), swimming lane E is to D and F) though content seldom.

HIV-1 core protein by infected B SC-40 cell expressing has been carried out similar analysis.In the P part of (Fig. 3 (1), relatively swimming lane E and F) cell culture supernatant liquid of (Fig. 3 (1), relatively swimming lane H and I) that v-gag2 infects and double infection, all observe all detectable core protein-p24, p55, p45, p39 and p17.Also separate the P part that derives from the double infection cell again by the continuous sucrose density gradient of 15-60% with centrifugal, and by above estimating the antigenic existence of p24 with the EIA method described in the 6.1.2. joint.Result shown in Fig. 3 (2) shows that p24 and 36%-40% sucrose part together are separated into a significant simple spike.These results suggest are worked as homogeneous granules by the gag protein of infected B SC40 cell expressing as granulometric facies and are released.In addition, there are gp120 and gp41 in the cell conditioned medium P of the double infection part, show that the particle that discharges from the coinfection cell combines the gp120/gp41 mixture.This conclusion further obtains the result's of immuno-electron microscope analysis support (6.2.3 that sees below joint).

6.2.3. with thin section electron microscopic microscopy method to recombinant chou

The HIV particle of making carries out microstructure analysis

HIV-1 particle for the manufacturing of direct analysis recombinant chou, separate the P part in the culture supernatant by the BSC40 cell of v-env5 and v-gag2 coinfection, and make it and gp120(MAb110-4) or gp41(MAb41-1) monoclonal antibody specific (MAbs) reaction.Basic antigen-antibody complex reacts with the second antibody-colloidal gold binding substances that is embedded in the plastics again, and by above observing it with thin section electron microscopic microscopy art described in the 6.1.3. joint.These analysis revealeds, contain electron dense cylindricality (Fig. 4, " a " and " c ") or spherical (Fig. 4, " b ") particle of the about 100-120nm of diameter of core on morphology similar in appearance to EM video (the Gelderblom et al. of separated HIV-1 virus particle, 1988, Micron and Microscopia, 19:41; Gelderblom et al., 1987, Virology, 156:171).In addition,, show on their surface, to present gp120(Fig. 4, " a " and " b " with colloidal gold binding substances marking particle) and gp41(Fig. 4, " c ").

Use anti-gp120MAb to being carried out similarity analysis by the complete BSC40 cell of two reorganization vaccinia virus coinfections.Observe the 100-120nm particle that in a large number gp120 antigen is positive.Suppose that the particle that great majority are associated with these cells has one of two kinds of the unique forms, an one feature is dispersive form of vesicles (Fig. 4, " e ", open arrow), another kind of different form is the two films district (Fig. 4, " d ", double-headed arrow) that has delocalization localized, thick.The applicant infers that these different structures represent immature particulate various forms, and this can be parallel with the person of appearance during the HIV-1 viral particle morphology forms.Sometimes can see the particle of the bar-shaped shell structure that contains abundant formation near cell surface, this tracks in the electron microscope image shown in " e " (open arrow) as Fig. 4.There are not in conjunction with gp120(Fig. 4 " d ", double-headed arrow by blastogenic vaccinia virus particle on the cytolemma).

6.2.4. the HIV-1 particulate nucleic acid content of recombinant chou generation

Carry out dot blotting hybridization with rna probe, to detect the HIV-1 particulate nucleic acid content of recombinant chou generation to gag or responding property of env sequence.According to the method for manufacturer recommendation, use Promega Riboprobe in-vitro transcription test kit, in the presence of radiolabeled Nucleotide, carry the dna profiling of gag or env sequence with synthesising probing needle through in-vitro transcription.Briefly, the recombinant chou particle that will be equivalent to 300ng p24 is dissolved in the RNA preparation dissolving damping fluid [2M guanidinium isothiocyanate, 125mM Trisodium Citrate (pH7.0), 0.125%Sarkocinate, 50% dimethyl sulfoxide (DMSO)], and be transferred on the nitrocellulose filter, the HIV(of the similar dissolved psoralene of various concentration inactivation is equivalent to 2600ng, 260ng, 26ng and 2.6ng P24) do parallel detection.Preparation respectively with the filter membrane of gag specificity or env specific probe reaction.Under 42 ℃, insulation is 2 hours in hybridization buffer (3 * ssc, 50% methane amide, 5 * DenhardtShi solution and the non-specific RNA of 150 μ g), adds each probe then with nitrocellulose filter.Filter membrane and probe are used 0.1 * ssc/0.1%SDS solution thorough washing then in 42 ℃ of following incubated overnight, dry air, and analyze it with radioautography.

The radioautograph collection of illustrative plates that has shown dot blotting hybridization among Fig. 5.Have only gag probe and nucleic acid reaction (the A group is to the B group) in the recombinant chou particulate samples.On the contrary, two probes all react with the control group virus of psoralene deactivation.The HIV-1 particle that this results suggest recombinant chou produces has wrapped up gag RNA, and does not wrap up env RNA.This conclusion meets this fact, has promptly comprised 5 of the parcel signal that is accredited as the HIV viral RNA recently ' do not translate sequence (Fig. 5, C organizes, arrow) in order to the gag gene that produces v-gag2 cowpox recombinant virus.

These results show that the HIV-1 particle that recombinant chou makes does not have replication.This conclusion has obtained the hereinafter support of experiment described in 12 joints, and its proof is with the long-time CD4 that is incubated of the HIV-1 particle of recombinant chou generation ⁺There is not HIV antigen in the cell in the cell.

7. embodiment: use single recombinant vaccinia virus vector to produce non-replicability

The HIV-1 particle that recombinant chou is made

As an alternative of the HIV-1 particle that produces the recombinant chou manufacturing with two kinds of recombinant vaccinia virus vectors (seeing the 6th joint), can use to comprise the single virus carrier system that makes up the recombined vaccinia virus that contains HIV-1 env and gag gene.In the following example, use the recombinant vaccinia virus infection BSC40 cell that contains HIV-1 env and gag gene.Infected cells is expressed capsid and the cAg of HIV-1, and it is assemblied in the infected cell to produce recombinant chou HIV-1 particle.By Recombinant HIV-1 particle of purifying in the substratum of infected cell be have in the body immunogenic.

7.1. general method

7.2. make up V-G2E5: a kind of contain HIV ENV and

The recombined vaccinia virus of GAG gene

See common pending U.S. Patent Application No.07/593 with restriction endonuclease EcoRI by plasmid pv-env5(, 401, the October 5 nineteen ninety applying date) last 3.2kbp fragment (Nucleotide 5707-8608) of downcutting the complete env encoding sequence (it is subjected to the control of vaccinia virus 7.5K promotor) that contains HIV-1.This fragment is inserted this plasmid of plasmid pv-gag2N(and common pending U.S. Patent Application 07/593, the person is basic identical described in 401, and just having inserted one in the Sma I site in gag encoding sequence downstream contains the synthetic linker of translating termination signal) the EcoRI site in.Plasmid pv-gag2N also contains the HIV-1 gag gene that is under the control of vaccinia virus 7.5K promotor.Provided the flow process that makes up plasmid pv-G2E5 among Fig. 6.With common pending U.S. Patent Application No.07/593, recombination method in the body described in 401 is introduced HIV-1 gag and env gene in the thymidine kinase gene of vaccinia virus, contains the gag of HIV-1 and the recombined vaccinia virus V-G2E5 of env gene thereby produce.

7.3. coexpression in V-G2E5 infected B SC-40 cell

HIV-1 capsid and cAg

With the superinfection number of 5pfu/ cell, V-G2E5 infects African green monkey kidney cell (BSC-40) with reorganization.Infect and washed cell and on the 7-15% gel, separated lysate in back 24 hours through SDS-PAGE.Protein transduction in the cellular lysate moved on on the nitrocellulose filter and with HIV positive human sero-reaction, then with the goat anti-human immunoglobulin antibody's reaction that is connected on the sour jujube root peroxidase.Result shown in Figure 7 shows that the capsid of HIV-1 and cAg are all expressed and processed in the cell that V-G2E5 infects.Two kinds of antigenic levels are with single similar with containing env or containing the recombined vaccinia virus expresser of gag.

7.4 the BSC-40 cell of V-G2E5 infection

Recombinant HIV-1 particle that produces

Use reorganization V-G2E5 by producing HIV-1 sample particle in the BSC-40 cell.On Cytodex 3 globules (Pharmacia LKB Biotechnology), and the method for recommending by manufacturer is cultivated it in rolling bottle in the EagleShi substratum of the Dulbecco improvement that contains 5% foetal calf serum with the BSC-40 cell inoculation.When cell reaches when merging, infect it with the MOI number of 5pfu/ cell with recombined vaccinia virus V-G2E5.Adsorb after 1 hour, wash cell twice, to remove excessive inoculum with fresh culture.37.5 ℃ insulation after 24 hours, is collected substratum and is removed cell debris with low-speed centrifugal.Then in 19 type rotors (Sorvall) with 19,000rpm was with centrifugal 3 hours of the substratum of predefecation.This ultracentrifugal throw out is suspended in PBS again, merges the back and use SW55Ti rotor (Beckman) with 32,000rpm precipitated it in centrifugal 1 hour once more.With last throw out resuspending in cold PBS and be stored in-70.5 ℃ down standby.Basically save described method according to 6.1.2. above, capture with p24 antigen respectively and the p24 and the gp120 antigenic content of this granular preparation of immunoblotting quantitative analysis.

Provided particulate radioimmunoprecipitation assay result among Fig. 8 by the cell generation of V-G2E5 infection.These results show, behind the high speed centrifugation, have gp120 and p24 antigen in the precipitation agglomerate of the infected cell culture medium part, and this shows that these sophisticated virion protein matter optionally are assembled into particle form.In these particles relatively enrichment p24 and gp120, the content in the HIV-1 virus particle preparation, this is determined by structure and biological similarity between the particle of recombinant chou manufacturing and the intrinsic HIV-1 virus particle.

8. embodiment: with plasmid DNA transfection in mammalian cell

Produce the HIV-1 particle that non-replicability recombinant chou is made

Except that the use recombinant vaccinia virus vector described in 6 and 7 joints above produces the Recombinant HIV-1 particulate system, also can use the system that comprises with the DNA transfection mammalian cell of coding HIV-1 env and gag gene.

Can make multiple scheme produce recombinant chou HIV particle with system described herein, it includes but are not limited to the compound plasmid vector that (1) transfection contains a plurality of HIV structures and regulatory gene, (2) cotransfection contains the multiple plasmid vector of different HIV genes, (3) use necessary and adjustable enhancers/promoters element simultaneously, to drive the HIV protein expression, (4) use the HIV that comprises tat and/or rev to regulate the expression of albumen with indirect control HIV gag and env structural protein, (5) use different expression of cell lines HIV protein and Recombinant HIV particle, (6) use coding by the proteinic plasmid vector of the HIV of modification.Can do some and change,, reach optimizing, and handle different kinds of protein and structures in the Recombinant HIV particle so that this system is applicable to different cell types.

8.1. plasmid structure

The many plasmid vectors that are used for this system all contain the heterozygosis CMV:HIV enhanser that is derived from expression vector pH3MPy: promotor (being called CmHi), said carrier contain the enhanser (Nucleotide-69 is to+78) of the direct early gene of cytomegalovirus that merges with enhanser and the tat district of HIV.Wherein some plasmid vector contains the available enhanser that derives from the direct early gene of cytomegalovirus (being called CMV) or mouse metal sulfur hydrochloride-I gene (Mt): promoter element.These regulatory elements are connected on the proteinic gene order of coding HIV, being to contain the Hpa I of adenovirus 2 of Ela gene poly A attachment site to Hha I (Nucleotide 1569-1680) fragment then, next is to be cloned into plasmid Bluescribe+(Strategene) in BamHI to the Sph I fragment of pBR322.Indivedual HIV protein by these plasmid vector codings are shown in the table I, and the structure flow process of these plasmids then is shown among Fig. 9.

The table I

HIV gene by the plasmid vector coding

The vector HIV gene

Gag+Pro Tat Rev Env other

CmHiEnv5（1104-（b1） +

CmHiTgfbEnv5（1113-a1） +

CmHiGag2Rre（1158-a1） +

CmVGag2Rre（1159-a1） +

CmHiRev（1132-c1） +

CmvRev（1152-a1） +

CmHiTat（1132-b1） +

BsMtRev（1202-2） +

BsMtTAT（1203-1） +

CmHIVdelXmn（1133-a1） + + + Vif，Vpr，

Vpu

CmHIVdelKpnAvr（Gag2TRE） + + + + Vpu

（1160-a1）

Plasmid CmHiEnv5(1104-b1) has the Nru I of pH3MpY to Hind III fragment, it contains the direct early gene enhanser of cytomegalovirus and HIV promotor and tat element, and be connected on the HIV capsid Env5 gene magazine (5671-8572, the Ava II is to the Kpn I).

Plasmid CmHiTgfbEnv5(1113-a1) contain and be connected to chimeric TGF-β: the Nru I of the pH3MPy on the HIV env gene is to Pst I enhanser: promoter fragment; Provide 5 ' non-monkey TGF-β gene of translating district and signal peptide directly to be merged on the HIV env gene signal cracking site in the Kpn I site that extends to Nucleotide 8572 places by Nucleotide 5856.

Plasmid CmHiGag2Rre(1158-a1) contains to be connected to and extend to Nucleotide 3372 Asp of place from Nucleotide 257 BssH II sites ⁷¹⁸Nru I on the HIV gag in site and the Pol encoding sequence, pH3MPy is to Xba I enhanser: promoter fragment.This be one with the same zone of gag encoding sequence that is included among the recombined vaccinia virus v-gag2, and comprise that complete gag reads sign indicating number, be thereafter that only about half of pol reads sign indicating number.The sequence that also comprises the hiv protease of encoding in this zone, and most of but not every ThermoScript II coding region.Provide the Xba I joint of rotaring inter-transtating terminating cipher to be connected to Asp ⁷¹⁸On the site.By Nucleotide 7178 place BgI II site be extended down to 7698 place Hind III site, contain the HIV env gene fragment of Rev reactive component thereafter.Plasmid CmvGag2Rre(1159-a1) basic identical with it, different being to use derives from expression vector CDM8(Invitrogen) NruI to Xba I cytomegalovirus enhanser and promoter fragment.

Plasmid CmHiRev(1132-c1) the Nru I of pH3MPy upstream that contains intronless HIV Rev gene is to Pst I enhanser: promoter fragment, its Bsu36 I site by Nucleotide 5500 places extends to the Kpn I site at Nucleotide 8572 places, and has lacked Nucleotide 5590-7935.Plasmid CmvRev(1152-a1) roughly the same with it, different being to use derives from expression vector CDM8(Invitrogen) the Nru I to Xba I cytomegalovirus enhanser and promoter fragment.

Plasmid CmHiTat(1132-b1) the Nru I of pH3MPy upstream that contains intronless HIV tat gene is to Bg1 II enhanser: promoter fragment, its Sa1 I site by Nucleotide 5331 places extends to the Kpn I site at Nucleotide 8572 places, and has lacked Nucleotide 5590-7935.

Plasmid BsMtRev(1202-2) contains the 1.7kb fragment that extends to mouse metal sulfur hydrochloride 1 gene of transcription initiation site by EcoR I site approximately-1700, be extended down to the upstream of intronless HIV rev gene in the Kpn I site at Nucleotide 8572 places by the Bsu36 I site at Nucleotide 5500 places, and disappearance Nucleotide 5590-7935.

Plasmid BsMtTat(1203-1) contain the 1.7kb fragment that extends to the mouse metal sulfur hydrochloride I gene of transcription initiation site from EcoR I site approximately-1700, extend to the upstream of intronless HIV tat gene in the Kpn I site of 8572 in Nucleotide by the Sal I site of Nucleotide 5331.

Plasmid CmHIVdelXmn(1133-a1) contains the same heterozygosis CMV:HIV enhanser that derives from the expression vector pH3MPy that is used for above-mentioned plasmid: promotor.This plasmid mixes HIV5 ' guide RNA sequence just in the Xmn I site at Nucleotide 384 places in gag reads the N-terminal in sign indicating number district; This fragment includes has plenty of the cut-grafting donor site, and this site is become to be positioned at the acceptor site of many HIV upstream region of gene by cut-grafting, and said HIV gene comprises coding tat, rev and the proteinic gene of env.The Xmn I site that is positioned at Nucleotide 384 places is connected near pol by an Xba I joint (it contains TAG rotaring inter-transtating terminating cipher, stops the process of translating of the gag N-terminal peptide behind about 20 amino-acid residues) and reads on the Xmn I site at Nucleotide 4034 places of yard C-terminal.The HIV sequence is extended to the Kpn I site at Nucleotide 8572 places by the zone of coding HIV adjusting albumen and less structural protein (comprising vif, vpr, tat, rev, vpu and HIV env albumen) afterwards.By at 5 ' cut-grafting donor site of the N-terminal front of gag gene with between the cut-grafting acceptor site of reading the Ma Qu upstream of coding range protein, carry out cut-grafting, each HIV protein that this plasmid can be encoded and be listed above.

Plasmid CmHIVdelKpnAvr(Gag2TRE) (1160-a1) contain and drive that whole gag reads sign indicating number and pol reads the heterozygosis CMV-HIV enhanser of yard expression of N-terminal part: promotor, it directly is connected among the HIV genomic 3 ' partly of the encoding sequence that contains tat, rev and env protein.In this plasmid, the Nru I of expression vector p3MPy to the direct early gene enhanser of EcoR I cytomegalovirus fragment is connected on the HIV promoter sequence that starts from Nucleotide-69 place; The leading RNA sequence of HIV is by the gag gene and enter the pol gene, and is extended to the Kpnl site at Nucleotide 3372 places, and it is introduced into a poly joint that comprises Nhe I site, and this Nhe I site contains TAG rotaring inter-transtating terminating cipher that pol reads sign indicating number.The HIV sequence is extended to the Kpnl site at Nucleotide 8572 places then.This carrier contain it is believed that for proteinic synthetic, the cut-grafting of HIV gag, proteolytic enzyme, tat, rev and env, kytoplasm transport, translate, processing and all very important all sequences element of function.This plasmid can be by carrying out cut-grafting between the cut-grafting acceptor site of reading the Ma Qu upstream of 5 ' cut-grafting donor site of gag gene N-terminal front and each protein of encoding, the HIV protein of cut-grafting mRNA reaches each other protein of listing above and can encode not.It also comprises the tar element (its tat deactivation effect for the HIV promotor is necessary) that is positioned at 60 Nucleotide before the HIV one-level mRNA transcript, and the Rre(rev reactive component between Nucleotide 7315 and 7559, it is necessary for location in the endochylema of the mRNA of the catalytic coding of rev HIV structural protein).Do not contain half pol that to encode in this plasmid vector and read the sign indicating number genomic central area of HIV of (comprising that partial inversion record zymoprotein and all integrase protein matter, all vif read the N-terminal that sign indicating number and vpr read sign indicating number); This carrier also lacks most of 5 ' LTR and all 3 ' LTR and most nef and reads sign indicating number.

8.2. transfection is to produce recombinant chou HIV-1 particle in stable Chinese hamster ovary celI

With plasmid vector CmHIV del KpnAvr(Gag2TRE) and the CmHiEnv5 transfection to dhfr ^-In the Chinese hamster ovary celI.Through selecting transfected clone with plasmid pSV2dhfr cotransfection.CHO transfection body contains excretory immune response sexual maturity gag and env protein in particle forms, and its sedimentation character is similar to the HIV-1 particulate protein of above HIV virus particle described in the 6th joint and recombinant chou manufacturing.

8.2.1. the transfection of cell and selection

In 60mm tissue culture ware, make Chinese hamster ovary celI in the HamShi F12 nutritional blend (not containing xanthoglobulin) that is added with 10% foetal calf serum and 150 μ g/ml proline(Pro), grow to 50% and merge, move in the serum free medium then.With Lipo-fectin(BRL), HIV expression plasmid CmHIVde1KpnAvr(Gag2TRE) (1160-a1) and CmHiEnv5(1104-b1) and the mixture insulation 5 hours that can select plasmid pSV2dhfr, remove the Lipofectin:DNA mixture and retract and contain in the blood serum medium, thus transfection CHO cell.After the transfection 2-3 days, culture is moved on in the selective medium (DMEM adds 10%FBS and proline(Pro)).After 2 days, use the trypsin treatment culture, and the aliquot sample that will contain 0.08% and 0.4% cell of having an appointment (is estimated respectively to contain 1 and 5 * 10 ³Individual transfectional cell) moves in the 96 The Small Well tissue culture wares.The aliquot sample that will contain 7.8% cell moves in the 6 The Small Well tissue culture wares.

In selective medium, continue to cultivate after 13 days, one group of 6 The Small Well plate fixed, and through with AIDS patients serum (TriMar), then with the goat anti-human antibody of coupling peroxidase and sour jujube root peroxidase substrate (3-amino-9-ethyl-carbazole) insulation, and pair cell carries out immunostaining to detect the HIV protein expression.Every The Small Well has the colony of the cell of 15-20 selection; Through the immunostaining analysis, find wherein to have approximately 10% colony to contain HIV protein.This analysis revealed has been inoculated in the 96 The Small Well plates of less number cell, has only about 10 to 15 The Small Wells to contain the colony of the cell of transfection, and wherein a part may be expressed HIV protein.

Collect then from the every The Small Well inner tissue substratum of 96 The Small Well plates, and detect excretory gag protein (above 6.1.2. joint) by gag proteantigen EIA method.Identify many candidate's The Small Wells with this detection method.Select 12 potential male The Small Wells (by respectively obtaining 4 in three independent transfections) to carry out the cell dispersion treatment then.This moment the positive The Small Well that detects by an unaided eye, sending out existing The Small Well merges basically, the microcolony that other then have only grown cell, the cell that obviously not having of also having survived.The cell that derives from all three potential positive The Small Wells is scatter; Move in the 6 The Small Well plates with tryptic digestion and with cell and to expand.

In addition, the small portion cell inoculation that is with each candidate cell is in multiple 24 The Small Well plates.Grow after 5 days, in each The Small Well, add wild-type HeLa cell or Hela T4 cell (transfected and expression CD4 protein).Second day, detect gag expression and env function with focusing on immunodetection.Briefly, fixed cell and be incubated with the anti-HIV serum of people, the goat anti-human antibody who is connected sour jujube root peroxidase and peroxidase substrate (3-amino-9-ethyl-carbazole).After this method detected, it was male that 4 clones (being divided into two classes) are arranged.Two clones (3010-C1 and 3010-C6) have only a few cell, but its dyeing is very dense, and have formed huge synplasm effectively with Hela T4 cell, but then do not form synplasm with wild-type Hela cell.Two other clone (3010-C5 and 3010-C13) has many positive cells, but its dyeing is not too strong, and only some and Hela T4 cell have formed synplasm.The cell of other clones is not having to dye.The antibody staining of positive cell is shown that they have synthesized gag protein, form synplasm with Hela T4 cell and prove that then functional gp120 and gp41 capsid protein matter are arranged on these cell surfaces.

After further expanding each candidate cell system, with the HIV protein synthesis of Western blotting analysis of cells lysate.With PBS the cell in 60mm and the 100mm tissue culture ware is washed twice and directly collected in 300 μ l or the 750 μ l Laemmli sample buffers.After boiling,, the total cell protein matter in the sample is decomposed through electrophoresis on 10% polyacrylamide gel or 8-16% gradient polypropylene enzyme amine gel.The cem cell of HIV, HIV infection and the aliquot sample (organizing in contrast) of cowpox reorganization v-gag1, v-gag2 or v-env5 infected B SC-40 cell are incubated.With the content electrophoretic transfer of gel to a slice nitrocellulose filter.Filter membrane and AIDS patients serum or derive from the rabbit anteserum reaction of gp160 immunity of the cell of v-env5 infection, thorough washing, again with ¹²⁵The reaction of the albumin A of I mark, and thorough washing once more.Manifest reaction product with X line film through radioautography then.

Find that also four positive cell lines identifying in the detection formerly are positive to gag protein.3010-C1 and 3010-C6 lysate contain several times the gag albumen of Duoing than 3010-C5 and 3010-C13 lysate.The clone that is negative in formerly detecting also is negative to gag in the Western engram analysis.Most of immunoreactivity materials all move altogether with the p55 precursor, and as slightly little product, and and HIV, or the principal product in the cell of HIV or VacGag1 infection does not conform to; Measure ripe p24 and p17gag protein very in a small amount in the cellular lysate only.This analysis proves that also the molten born of the same parents of positive cell line exist gp160, gp120 and gp41 capsid protein matter in producing.

8.2.2. the HIV-1 particulate characteristic of recombinant chou manufacturing

For proving in these cells synthetic gag and env protein assembling assembly virus-like particle and being secreted out, collect the culture supernatant and clarify it with low-speed centrifugal.Centrifugal (in the SW55 rotor 32,000rpm, in the SW41 rotor 27,000rpm, in 19 type rotors 19,000rpm) collecting granules part.The Western engram analysis proves that this material contains gag and env protein.The basic gag protein that measures in particle is sophisticated p24 and p17 albumen; Also can measure in a small amount p55 and p40 precursor protein.Otherwise major part is a precursor protein in the cellular lysate.Gp160, gp120 gp41 capsid protein all can detect in particle.

Compare the Western trace, small portion gag that contains in the cell as can be known and env protein are as granule secretion.Quantitative gag antigen EIA analysis revealed to the granular preparation of many 3010-C6 cell generation is done produces 3-17ng gag in every milliliter of culture.The also sedimentation by in saccharose gradient (in the SW55 rotor, by 15-60% sucrose with 50, centrifugal 2 hours of 000rpm) analyzed the particle that 3010-C5 clone produces.As shown in figure 10, EIA and the Western engram analysis to the gradient centrifugation each several part shows that particle has formed unimodal band at about 35-40% sucrose place.

8.3. in mammalian cell, express ENV and GAG protein

And generation recombinant chou HIV-1 and other suitable strategies of particulate

Be described as in cell transformed expressing the multiple different strategy that substitutes that the HIV particle of HIV protein and recombinant chou manufacturing is adopted in this section.Possible change includes but are not limited to the compound plasmid vector that (1) transfection contains a plurality of HIV structures and/or regulatory gene, (2) a plurality of plasmid vectors that contain different HIV genes of cotransfection, (3) utilization structure and adjustable enhancers/promoters element are expressed to drive the HIV particulate, (4) utilize adjusting to comprise that the HIV of tat and/or rev regulates protein expression, with the expression of indirect control HIV gag and env structural protein, and (5) are at dissimilar cell inner expression HIV protein.These changes can be used for making each parameter optimization of system, and can control independently particle protein ingredient and at dissimilar intracellular expression levels.

Use the standard calcium phosphate transfection method, use BSC-40 and Vero sometimes through transient transfection Hela() cell tests plasmid and plasmid makes up.Total lysate or Recombinant HIV particle (collecting it with high speed centrifugation from substratum) product are done the polyacrylamide gel electrophoresis analysis, the product electrotransfer is detected it to nitrocellulose filter and with specific antisera.When analyzing env protein, use ¹²⁵The monoclonal antibody 110-4 of I mark detects filter membrane (6.1.2. that sees above joint), and the latter can be attached to the V of gp160 and gp120 ₃On the epitope in zone; When analyzing gag protein,, use then with AIDS patients serum (TriMar) ¹²⁵The a-protein of I mark is detected filter membrane.Manifest product with X line film with radioautography.

8.3.1. can utilize the combination of many different plasmids and plasmid to realize

The expression of HIV protein in the Hela cell

Can be through the transfection multiple HIV protein of encoding, comprise structure and regulate proteic complex plasmid and realize HIV gag and env protein expression; In addition, the also transfection proteinic multiple plasmid of different HIV of encoding together.

With absolute coding env(CmHiTgfbEnv5 or CmHiEnv5), or gag(CmHiGag2Rre) plasmid, in conjunction with encoding tat and the proteinic plasmid transfection of rev in the Hela cell, can cause the expression of immunoreactivity gp160 and gp120HIV capsid protein, or cause comprising the basic translational product of p55, p47 and p39 processing intermediate product, and the ripe gag protein of p24, p17 is in the expression of interior immunoreactivity HIV gag related protein.

In addition, can regulate albumen and env with in single transcription unit, comprising tat and rev, or plasmid vector (CmHIVdelXmn or the CmHIVdelKpnAvr(Gag2TRE) transfection of the functional encoding sequence of env and gag, structural protein also can cause precursor and ripe env or gag and env proteinic synthetic in the Hela cell.Also cause gag and env proteinic synthetic with CmHIVdelXmn and CmHiGag2Rre or CmVGag2Rre cotransfection.Use each plasmid or unite and use CmHIVdelKpnAvr(Gag2TRE) (1160-a1), CmHIVdelXmn(1133-1a)+CmHiGag2Rre(1158-a1), and CmHIVdelXmn(1133-a1)+CmvGag2Rre(1159-a1) also can cause recombinant chou HIV granule secretion in substratum.

Therefore, available number of ways uses this system to realize that the expression of gag and env structural protein and recombinant chou HIV particulate produce.Compare the expression level that is reached with different plasmid combinations in the table II, but proved the protein that the various combination optimum expression is different.For example, when when encoding tat and the proteinic plasmid co-transfection of rev respectively, can more effectively express env.On the other hand, can be by being included in the single transcription unit the two kinds plasmid CmHIVdelKpnAvr(Gag2TRE that regulate the functional encoding sequence of albumen) (1160-a1) more effectively express gag.Use complex plasmid CmHIVdelKpnAvr(Gag2TRE) when (1160-a1) encoding plasmid co-transfection, can obtain two kinds of protein of medium level with isolating env.

The table II

Share resulting env of different plasmids and gag protein expression

Level relatively

Plasmid env gag

CmHiEnv5+CmHiTat+CmHiRev ++++ -

CmHiGag2Rre+CmHiTat+CmHiRev - ++

CmvGag2+CmvRev - +

CmHIVdelXmn + -

CmHiGag2Rre+CmHIVdelXmn + ++++

CmHIVdelKpnAvr（Gag2TRE） + ++++

CmHiEnv5+CmHIVdelKpnAvr（Gag2TRE） +++ +++

Share the gag that obtains behind the various different plasmid transfections and relative expression's level of env indicates with arbitrary unit; Measure gag and env expression level respectively, and needn't use same canonical representation.

In these embodiments, rev is very important for location in the endochylema effectively of the mRNA of coding HIV structural protein, and depend on cis-acting regulatory element part, that be called rev reactive component (Rre) of the HIV env gene that is positioned at coding gp41 molecule N-terminal part, it is introduced in the gag plasmid as the Bg1 II of capsid gene to Hind III fragment, and may be for expressing HIVgag effectively and env protein is necessary.Tat albumen by be called tar, be positioned at interaction of HIV rna transcription cis-acting regulatory element of 75 Nucleotide before this, can strengthen the transcription that begins by the HIV promotor.This tar gene is present in by CmHiGag2Rre, CmHiTgfbEnv5, and in the CmHi heterozygosis enhancer element of CmHiRev utilization.

Can not rely on tat and express gag or env, because these plasmids do not contain the tat district through cotransfection plasmid such as CmvGag2Rre and CmvRev.But during with the CmvGag2Rre+CmvRev transfection, the expression level of gag is than low with the CmHiGag2Rre+CmHiTat-CmHiRev transfection.Because the enhanser of the direct early gene of known cytomegalovirus: promotor is the very strong element of transcribing, so proof CMV:HIV enhanser: it may be strong especially expression system that promoter element adds HIV tat gene.The env protein expression has also been done similar research.Heterozygosis between other enhancer elements and the HIV promotor/tat element can have other useful properties.

These results show, different HIV structural proteins is to reach maximum the expression and requirement that HIV is regulated albumen or other factors difference to some extent, and can be by selecting suitable plasmid or share these plasmids and control gag to the proteinic ratio of env.For example, can produce recombinant chou HIV particulate clone with the further transfection of the plasmid of coded auxiliary HIV albumen such as vpu or vif, to change recombinant chou HIV particulate production level and/or character.Another example is can select with coding tat, rev and the proteinic plasmid transfection clone of gag (thereby it can produce the proteinic particle of an encoding gag), and the different env of the usefulness that continues coding plasmid transfection it, contain from the different virus strain or have the proteinic particle of env of other structural modifications with generation.For example, to comparing, as seen have equally effectively and express by the natural signals sequence (CMHIEnv5) that contains HIV env gene or the env protein of genetic expression that comprises the fusion structure (CmHiTgfbEnv5) of monkey TGF-β-1 signal sequence and ripe gp160N end.

Regulate HIVenv 8.3.2. use adjustable promotor

Protein expression

In another embodiment, use the plasmid that combines CmHiTgfbEnv5+CmHiTat+CmHiRev, with selecting plasmid pSV2dhfr transfection dhfr ^-Chinese hamster ovary celI.The first piece inspection of the clone of selection in this experiment is not found that the HIV env of the level of can surveying expresses.Cell is grown in the substratum of cumulative methotrexate, the alternative pSV2dhfr plasmid of the transfection of increasing, and coamplification HIV tat, rev and env coding plasmid is to select cell, the low-level HIV env expression thereby expectation can be selected to increase.The result shows and has synthesized HIV env protein in the clone.Growth can not cause the raising gradually of env protein expression level with the cell of the HIV encoding histone plasmid of select further to increase dhfr gene and cotransfection in the methotrexate of cumulative concentration.For keeping the env protein expression level preferably to proceed to select,, then capsid protein matter expression level can occur in the cell growth and reduce because if further do not select.As if the proteinic level of cell expressing env is high more, and its growth is slow more, and lower than those clone density that return back to low env protein expression level.These results show that the expression of capsid protein matter is virose in these cells, and can therefore the env protein expression be limited on the accessible level.

Can use the plasmid that can regulate promotor to overcome this restriction with control HIV protein expression.Can be in initial cell, and then induce and make it to express HIV protein high-levelly with not induction state growth with these plasmid transfections, thus be subjected to toxic action make before attacking its at short notice high level produce expression product.

Example as a kind of like this method, tat of coding respectively and the proteinic plasmid of rev under the mouse metal sulfur hydrochloride promotor control that is in the metal adjusting have been made up, share following plasmid cotransfection Hela cell: (a) CmHiTgfbEnv5+CmHiTat+CmHiRev, (b) CmHiTgfbEnv5+BsMtTat+CmHiRev and (c) CmHiTgfbEnv5+CmHiTat+BsMtRev, use the total lysate of Western blotting analysis of cells then, prove that all cells has all produced immunoreactivity gp160 and gp120 HIV capsid protein matter (Figure 11).Produced the env protein expression of highest level with the CmHiTgfbEnv5+CmHiTat+CmHiRev transfection.More meaningfully, find in the transfection of carrying out with the plasmid that utilizes the Mt promotor, can be through in tissue culture medium (TCM), adding zinc and make expression level improve several times (seeing Figure 11) the day before yesterday in harvested cell.

These results show, can utilize adjustable promotor to regulate the HIV protein expression, and can regulate the expression that protein is regulated the HIV structural protein by regulating HIV tat and rev.Another example of this method comprises the expression of the complex plasmid that uses the multiple HIV protein of adjustable promotor control coding, perhaps make up new derivable regulatory element, as contain regulatory element (this structure links to each other with the tar district with the promotor of HIV) the heterozygosis Mt:Hi enhanser promotor of metallothioneins promotor.

8.3.3. in BSC-40 and VERO cell, express HIV protein

In the present embodiment, share coding HIV tat, rev, env and the proteinic various plasmids of gag (comprising CmHiGag2Rre+CmHiEnv5+CmHiTat+CmHiRev), and CmHIVdelKpnAvr(Gag2 TRE) transfection Hela, BSC-40 and Vero cell.With regard to product manufacturing and expression efficiency, the primary expression feature of each clone gag and env all is similar.But for the highest, low 3 times approximately in BSC40 cell then hangs down 5 times in the Vero cell to total expression level at the Hela cell.

These results and above-mentionedly together prove with the result that Chinese hamster ovary celI performed an analysis can use the various kinds of cell system that derives from not of the same race and Different Organs to express HIV protein, thereby can be the cell that specific purpose is selected specific desired characteristic for use.

9. embodiment: use the reorganization of the HIV-1 capsid antigen of expressing clipped form

Vaccinia virus produces Recombinant HIV-1 particle of the constitutional features that modification is arranged

Two strain recombined vaccinia virus of the HIV-1gp160 expression of instructing brachymemma in infected B SC-40 cell are described below.Can use these recombined vaccinia virus to make carrier, and share above the 6th joint of v-gag2() or the carrier of other coding cAgs, produce Recombinant HIV-1 particle that has improved antiviral and/or immunogenic properties with the described system of the 6th joint above.

9.1. recombined vaccinia virus V-ED2

Structure contains the recombinant plasmid of HIV-1 env encoding sequence, and said sequence is the Nucleotide 5705 to 8068 in cowpox recombinant vectors pGS62 is inserted in BamHI site, 7.5K promotor downstream.The HIV-1 sequence is to see common pending U.S. Patent Application No.07/593 as plasmid pv-env5(, 401, October 5 nineteen ninety applying date) 2.36Kbp BamHI fragment obtain.This fragment contains complete gp120 encoding sequence, and 241 amino acid of the N-terminal of gp41, is included in preceding 49 amino-acid residues of the gp41 cytoplasmic domain of the possible C-terminal of striding the film sequence.By method described in the above-mentioned common pending U.S. Patent Application, the mosaic gene that will contain 7.5K promotor and HIV-1 env sequence inserts in the vaccinia virus thymidine kinase gene.

Gained recombinant virus v-ED2 instructs the expression of the gp160 that is cut short, and the latter is with the same gp120 and the gp41(Figure 12 of being cracked into effectively of wild-type gp160).The HelaCD that is infected by v-ED2 ⁺ ₄Cell is easier to form full cell space (the multinuclear giant cell is the feature of HIV-1 infection) than v-env5 the infected, and it can express total length gp160 capsid glycoprotein precursor.These results disclose, and capsid glycoprotein comparable " wild-type " the capsid glycoprotein that is produced by v-ED2 has better functionally active or can more effectively present it.

9.2. recombined vaccinia virus V-ENV5DCT

Construction of recombinant virus v-ENV5DCT is to contain whole env encoding sequence (the BRV isolates of HIV-1v except that 13 amino acid of C-terminal of gp41; Wain-Hobson et al., 1985, Cell, 40:9317).By the oligonucleotide mutafacient system of having stated (Kunkel et al., 1987, Meth.In Enzymol., 154:367-382) introducing deletion mutantion.By pending U.S. Patent Application No.07/593, the 5 days October nineteen ninety applying date of 401() method described in will contain in the mosaic gene insertion vaccinia virus thymidine kinase gene of HIV env sequence of 7.5K promotor and sudden change.

10. embodiment: use the recombined vaccinia virus of expressing undressed GP160 to produce

Have by the Recombinant HIV of the constitutional features of modification-1 particle

Below describe two recombined vaccinia virus that instruct gp160 precursor capsid P-glycoprotein expression, wherein said glycoprotein has sudden change in the proteolytic cleavage site (S) between gp120 and gp41.Can use these recombined vaccinia virus to make carrier, and in conjunction with v-gag2 or other coding core-antigenic carriers, the system described in the 6th joint produce Recombinant HIV-1 particle that has improved antiviral and/or immunogen character in order to using above.

10.1. recombined vaccinia virus v-160NG

Construction recombination plasmid (pv-160NC), this plasmid contain insert 7.5K promotor downstream among the cowpox recombinant vectors pGS62, from the gp160 encoding sequence of the sudden change of Nucleotide 5803 to 8495.Sudden change be roughly by state method (Kunkel et al., 1987, Meth.Enzymol. 154:367-382) introduces through the oligonucleotide specific mutant.Mutant nucleotide sequence ("+" represents the cracking site between gp120 and the gp41) as follows:

HIV-1?BR?env gp120 ↑gp41

Gln?Arg?Glu?Lys?Arg ↑Ala

" wild-type

" ... CAG AGA GAA AAA AGA ↑ GCA...

v-160ONC ...CAG?ATA?GAA?GAA?TTC ↑GCA...

Gln IleGlu Phe↑Ala

According to common pending U.S. Patent Application 07/593, the 5 days October nineteen ninety applying date of 401() method described in, reorganization is inserted mosaic gene in the vaccinia virus genome at the thymidine kinase gene place in body.The gained recombinant virus instructs the expression of the gp160 precursor capsid glycoprotein that is not cracked into gp120 and gp41.

10.2. recombined vaccinia virus v-11K160NC

Structure contains 10.1 joints that see above with v=160NC() same but be in the recombined vaccinia virus of the sudden change HIV-1 env gene under the control of vaccinia virus 11K promotor.Go up cutting-out 2.26kbp BamHI fragment by plasmid pv-160NC, and insert cowpox recombinant vectors pSC10(Chakrabarti et al. in the EcoRI site in 11K promotor downstream, 1985, Mol.Cell.Biol. is in redundant organism 5:3405-3409).So just produced the mosaic gene that contains vaccinia virus 11K promotor and complete sudden change gp160 encoding sequence.This mosaic gene inserts in the vaccinia virus genome at the thymidine kinase gene place.The recombinant virus of gained instructs the HIV-1gp160 of high level generation sudden change in the cell that infects.

11. embodiment: the recombined vaccinia virus of expressing HIV-1 vpu gene

Except that coexpression HIV-1 capsid and cAg, assembling of Recombinant HIV-1 particulate and generation do not need the HIV-1 specific protein.But this process can be participated in and quicken to other factors in virus and cell source.A kind of factor is the virus protein by the vpu coding.This protein is the glycosylated polypeptides of obvious and the related 16kb of endochylema film internal surface.Though it is not necessary that vpu forms for particle, the virus particle that the sudden change among the vpu can cause being discharged by infected cells reduces (Terwilliger et al., 1989, Proc.Natl.Acad.Sci.USA, 86:5163-5167; Strebel et al., 1989, J.Virol., 63:3487-3791).The mechanism of vpu effect still do not understand, and do not determine its in recombination system as herein described to granuloplastic effect.Expection accessory molecule such as vpu can promote virus particle assembling and/or dispose procedure.The following example is described one and be can be used for detecting these accessory molecule effects, and proves the system of their practicality in the reorganization particle produces.

Structure one contains the recombined vaccinia virus of complete HIV-1 vpu encoding sequence.The 290bp fragment of the HIV-1cDNA of Nucleotide 5636 to 5927 is inserted among the cowpox recombinant vectors pGW62 between the Sma I in 7.5K promotor downstream and EcoR I site.This fragment contains the vpu encoding sequence and 7bp5 ' does not translate sequence and 37bp3 ' does not translate sequence.Reorganization is inserted this mosaic gene in the vaccinia virus genome in body then.Press above method described in the chapters and sections such as the 7th joint, proof vpu is to the effect of formation of reorganization particle and release in the cell of v-vpu and v-G2E5 cotransfection.

12. embodiment: Recombinant HIV-1 particulate antivirus action

Present embodiment is intended to prove that Recombinant HIV of the present invention-1 particle is in external reduction or eliminate HIV to CD4 ⁺Infective ability of lymphocyte.The result shows that Recombinant HIV-1 particle can dosage dependence mode suppress the HIV-1 infection effectively.

12.1. infectious detection method

With every The Small Well 4 * 10 ⁴Individual cell is inoculated into the T lymphoblast (CEM) in the 0.4ml substratum (adding the RPMI-1640 of 10% foetal calf serum) in the 24 The Small Well culture plates.Recombinant HIV particle in the 0.4ml substratum of every pair of each different amount of acceptance (seeing Table 1) of repetition The Small Well.According to see above 6.1.2. joint of EIA() the quantitative particle that adds of p24 gag concentration of equal value that records.The contrast The Small Well does not add particle, only adds the 0.4ml substratum and supplies volume.Cell and particle are incubated 3 hours down in 37 ℃, add HIV then.One group of each acceptance (1) of repetition The Small Well that is equivalent to variant granule density does not add HIV, and (2) are equivalent to the 40TCID of 5pg p24 gag ₅₀(the infectious dose unit 50 of tissue culture) low viral dosage, or (3) are equivalent to the 400TCID of 50pg p24 ₅₀High viral dosage.Infected the back the 3rd day, and discarded substratum and change the fresh culture that contains suitable primary particles concentration into by each The Small Well.In infecting the back in the time of the 5th and 6 day, collect the cell sample of five equilibrium by each The Small Well, use the indirect immunofluorescence detection method, detecting the interior antigenic expression of HIV of cell determines infectious, wherein utilize the anti-HIV serum of people's polyclone to make first antiserum(antisera), the goat anti-human igg with the combined with fluorescent element makes second antiserum(antisera) then.

12.2. result

The infectious result that table I bottom provides shows that Recombinant HIV-1 particle can block the infectivity of HIV virus particle to cem cell in dosage dependence mode.Also deducibility, reorganization particle right and wrong own are infective, and can not be in time multiplexed cell system because with the cell of high density Recombinant HIV-1 particle heat-insulating in do not detect fluorescence in the cell (Figure 13, A group).In addition, although observe a plurality of synplasms in the cell culture (Figure 13 .C group) that adds HIV, the reorganization particle can not induce synplasm to form.

The table III

The infectivity that suppresses HIV virus with Recombinant HIV-1 particle

Cem cell Recombinant HIV-1 particle virus ²Fluorescence %

(excessive multiple) ¹(TCID ₅₀) 5 days 6 days

+ 0 0 0 0

+ 60 0 0 0

+ 600 0 0 0

+ 0 40 20 ＞90

+ 60 40 10-20 ＞90

+ 600 40 ＜1 10

+ 0 400 90 ＞90

+ 60 400 60-80 ＞90

+ 600 400 30 ＞90

1 particle for the excessive multiple of virus with respect to equivalent p24 protein.

2 human immunodeficiency virus type 1s (HIV-1), the LAV-1 virus strain.

13. embodiment: Recombinant HIV-1 particle suppresses by HIV-1 serum

The HIV-1 of the peripheral blood lymphocyte of positive donor separation infects

Above the embodiment described in the 12nd joint proves that Recombinant HIV-1 particle relies on mode in the infection of vitro inhibition HIV-1 to T lymphoblast (CEM) with dosage.Following embodiment uses peripheral blood lymphocyte (PBL) system of cultivation further to confirm Recombinant HIV-1 particulate antivirus action.The PBL that is used for this analytical system is isolating by the seropositivity donor of HIV-1 infection, and the infection frequency scope of infected cell is 0.04%-1.3%(Psallidopoulos among the isolating PBL, M.C., et al., 1989, J.Virol., 631:4626-4644).Between incubation period, the CD4 that virus infection is broadcast rapidly by acellular and cell-cell propagation approach and do not infected ⁺Cell.Nearest result (the Daar that obtains by other research groups, E.S., et al., 1990, Proc.Natl.Acad.Sci.USA, 87:6574-6578) prompting, with regard to the reaction to the effect of neutralization effect and enantiopathy toxic agent (as solubility CD4), the laboratory isolate of HIV-1 is different from initial patient's isolate.Therefore, the peripheral blood lymphocyte system appraisal Recombinant HIV-1 particulate antivirus action of use cultivating may have better biology dependency than the described cem cell of the 12nd joint system above.

13.1. infectious detection method

Separate the faint yellow clad material on the Hypaque ficoll buffer reagent, gather in the crops peripheral blood lymphocyte (PBL) in the blood by HIV-1 seropositivity donor.Then cell is placed substratum (adding the RPMI-1640 of 10% foetal calf serum) insulation to handle 1 hour with CD8, CD16 and CD20 monoclonal antibody specific down for 37 ℃, add the rabbit complement again and handled 1 hour down, to remove CD8 in 4 ℃ ⁺Lymphocyte (Zarl-ing, J.M., et al., 1990, Nature(London) 347:92-95).The gained cell product is mainly by CD4 ⁺Lymphocyte, bone-marrow-derived lymphocyte and scavenger cell are formed.The concentration of cell in the 0.5ml substratum with 1M cell/The Small Well is inoculated in the 24 The Small Well culture plates.Add the Recombinant HIV particle of different concns (seeing Table IV and V), the HIV-1 virus particle of psoralene-UV-light inactivation or the HIV-1 capsid glycoprotein precursor gp160 of purifying in each multiple The Small Well respectively.All The Small Wells all add 0.5ml and contain and the respond MAb G19.4(Hoxie of activity of T cell CD3 antigen then, J.A., et al., 1986, Science, 234:1123-1127) substratum of (final concentration is 1 μ g/ml) and interleukin II (IL-2) (final concentration 5%).Shown duplicate (Zarling, J.M.et al., 1990, the Nature(London) 347:92-95) that can induce HIV-1 virus in latent period with solubility CD3 MAb activating T cell.

4 or 5 days (being respectively donor Z29, Z30 or donor Z31, Z39) in activation back by extracting substratum in the The Small Well, washed cell and changed fresh culture into the PBS that adds 10% human serum.The viral insulation of HIV-1 that makes identical sample list and fresh culture (seeing below) or add Recombinant HIV-1 particle or the inactivation of proper concn again.After for the second time adding particle or inactivation virus, with certain interim by results media samples in the The Small Well and with specificity EIA method (6.1.2 that sees above joint) analysis p24gag content.In addition, the monoclonal antibody mixture of collecting cell and use and gp120 and gp24gag reaction simultaneously detects the HIV-1 protein expression.

Wash out the cell of the virus of reorganization particle or inactivation in the time of the 4th or 5 day, moved in the 8th day and contain in the new The Small Well of the anti-CD3 MAb of immobilization, continue to duplicate with inducing cell.Generation with p24gag in the EIA method detection culture supernatants sample in the 11st day, (seeing Table V).Designing the experiment of this part is reversible phenomenon with the restraining effect to the virus generation whether appraisal records after with Recombinant HIV-1 particle disposal.

13.2. result

Provided the result of analysis of infection in table IV and the V.The inhibition of Recombinant HIV-1 particle is passed through to infect from the culture transmitted virus of the sample of four different seropositivity patient's acquisitions.This inhibition is a dose-dependently, and can come out with the proteinic flow measurement of 0.1 μ g/ml p24gag of equal value in the sample of Recombinant HIV-1 particle disposal.Though the virus of psoralene/UV-light inactivation can similarly suppress the diffusion (table IV, donor Z29, the 6th day sample) of virus infection in some cases, compares with Recombinant HIV-1 particle, needs many 10 times inactivation virus.

The cell of washing Recombinant HIV-1 particle or inactivation virus at the 4th day off still continues to suppress p24gag and produces, and prompting removes degranulation and is not sufficient to make this phenomenon to reverse (table V).Interesting is that data are also pointed out the HIV-1 virus particle more effective (table V, two donors) of Recombinant HIV in this restraining effect-1 particle than inactivation.

The table IV

Recombinant HIV-1 particle is to the restraining effect of HIV-1 viral infection

Donor treatment dosage (μ g/ml) ¹The percent inhibition that p24gag produces

The 9th day

Z31 particle 1 86

0.1 18

0.01 0

Z39 particle 1 99

0.1 5

0.01 0

The 6th day

Z29 particle 10 96

1 97

0.1 73

0.01 15

The HIV 10 90 of deactivation

1 78

0.1 0

Z30 particle 10 86

1 84

0.1 22

0.01 0

The HIV 10 84 of deactivation

1 78

0.1 0

¹The concentration of Recombinant HIV-1 particle and inactivation virus is listed with respect to p24gag concentration.The relevant capsid glycoprotein content of these preparations is the 5-10% of p24gag.

The table V

Recombinant HIV-1 particle is irreversible to the restraining effect that HIV infects

Donor treatment dosage (μ g/ml) ¹The percent inhibition that p24gag produces

Z29 particle 10＞79

1 ＞51

0.1 0

0.01 0

The HIV 10＞44 of deactivation

1 0

0.1 0

Z30 particle 10 99

1 91

0.1 52

0.01 4

The HIV 10 50 of deactivation

1 15

0.1 1

1 concentration of handling is with shown in the table IV.

2 in infecting the restraining effect that the back was detected the p24gag generation on the 11st day.Wash cell on the 4th day and make it to recover the situation that is untreated.For guaranteeing the cell growth, in the following culturing cell of 5%IL-2 existence and in the immobilised CD3 MAb of the 8th angel's cells contacting.

14. embodiment: immunogenicity in the Recombinant HIV-1 particulate body

Use small animal model to carry out following experiment, to evaluate immunogenicity in the non-replicability Recombinant HIV-1 particulate body.Selecting rabbit for use in these researchs, is to have neutralizing antibody because once be reported in the past in the rabbit body of accepting various forms HIV base immunogen immune.

Evaluate each by the humoral immune reaction of immunize rabbit with ELISA and Western engram analysis method.Available ELISA method detects total HIV specific antibody titre, and with the Western engram analysis method estimation antibody response with indivedual virus proteins.In addition, identified feature at two cell immune responses that cause in by the immunize rabbit body.These result of experiment have confirmed that further Recombinant HIV-1 particle produces the ability of HIV-1 specific immune response in the immunized animal body.

14.1. immunogenicity detection method

Two female New Zealand white rabbits of HIV-1 virus immunity with Recombinant HIV-1 particle (above the 6th joint) or psoralene deactivation.Used immunogenic amount is according to being able to standardized to capture the relative p24gag protein concentration that EIA method (above 6.1.2. joint) records.Immune programme for children is shown in down in the tabulation VI.For fundamental immunity (1 °), add complete freund's adjuvant preparation immunogen and to be equivalent to 120 μ g p24gag and the proteinic amount of 4-6 μ g gp120env through the intramuscular injection immune animal with 1: 1 ratio.After 5 weeks, add incomplete Freund's adjuvant preparation immunogen, rabbit is done immunity (2 °, booster immunization) for the second time through subcutaneous route with 1: 1 ratio.Used immunogenic amount is equivalent to 190 μ g p24 and 10-20 μ g gp120 protein in the booster immunization.

At interval by collecting serum in the animal body, use the virus or the immobilised pure gp120 of immobilization completely destroy to detect anti-HIV reactivity with different time with ELISA method and/or Western blotting.In addition from beginning to test the serum of collecting each animal the last week, as pre-immunity contrast.For the Western engram analysis, the LAV-1 virolysis of purifying in the SDS-PAGE sample buffer, and is carried out electrophoretic separation in acrylamide gradient (7%-15%) plate gel.With standard technique isolating virus protein is transferred on the nitrocellulose filter.Then filter membrane is cut into identical and be used for the Western engram analysis.Briefly, with blocking damping fluid (3% exsiccant milk is dissolved among the PBS bovine lacto transfer technique optimizer) under 22 ℃ the blocking-up of filter membrane bar after 3 hours, being made filter membrane bar and 4 ℃ of following incubated overnight of specificity rabbit anteserum sample of diluting at 1: 50 in bovine lacto transfer technique optimizer 0.2%NP40.These filter membrane bars of thorough washing then are with in bovine lacto transfer technique optimizer 0.2%NP40 ¹²⁵I albumin A (ICN Radiochemicals) is incubated 2 hours down for 22 ℃.Manifest protein band through radioautograph.As the indication that virus produces and discharges, also checked and to have reduced among the p24gag generation level in the serum and the existence of antibody.

Use the infectious detection method of external cem cell to detect by in the immunize rabbit serum and the existence of the antibody of LAV-BRU isolate.Heat inactivation serum, then in substratum (adding the RPMI-1640 of 10% foetal calf serum) serial dilution it.The serum and the 30TCID that mix the equal-volume dilution ₅₀The virus inoculation thing also is incubated 45 minutes under 37 ℃.0.1mlCEM cell culture (2 * 10 in being present in 96 The Small Well microtiter plates ⁴Individual cell) virus formulation of add attacking in (0.1ml), 37 ℃ of insulations and mix it at any time down.Be incubated after 1 hour, by removing virus-serum mixture in the The Small Well, and change the substratum that contains beginning rabbit anteserum at the beginning of the suitable extent of dilution into.Use the p24gag level in the EIA method detection supernatant after 5 days, with the progression of infection in the monitoring culture.

The table VI

The rabbit immune programme for children

238,239 0 weeks of rabbit: 1 ° of immunity

5 weeks: 2 ° of immunity

33 weeks: 3 ° of immunity (having only rabbit 238)

241,243 0 weeks: 1 ° of immunity

4 weeks: 2 ° of immunity

18 weeks: 3 ° of immunity (having only rabbit 241)

Immunity ¹

The rabbit immunogen

1° 2° 3°

238 Recombinant HIV-1 particle, 120 μ g, 190 μ g, 140 μ g

120μg 100μg 120μg

The HIV-1 120 μ g 190 μ g of 239 deactivations

120μg 100μg

¹The preparation fundamental immunity is former and through the intramuscular injection administration in complete Freund's adjuvant; Preparation first and immune for the third time immunogen and in incomplete Freund's adjuvant through the subcutaneous injection administration.

14.2. result

14.2.1. the antibody titers that records with the ELISA method

Figure 14 has provided the rabbit 238 that records with the ELISA method and 239 before 1 ° of immunity and the relative antibody titers in the serum sample of immunity back 2,5 collection during with 7 weeks.Rabbit 238(Figure 14 A) finds expression in and has improved the proteinic immune response of HIV (seeing 7 weekly datas, i.e. 2 weekly datas behind the booster immunization) 5 weeks of immunity back (being maintained to 2 ° of immunity backs).On the contrary, in rabbit 239 samples 1 ° of immunity back recorded in 2 weeks with the proteinic significant reaction activity of HIV (Figure 14, B), and in 5 week the back reduce.But can be observed the remarkable booster action of antibody titers in the serum sample of collecting in by rabbit 239 bodies in the 7th when week.

A kind of explanation to reinforcement Low Response in the rabbit 238 is that (seeing Table the bloodletting program of VI) carried out in 2 ° of immunity of animal during the peak value immune response.Otherwise, descending when 2 and 5 weeks (relatively bloodletting detected result) when imposing 2 ° of 239 intravital antibody titerss of rabbit when immune, it is more remarkable therefore to strengthen reaction.

Be to use immobilization the to break full capsid glycoprotein gp120 of virus (group 1 and 2) or immobilization purifying carries out the ELISA test to all rabbit anteserums the result who provides among Figure 15.Shown in data are terminal point titres during the different interval after the fundamental immunity.The abscissa value is represented all numbers when collecting serum sample after the fundamental immunity.The arrow indication second time and immune for the third time time (seeing Table VI).As shown in the figure, all produced the antigen reactivity figure that is associated with the reinforcement program with the rabbit (R238 and R241) of Recombinant HIV-1 virus particle immunity and with the rabbit (R239 and R243) of the HIV-1 virus particle immunity of psoralene/UV-light deactivation.Mainly reflected reactivity with p24gag protein (its constitute in Recombinant HIV-1 in the HIV-1 virus particle about 90% protein content) with the respond titre of antibody in active each rabbit anteserum of the totivirus of fragmentation.With the reactivity of totivirus approximately than with reactive high 2 to 3 orders of magnitude of gp120, this has represented directly and low-level (5-10%) relevant otherness of grain pattern underpants chitose albumen.

14.2.2. with in the rabbit anti-serum and the infectivity of HIV-1

Figure 16 show with the immunity of Recombinant HIV-1 particle produced anti-homology HIV-1(LAV-1 virus strain, BRU isolate) neutralizing antibody.As to the global reactivity of virus protein, the titre of neutralizing antibody is relevant with the booster immunization program.Fig. 7 C also shows, in the rabbit 238 of Recombinant HIV-1 particle immunity and 241 serum, occurred senior middle school and titre (Figure 16, A).The titre of being reported is relevant with 75% inhibiting rate that p24gag produces.

14.2.3. cell immune response

Summed up the cell immune response that causes with after the immunity of reorganization particle in the VII of tabulating down.These results show, by being bred in the reaction to the differential stimulus of HIV-1 antigen by isolating lymphocyte in the immunize rabbit body, this is in the indicator cells immunity the necessary active a kind of reaction of memory t cell of institute.Two with the rabbit of Recombinant HIV-1 particle immunity through immunity back stimulation index is more meaningful for the third time.Very high stimulation index and measured total height immunoreactivity relevant (Figure 15, B that rabbit 243 is recorded; Figure 16).

The table VII

By the HIV specific T-cells proliferative response of immunize rabbit

Immunogen animal stimulator antigen stimulation index

Strengthen for the first time strengthening for the second time

The HIV 3.0 15.0 of reorganization particle 238 deactivations

Reorganization particle N.T. 8.4

The HIV 2.2 5.4 of 241 deactivations

Reorganization particle N.T. N.T.

HIV 2.6 N.B. of UV-light/benefit bone 239 deactivations

The reorganization particle N.T. N.B. of the plain deactivation of fat

HIV 95.0 N.B. of 243 deactivations

Reorganization particle 33.0 N.B.

N.B.: do not strengthen.

N.T.: not test (N.T.).

Stimulation index: mix among the PB1 that is stimulated ³[H] TdR cpm number is divided by mixing among the PBL that is stimulated ³[H] TdR cpm number.

14.2.4. the reactive Western engram analysis of antagonist

Provided the result of Western engram analysis among Fig. 8.The reaction of the fundamental immunity that occurs in the 5th all serum of rabbit 238 is that antagonism gag is proteinic, and only with the gp41env protein activity (Figure 17, swimming lane 1) that responds.Do not measure clear and definite stiffening effect though detect the 7th all serum samples of rabbit 238 with the ELISA method, but the Western engram analysis then shows redistributing of antibody response arranged between all kinds protein, and the proteinic new antibodies reactivity of appearance and higher molecular weight HIV (Figure 17, swimming lane 2).Otherwise, then mainly be owing to improved and gag protein and the proteinic reactivity of env gp120 by the virogenetic remarkable reinfocing effect of inactivation (Figure 17, swimming lane 3).The reactivity of the individual human serum of the seropositivity of the merging of the swimming lane 5,6 of Figure 17 and the 7 various concentration of representative (being respectively 1: 100,1: 1000 and 1: 10000), and as control antibodies.

15. embodiment: Recombinant HIV-1 particle is incorporated into CD4 ⁺

On the cell and enter CD4 ⁺In the cell

Design this experiment with check Recombinant HIV of the present invention-1 particle whether with CD4 ⁺The cell combination, and warp merges with the endochylema film and enters in the cell as the HIV virus particle.

15.1. internalization detection method

Used Hela cell in this detection method by the gene transfection of coding CD4 molecule.Determined the characteristic of these cells, and shown that its support productivity HIV-1 infects.Make cell in the Eagles substratum of the DulbeccoShi improvement of adding 10% foetal calf serum, on slide, be monolayer growth.Wash cell and be incubated 2 hours down in 37 ℃ with PBS/1%FCS with 50 μ g/ml Recombinant HIV particles.The thorough washing cell is also fixed it with 3.7% paraformaldehyde solution then.Fixed cell and 1: 100 dilution, can with the monoclonal antibody mixture of the gp120 of HIV-1 and p24gag proteins react in 22 ℃ of insulations 30 minutes down.Once more the thorough washing cell and with the goat anti-mouse antibody insulation that is attached to the protein affinity purification on the FITC of dilution in 1: 50.22 ℃ are descended insulation after 30 minutes, wash cell and are fixed in to prepare against in the sealing matrix with PBS/1%FCS and observe.With examining method (CLSM) analytic sample to dye in the showed cell with burnt laser scanning microscope.

15.2. result

CLS Photomicrograph shown in Figure 18 is promptly represented above-mentioned assay.The Hela CD4 that presents positive fluorescence behind Figure 18 A demonstration and Recombinant HIV-1 particle heat-insulating ⁺Cell.Figure 18 B shows the result that same cell is carried out the inspection of optics subdivision with the CLSM method, begins from the sample top and pushes surface of glass slide gradually to 1 μ m increment.These Photomicrographs show that fluorescence manifests more along with intracellular environment and increases, and this shows that Recombinant HIV-1 particle is combining back and then internalization with cell.

16. embodiment: Recombinant HIV-1 particle is the non-human primate

In immunogenicity

How following embodiment explanation detects the non-human primate intravital immunogenicity of Recombinant HIV-1 particle in the recombined vaccinia virus immunity of the HIV-1 virus particle of using Recombinant HIV-1 particle, psoralene/UV-light deactivation and expression HIV-1gag and env protein.The result proves that Recombinant HIV-1 particle has caused cellular immunization and comprised the humoral immune reaction that the anti-HIV-1 neutralizing antibody produces.

16.1. immune programme for children

HIV-1 virus particle or expression HIV-1gag and the proteinic recombined vaccinia virus immunity of env 12 macaques (Macaca fascicularis) by following program Recombinant HIV-1 particle, psoralene/UV-light deactivation:

Group number (n=2) fundamental immunity is former/and the adjuvant second immunisation is former/the adjuvant booster immunization

1 V-G2E5/ does not have the 8th week of particle/IFA

2 V-G2E5/ do not have the 8th week of rgp160/IFA

3 particles/the 4th week of IFA particle/IFA

4 particles/the 4th week of DETOX particle/DETOX

The 4th week of HIV/IFA of the HIV/IFA deactivation of 5 deactivations

The 4th week of HIV/DETOX of the HIV/DETOX deactivation of 6 deactivations

Use the cutaneous scarification method to be inoculated animal 1 * 10 with every ⁷The plaque forming unit of individual V-G2E5 (above the 7th joint) carries out immunity with the recombined vaccinia virus of living.Capture with p24 antigen that detection method (Genetic Systems) detects every dose of Recombinant HIV sample particle and the psoralene/HIV virus particle of UV-light deactivation is equivalent to contain the p24 of 200 μ g, and detect the gp120 that it approximately contains 6 μ g with immunoblotting.By with purification of Recombinant gp160 in the BSC-40 cell of expressing same antigenic recombinant vaccinia virus infection, and use it with the dosage of the each immune 6 μ g of every animal, this dosage level is similar in appearance to Recombinant HIV-1 particulate dosage level.At incomplete Freund's adjuvant (Difco) or DETOX(RIBI Immunobiochm) all particles of preparation or subunit's immunogen in (it contains toxic monophosphoryl lipid A and bacteria cell wall skeleton).All finish with all immunity that the HIV-1 virus particle of reorganization particle, deactivation or the gp160 that recombinates carry out with intramuscular injection.

16.2. result

Detect the anti-HIV-1 immune response that these macaques produce with following method: (1) detects total HIV-1 specific antibody with totivirus particle ELISA method; (2) detect HIV-1 capsid specific antibody with the gp120ELISA method; (3) detect neutralizing antibody with focusing on immunodetection; (4) detect cell-mediated immunity according to Lymphoid tissue proliferative response to the HIV-1 hormesis.The results are summarized in down in the tabulation VIII.

The table VIII

By the anti-HIV-1 immune response of the macaque of immunity generation

Group number and immune animal antibody response ^a

Among the HIV gp120 and S.I ^b

1 v-G2E5/ 89133 ＞25600 ＞1600 ＞400 2.1

Particle 89,149 12800＞1600＞400 115.4

2 v-G2E5/ 89147 1600 400 25 74.3

gp160 89138 0 800 100 69.7

3 particle 88079＞51200＞800 25 3.9

（IFA） 89068 6400 ＞1600 25 50.0

4 particles 88,116 800 1,600 25 2.8

（DETOX） 89072 0 ＞25 0 4.6

5 deactivation HIV 89,150 12,800 800＞25 4.7

（IFA） 89086 3200 ＞25 0 43.3

6 deactivation HIV 89,151 1,600 1,600 25 5.7

（DETOX） 89156 50 800 0 3.9

Contrast monkey 000 0.9

The a antibody response is in 4 weeks of back of immunity for the second time, i.e. the 1st and 2 treated animals 12 weeks after immunity, the 3rd to 6 treated animal detects during 8 weeks after immunity.

In b Lymphoid tissue proliferative response 4 whens week after fundamental immunity, detected.Stimulation index (S.I.) is to mix intracellular that HIV-1 stimulates ³H-thymidine c.p.m number is divided by mixing do not stimulate intracellular ³H-thymidine c.p.m. number and definite.

The result shows shown in the table VI: (1) Recombinant HIV-1 particle is immunogen and can causes the HIV specific immune response, particularly when being used for having used the animal of expressing env and the pre-immunity of the antigenic recombined vaccinia virus of gag before this; (2) strengthen the previous immunizing power of using the animal of the pre-immunity of Recombinant HIV-1 particle, can make it than more effectively causing immune response with equivalent solubility gp160 booster immunization, this is likely because Recombinant HIV-1 particle can transmit capsid antigen effectively; (3) when being used for basis and second immunisation as single immunogen, Recombinant HIV-1 particle can be in great majority be accepted the macaque body of immunity, with the level of the animal of waiting the immunity of dosage deactivation HIV-1 virus particle similar in appearance to usefulness, cause the t cell immune response of HIV specific antibody.

17. microbial preservation

Following recombinant vaccine virus be deposited in American type culture collection (Rockville, MD), and specified preservation registration number:

The recombinant virus registration number

v-env5 VR 2113

v-gag2 VR 2265

v-G2E5

Following recombinant plasmid has been deposited in NRRL(Peoria, Illinois) and specified preservation registration number:

Plasmid host registration number

CMHiEnv5（1104-b1） E.coli DH5a

CmHIVdelKpnAvr E.coli DH5a

（Gag2TRE）（1160-a1）

The present invention is not limited only to the scope of the virus of preservation, because the example of preservation virus is just as indivedual examples of one of the present invention aspect, its any function equivalent is all within the scope of the invention., the shown and description person various changes of the present invention are seen for those skilled in the art except that this paper, all become conspicuous with reference to above explanation and accompanying drawing.These are changed and all to fall into the present invention and await the reply in the scope of claim.

Also should be clear and definite be that it all is proximate relating to all base pairs of Nucleotide and peptide and amino-acid residue number and given magnitude range, and the purpose in order to describe just.

The nucleotide position of given HIV-1 gene all is based at Wain-Hobson et al., and 1985, the genome sequence of the LAV-BRU isolate of delivering among the Cell 40:9317 and fixed.

Claims (151)

1, the HIV particle of the non-replicability reorganization preparation of a kind of core that contains assembling and capsid protein, it is combined with the nature HIV (human immunodeficiency virus) manyly causes immunogenic epitope.

2, the HIV particle of the non-replicability reorganization preparation of a kind of core that contains assembling and capsid protein, it is combined with the many of nature HIV (human immunodeficiency virus) and causes immunogenic epitope, and has and the essentially identical form of natural HIV (human immunodeficiency virus).

3, the HIV particle of non-replicability reorganization preparation as claimed in claim 1 or 2, core protein wherein contains HIV (human immunodeficiency virus) 1 type core protein.

4, the HIV particle of non-replicability reorganization preparation as claimed in claim 3, HIV (human immunodeficiency virus) 1 type core protein wherein contains p24.

5, the HIV particle of non-replicability reorganization preparation as claimed in claim 3, HIV (human immunodeficiency virus) 1 type core protein wherein contains p24 and p17.

6, the HIV particle of recombinating and preparing as the non-replicability of claim 1 or 2, capsid protein wherein contains HIV (human immunodeficiency virus) 1 type capsid protein.

7, the HIV particle of non-replicability reorganization preparation as claimed in claim 6, HIV-1 capsid protein wherein contains gp41.

8, the HIV particle of non-replicability reorganization preparation as claimed in claim 6, wherein HIV (human immunodeficiency virus) 1 type capsid protein contains gp41 and gp120.

9, the HIV particle of non-replicability reorganization preparation as claimed in claim 6, wherein the HIV-1 capsid protein contains gp160.

10, the HIV particle of non-replicability reorganization preparation as claimed in claim 9, wherein gp160 can not cut and produces gp120 and gp41.

11, the HIV particle of non-replicability reorganization preparation as claimed in claim 9, wherein gp160 is by brachymemma.

12, the HIV particle of recombinating and preparing as the non-replicability of claim 10, wherein gp160 is by brachymemma.

13, the HIV particle of recombinating and preparing as the non-replicability of claim 1 and 2, wherein core protein contains the HIV-2 core protein.

14, the HIV particle of recombinating and preparing as the non-replicability of claim 1 and 2, wherein this capsid protein contains the HIV-2 capsid protein.

15, the HIV particle of recombinating and preparing as the non-replicability of claim 1 and 2, wherein this capsid protein contains HIV-1 and HIV-2 capsid protein.

16, a kind of HIV-1 particle of non-replicability reorganization preparation of capsid protein that contains the HIV-1 core of assembling and be combined with a plurality of immunogenicity epitopes of natural HIV-1.

17, a kind of HIV-2 particle of non-replicability reorganization preparation of capsid protein that contains the HIV-2 core of assembling and be combined with a plurality of immunogenicity epitopes of natural HIV-2.

18, a kind of HIV particle of non-replicability reorganization preparation of capsid protein that contains the HIV core of assembling and be combined with a plurality of immunogenicity epitopes of natural HIV-1 and natural HIV-2.

19, a kind of vaccine preparation, immunogen wherein contain the HIV particle of the non-replicability reorganization preparation of claim 1.

20, a kind of vaccine preparation, immunogen wherein contain the HIV particle of the non-replicability reorganization preparation of claim 2.

21, a kind of vaccine preparation, wherein immunogen includes the HIV particle of the non-replicability reorganization preparation of claim 3.

22, a kind of vaccine preparation, wherein immunogen includes the HIV particle of the non-replicability reorganization preparation of claim 4.

23, a kind of vaccine preparation, wherein immunogen includes the HIV particle of the non-replicability reorganization preparation of claim 5.

24, a kind of vaccine preparation, wherein immunogen includes the HIV particle of the non-replicability reorganization preparation of claim 6.

25, a kind of vaccine preparation, immunogen wherein include the HIV particle of the non-replicability reorganization preparation of claim 7.

26, a kind of vaccine preparation, immunogen wherein include the HIV particle of the described non-replicability reorganization preparation of claim 8.

27, a kind of vaccine preparation, immunogen wherein include the HIV particle of the non-replicability reorganization preparation in the claim 9.

28, a kind of vaccine preparation, immunogen wherein include the HIV particle of the non-replicability reorganization preparation in the claim 10.

29, a kind of vaccine preparation, immunogen wherein include the HIV particle of the non-replicability reorganization preparation in the claim 11.

30, a kind of vaccine preparation, immunogen wherein include the HIV particle of the non-replicability reorganization preparation in the claim 12.

31, a kind of vaccine preparation, immunogen wherein include the HIV particle of the non-replicability reorganization preparation in the claim 13.

32, a kind of vaccine preparation, immunogen wherein include the HIV particle of the non-replicability reorganization preparation in the claim 14.

33, a kind of vaccine preparation, immunogen wherein include the HIV particle of the non-replicability reorganization preparation in the claim 15.

34, a kind of vaccine preparation, immunogen wherein include the HIV particle of the non-replicability reorganization preparation in the claim 16.

35, a kind of vaccine preparation, immunogen wherein include the HIV particle of the non-replicability reorganization preparation in the claim 17.

36, a kind of vaccine preparation, immunogen wherein include the HIV particle of the non-replicability reorganization preparation in the claim 18.

37, a kind of vaccine preparation, immunogen wherein include the HIV particle that multiple different non-replicability reorganization prepares.

38, a kind of method that suppresses the acquired immune deficiency syndrome (AIDS) development in having infected HIV (human immunodeficiency virus) individual comprises that HIV particle that the non-replicability reorganization with the dosage of effective inhibition HIV (human immunodeficiency virus) infections prepares is to this individuality administration.

39, a kind of method that suppresses the lymphadenopathy development in having infected HIV (human immunodeficiency virus) individual comprises that HIV particle that the non-replicability reorganization with effective inhibition HIV (human immunodeficiency virus) infections dosage prepares is to this individuality administration.

40, a kind of method that suppresses the AIDS related complex development in the human body that has infected HIV (human immunodeficiency virus) comprises that HIV particle with the non-replicability reorganization preparation of effective inhibition HIV (human immunodeficiency virus) infections dosage is to this individuality administration.

41, a kind of CD4 that causes by HIV (human immunodeficiency virus) that reduces ⁺The method of lymphocyte infectivity comprises these lymphocytes of HIV particle disposal of the non-replicability reorganization preparation of available inhibition HIV infections effective dose.

42, a kind of retroviral particle of non-replicability reorganization preparation, it contains the retroviral core of assembling and is combined with the capsid protein of the retroviral a plurality of immunogenicity epitopes of natural human.

43, a kind of retroviral particle of non-replicability reorganization preparation, contain the retrovirus core of assembling and be combined with the retroviral a plurality of immunogenicity epitopes of natural human and with natural human retrovirus essentially identical glutelin on morphology.

44, the retroviral particle of recombinating and preparing as the non-replicability of claim 42 or 43, retrovirus core protein wherein contains the HTLV-1 core protein.

45, the retroviral particle of recombinating and preparing as the non-replicability of claim 42 or 43, retrovirus core protein wherein contains HTLV-II core protein.

46, the retroviral particle of recombinating and preparing as the non-replicability of claim 42 or 43, wherein retroviral capsid protein contains HTLV-I capsid protein.

47, the retroviral particle of recombinating and preparing as the non-replicability of claim 42 or 43, wherein retroviral capsid protein contains HTLV-II capsid protein.

48, the retroviral particle of recombinating and preparing as the non-replicability of claim 42 or 43, wherein retroviral capsid protein contains HTLV-1 and HTLV-II capsid protein.

49, a kind of HTLV-1 particle of non-replicability reorganization preparation contains HTLV-1 core that is assembled and the capsid protein that is combined with a plurality of immunogenicity epitopes of natural HTLV-1.

50, a kind of HTLV-II particle of non-replicability reorganization preparation contains the HTLV-II core of assembling and is combined with the capsid protein of a plurality of immunogenicity epitopes of natural HTLV-II.

51, a kind of HTLV particle of non-replicability reorganization preparation contains HTLV core that is assembled and the capsid protein that is combined with a plurality of immunogenicity epitopes of nature HTLV-1 and HTLV-II.

52, a kind of vaccine preparation, immunogen wherein include the retroviral particle of the non-replicability reorganization preparation in the claim 42.

53, a kind of vaccine preparation, immunogen wherein include the retroviral particle of the non-replicability reorganization preparation in the claim 43.

54, a kind of vaccine preparation, immunogen wherein include the retroviral particle of the non-replicability reorganization preparation in the claim 44.

55, a kind of vaccine preparation, immunogen wherein include the retroviral particle of the non-replicability reorganization preparation in the claim 45.

56, a kind of vaccine preparation, immunogen wherein include the retroviral particle of the non-replicability reorganization preparation in the claim 46.

57, a kind of vaccine preparation, immunogen wherein include the retroviral particle of the non-replicability reorganization preparation in the claim 47.

58, a kind of vaccine preparation, immunogen wherein include the retroviral particle of the non-replicability reorganization preparation in the claim 48.

59, a kind of vaccine preparation, immunogen wherein include the HTLV-1 particle of the non-replicability reorganization preparation in the claim 49.

60, a kind of vaccine preparation, immunogen wherein include the HTLV-1 particle of the non-replicability reorganization preparation in the claim 50.

61, a kind of vaccine preparation, immunogen wherein include the HTLV particle of the non-replicability reorganization preparation in the claim 51.

62, a kind of method that suppresses the development of mature T cellularity leukemia in having infected the HTLV-1 individuality comprises that HTLV-1 particle with the non-replicability reorganization preparation of effective inhibition HTLV-1 infective dose is to this individuality administration.

63, a kind ofly in having infected HTLV-1 individual, suppress the lymphadenomatous development method of HTLV-1 dependency, comprise that HTLV-I particle that the non-replicability reorganization with effective inhibition HTLV-1 infective dose prepares is to this individuality administration.

64, a kind of method that suppresses the development of HTLV-II related leukemia in having infected HTLV-II individuality comprises that HTLV-II particle with the non-replicability reorganization preparation of effective inhibition HTLV-II infective dose is to this individuality medication.

65, a kind of CD4 that causes by human reverse transcript virus that reduces ⁺The method of lymphocytic infectivity comprises with what effectively suppress human body retroviral infection dosage and can handle this lymphocyte with the retroviral particle of the non-replicability reorganization of cd4 cell surface receptor bonded preparation.

66, a kind of method for preparing the retroviral particle of non-replicability reorganization preparation comprises:

(a) the retrovirus core of will having encoded, the nucleotide sequence of proteolytic enzyme and capsid protein are incorporated into during mammalian host cell learns;

(b) in this mammalian host cell, express ripe reverse transcription core and capsid protein simultaneously;

(c) cultivate this mammalian host cell; With

(d) from substratum, reclaim the retroviral particle that this non-replicability reorganization prepares.

67, according to the method for claim 66, wherein be incorporated in this mammalian cell by the nucleotide sequence that infects to have encoded retrovirus core, proteolytic enzyme and capsid protein with a kind of live vector.

68, according to the method for claim 66, wherein be incorporated in this mammalian cell by the nucleotide sequence that infects to have encoded retrovirus core, proteolytic enzyme and capsid protein with multiple live vector.

69, according to the method for claim 67, live vector wherein includes a recombinant vaccine virus.

70, according to the method for claim 68, live vector wherein contains recombinant vaccine virus.

71, according to the method for claim 67, live vector wherein contains a recombinant retrovirus.

72, according to the method for claim 68, live vector wherein contains recombinant retrovirus.

73, according to the method for claim 66, wherein be incorporated in this mammalian cell by the nucleotide sequence that infects to have encoded retrovirus core, proteolytic enzyme and capsid protein with a dna vector.

74, according to the method for claim 66, wherein be incorporated in this mammalian cell by the nucleotide sequence that infects to have encoded retrovirus core, proteolytic enzyme and capsid protein with a plurality of dna vectors.

75, a kind of HIV particulate method for preparing non-replicability reorganization preparation comprises:

(a) with the recombinant vaccine virus of (ⅰ) a kind of HIV of carrying env gene and (ⅱ) recombinant vaccine virus of a kind of HIV of having gag and proteinase gene infect a kind of mammalian host cell simultaneously;

(b) in the mammalian host cell that infects, express the gene product that ripe HIV env and gag encode simultaneously;

(c) cultivate the mammalian host cell of this infection; With

(d) from this substratum, reclaim the HIV particle that this non-replicability reorganization prepares.

76, according to the method for claim 75, also comprise with carrying being selected from tat, rev, vif, the recombinant vaccine virus of at least a HIV gene of vpr and one group of gene of vpu infects this mammalian host cell.

77, according to the method for claim 75, also comprise with having being selected from tat, rev, rif, the multiple recombinant vaccine virus of at least two kinds of genes infects mammalian host cell in one group of gene of vpr and vpu.

78, according to the method for claim 75,76 or 77, HIV env gene wherein is a kind of HIV-1 env gene.

79, according to the method for claim 75,76 or 77, HIV env gene wherein is a kind of HIV-2 env gene.

80, according to the method for claim 75,76 or 77, HIV env gene wherein is a kind of HIV-1 gag gene.

81, according to the method for claim 75,76 or 77, HIV env gene wherein is a kind of HIV-2gag gene.

82, according to the method for claim 75,76 or 77, hiv protease gene wherein is a kind of HIV-1 proteinase gene.

83, according to the method for claim 75,76 or 77, hiv protease gene wherein is a kind of HIV-2 proteinase gene.

84, according to the method for claim 75,76 or 77, mammalian host cell wherein is a kind of cercopithecus aethiops cell.

85, a kind of HIV particulate method for preparing non-replicability reorganization preparation comprises:

(a) with a kind of HIV env that has, the recombinant vaccine virus of gag and proteinase gene infects a kind of mammalian host cell;

(b) in the mammalian host cell that infects, express the gene product that ripe HIV env and gag encode simultaneously;

(c) cultivate the mammalian host cell that infects; And

(d) from substratum, reclaim the HIV particle that non-replicability reorganization prepares.

86,5 method according to Claim 8 also comprises with being selected from tat a kind of having, rev, and vif, the recombinant vaccine virus of at least a HIV gene of vpr and one group of gene of vpu infects mammalian host cell.

87,5 method according to Claim 8 also comprises with having simultaneously being selected from tat, rev, and vif, the multiple recombinant vaccine virus of at least two kinds of HIV genes of vpr and one group of gene of vpu infects mammalian host cell.

88,5,86 or 87 method according to Claim 8, HIV env gene wherein is a kind of HIV-1 env gene.

89,5,86 or 87 method according to Claim 8, HIV env gene wherein is a kind of HIV-2 env gene.

90,5,86 or 87 method according to Claim 8, HIV gag gene wherein is a kind of HIV-1 gag gene.

91,5,86 or 87 method according to Claim 8, HIV gag gene wherein is a kind of HIV-2 gag gene.

92,5,86 or 87 method according to Claim 8, hiv protease gene wherein is a kind of HIV-1 proteinase gene.

93,5,86 or 87 method according to Claim 8, hiv protease gene wherein is a kind of HIV-2 proteinase gene.

94,5,86 or 87 method according to Claim 8, mammalian host cell wherein is a kind of cercopithecus aethiops cell.

95, a kind of HIV particulate method for preparing non-replicability reorganization preparation comprises:

(a) with a kind of HIV env that has, a kind of mammalian host cell of recombinant plasmid transfection of gag and proteinase gene;

(b) in the mammalian host cell of transfection, express the gene product that ripe HIV env and gag encode simultaneously;

(c) mammalian host cell of transfection is cultivated; And

(d) from substratum, reclaim the HIV particle that non-replicability reorganization prepares.

96, according to the method for claim 95, recombinant plasmid wherein also have at least a other be selected from tat, rev, vif, the HIV gene of vpr and one group of gene of vpu.

97, according to the method for claim 95, also comprise with a kind of having at least one and being selected from tat, rev, vif, the recombinant plasmid of the HIV gene of vpr and one group of gene of vpu carries out transfection to mammalian host cell.

98, according to the method for claim 95, also comprise with having at least two simultaneously being selected from tat, rev, vif, the multiple recombinant plasmid of the HIV gene in one group of gene of vpr and vpu carries out transfection to mammalian host cell.

99, according to claim 95,96,97 or 98 method, HIV env gene wherein is a kind of HIV-1 env gene.

100, according to claim 95,96,97 or 98 method, HIV env gene wherein is a kind of HIV-2 env gene.

101, according to claim 95,96,97 or 98 method, HIV gag gene wherein is a kind of HIV-1 gag gene.

102, according to claim 95,96,97 or 98 method, HIV gag gene wherein is a kind of HIV-2 gag gene.

103, according to claim 95,96,97 or 98 method, hiv protease gene wherein is a kind of HIV-1 proteinase gene.

104, according to claim 95,96,97 or 98 method, hiv protease gene wherein is a kind of HIV-2 proteinase gene.

105, a kind of HIV particulate method for preparing non-replicability reorganization preparation comprises:

(a) have the recombinant plasmid of at least one HIV env gene and (ⅱ) a kind ofly have at least that the recombinant plasmid of HIV gag and proteinase gene carries out transfection to a kind of mammalian host cell with (ⅰ) is a kind of;

(b) in the mammalian host cell of transfection, express the gene product that ripe HIV env gag encodes simultaneously;

(c) mammalian host cell of cultivation transfection; And

(d) from substratum, reclaim the HIV particle that non-replicability reorganization prepares.

106, according to the method for claim 105, also comprise with being selected from tat a kind of having, rev, vif, the recombinant plasmid of at least one HIV gene carries out transfection to mammalian host cell in one group of gene of vpr and vpu.

107, according to the method for claim 105, also comprise having simultaneously being selected from tat, rev, vif, the multiple recombinant plasmid of at least two HIV genes carries out transfection to mammalian host cell in one group of gene of vpr and vpu.

108, according to the method for claim 105,106 or 107, HIV env gene wherein is a kind of HIV-1 env gene.

109, according to the method for claim 105,106 or 107, HIV env gene wherein is a kind of HIV-2 env gene.

110, according to the method for claim 105,106 or 107, HIV gag gene wherein is a kind of HIV-1 gag gene.

111, according to the method for claim 105,106 or 107, HIV gag gene wherein is a kind of HIV-2 gag gene.

112, according to the method for claim 105,106 or 107, hiv protease gene wherein is a kind of HIV-1 proteinase gene.

113, according to the method for claim 105,106 or 107, hiv protease gene wherein is a kind of HIV-2 proteinase gene.

114, according to the method for claim 95 or 105, mammalian host cell wherein is a kind of Chinese hamster gonad cell.

115, according to the method for claim 95 or 105, mammalian host cell wherein is a kind of cercopithecus aethiops cell.

116, according to the method for claim 95 or 105, mammalian host cell wherein is a kind of Vero cell.

117, according to the method for claim 95 or 105, mammalian host cell wherein is a kind of HeLa cell.

118, according to claim 75,85,95 or 105 method, a kind of gp160 albumen that does not cut of HIV env genes encoding wherein.

119, according to claim 75,85,95 or 105 method, HIV env genes encoding wherein a kind of gp160 protein of brachymemma.

120, a kind of HIV-1 particulate method for preparing non-replicability reorganization preparation comprises:

(a) with the recombinant vaccine virus v-env5 and the V-gag2 that are deposited in ATCC a kind of BSC-40 host cell is infected simultaneously;

(b) cultivate infected B SC-40 cell; And

(c) from substratum, reclaim the HIV-1 particle that non-replicability reorganization prepares.

121, a kind of HIV-1 particle for preparing non-replicability reorganization preparation comprises:

(a) with (ⅰ) have respectively the multiple recombinant vaccine virus of different HIV-1 env gene and (ⅱ) recombinant vaccine virus of a kind of HIV-1 of having gag and proteinase gene simultaneously a kind of mammalian host cell is infected;

(b) cultivate the mammalian host cell that infects; And

(c) from substratum, reclaim the HIV-1 particle that non-replicability reorganization prepares.

122, a kind of HIV-2 particulate method for preparing non-replicability reorganization preparation comprises:

(a) with (ⅰ) have respectively the multiple recombinant vaccine virus of different HIV-2 env genes and (ⅱ) recombinant vaccine virus of a kind of HIV-2 of having gag and proteinase gene a kind of mammalian host cell is infected simultaneously;

(b) cultivate the mammalian host cell that infects; And

(c) from substratum, reclaim the HIV-2 particle that non-replicability reorganization prepares.

123, a kind of HIV particulate method for preparing non-replicability reorganization preparation comprises:

(a) in mammalian cell, express HIV env coding and structural protein HIV gag coding simultaneously; And

(b) from substratum, reclaim the HIV particle that non-replicability reorganization prepares.

124, a kind of HIV-1 particulate method for preparing non-replicability reorganization preparation comprises:

(a) infect a kind of BSC-40 host cell with the recombinant vaccine virus v-G2E5 that is deposited in ATCC;

(b) cultivate infected B SC-40 cell; And

(c) from substratum, reclaim the HIV-1 particle that obtains non-replicability reorganization preparation

125, a kind of HIV-1 particulate method for preparing non-replicability reorganization preparation comprises:

(a) with (ⅰ) recombinant vaccine virus v-ED2 and (ⅱ) recombinant vaccine virus v-gag2 infect a kind of BSC-40 host cell simultaneously;

(b) cultivate infected B SC-40 cell; And

(c) from substratum, reclaim the HIV-1 particle that non-replicability reorganization prepares.

126, a kind of HIV-1 particulate method for preparing non-replicability reorganization preparation comprises:

(a) with (ⅰ) recombinant vaccine virus v-ENV5DCT and

(ⅱ) recombinant vaccine virus v-gag2 infects a kind of BSC-40 host cell simultaneously;

(b) cultivate infected B SC-40 cell; And

(c) from substratum, reclaim the HIV-1 particle that non-replicability reorganization prepares.

127, a kind of HIV-1 particulate method for preparing non-replicability reorganization preparation comprises:

(a) with (ⅰ) recombinant vaccine virus v-160NC and (ⅱ) recombinant vaccine virus v-gag2 a kind of BSC-40 host cell is infected simultaneously;

(b) cultivate infected B SC-40 cell; And

(c) from substratum, reclaim the HIV-1 particle that non-replicability reorganization prepares.

128, a kind of HIV-1 particulate method for preparing non-replicability reorganization preparation comprises:

(a) with (ⅰ) recombinant vaccine virus V-11K160NC and (ⅱ) recombinant vaccine virus v-gag2 simultaneously a kind of BSC-40 host cell is infected;

(b) cultivate infected B SC-40 cell; And

(c) from substratum, reclaim the HIV-1 particle that non-replicability reorganization prepares.

129, a kind of HIV-1 particulate method for preparing non-replicability reorganization preparation comprises:

(a) with being deposited in the recombinant plasmid CmHIVdel KpnAvr(Gag2 TRE of NRRL) (1160-al) and CmHiEnv5(1104-b1) a kind of mammalian cell of transfection simultaneously;

(b) in the Chinese hamster ovary celI of transfection, express the gene product that ripe HIV env and gag encode simultaneously;

(c) Chinese hamster ovary celI of cultivation transfection; And

(d) from substratum, reclaim the HIV-1 particle that non-replicability reorganization prepares.

130, a kind of HIV-1 particulate method for preparing non-replicability reorganization preparation comprises:

(a) with a kind of recombinant plasmid CmHIV del KpnAvr(Gag2 TRE that is deposited in NRRL) (1160-al) a kind of mammalian cell of transfection;

(b) in the Chinese hamster ovary celI of transfection, express ripe HIV env and gag encoding gene product simultaneously;

(c) Chinese hamster ovary celI of cultivation transfection; And

(d) from substratum, reclaim the HIV-1 particle that non-replicability reorganization prepares.

131, a kind of HIV-1 particulate method for preparing non-replicability reorganization preparation comprises:

(a) with being deposited in the recombinant plasmid CmHiGag2Rre(1158-al of NRRL) and CmHIVdelXmn(1133-al) common a kind of mammalian cell of transfection;

(b) in the Chinese hamster ovary celI of transfection, express the gene product that ripe HIV env and gag encode simultaneously;

(c) Chinese hamster ovary celI of cultivation transfection; And

(d) from substratum, reclaim the HIV-1 particle that non-replicability reorganization prepares.

132, a kind of HIV-1 particulate method for preparing non-replicability reorganization preparation comprises:

(a) with being deposited in the recombinant plasmid CmHIVdelXmn(1133-al of NRRL) and CmVGag2Rre(1159-al) common a kind of mammalian cell of transfection;

(b) in the Chinese hamster ovary celI of transfection, express the gene product that ripe HIV env and gag encode simultaneously;

(c) Chinese hamster ovary celI of cultivation transfection; And

(d) from substratum, reclaim the HIV-1 particle that non-replicability reorganization prepares.

133, a kind of HIV-1 particulate method for preparing non-replicability reorganization preparation comprises:

(a) with the recombinant plasmid CmHiGag2Rre that is deposited in NRRL, CmHiEnv5, a kind of mammalian cell of the common transfection of CmHiTat and CmHiRev;

(b) in the Chinese hamster ovary celI of transfection, express the gene product that ripe HIV env and gag encode simultaneously;

(c) Chinese hamster ovary celI of cultivation transfection; And

(d) from substratum, reclaim the HIV-1 particle that non-replicability reorganization prepares.

134, according to claim 129,130,131,132 or 133 method, mammalian cell wherein is a kind of BSC-40 cell.

135, according to claim 129,130,131,132 or 133 method, mammalian cell wherein is a kind of HeLa cell.

136, according to claim 129,130,131,132 or 133 method, mammalian cell wherein is a kind of Vero cell.

137, according to claim 129,130,131,132 or 133 method, mammalian cell wherein is a kind of Chinese hamster ovary celI.

138, according to claim 129,130,131,132 or 133 method, mammalian cell wherein is a kind of dhfr-defective Chinese hamster ovary celI.

139, be deposited in ATCC, its specified preserving number is

Recombinant vaccine virus V-gag2.

140, be deposited in ATCC, its specified preserving number is

Recombinant vaccine virus V-G2E5.

141, recombinant vaccine virus V-ED2.

142, recombinant vaccine virus v-ENV5DCT.

143, recombinant vaccine virus v-160NC.

144, recombinant vaccine virus v-11K160NC.

145, plasmid CmHiVdelKpnAvr(Gag2 TRE) (1160-a1).

146, plasmid CmHiEnv5(1104-b1).

147, plasmid CmHiGag2Rre(1158-a1).

148, plasmid CmHIVdelXmn(1133-a1).

149, plasmid CmvGag2Rre(1159-a1).

150, plasmid CmHiTat.

151, plasmid CmHiRev.

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