CN1030256A - Methods and substances for detection and treatment of human immunodeficiency virus - Google Patents

Abstract

The invention discloses immuno active polypeptide, preferably antibody or antibody fragment, especially monoclonal antibody, they have activity to the proteic idiotype antibody of human lymphocyte T4, but and with in the donor and in external and the HIV mode and the HIV virus particle that infect react and the HIV particle of detection in biological fluid.Best embodiment comprises that with new small white mouse hydridization oncocyte be JT4C8 at present, JT4C12, JT4C16, JT1-1F3, JT1-1F3-E5, monoclonal anti--mono-clonal-Anti-Human's lymphocyte T4 antibody that JT1-1D7 and JT2-N15 produce.Automatic and passive inoculation method is also disclosed.

Description

Detect and treat method and the material of human immunodeficiency virus

The present invention generally speaking relates to human immunodeficiency virus (HIV) diagnosis of infection and used method and the material of treatment, more particularly, the present invention relates to have immunocompetent polypeptide, preferably antibody or antibody fragment, especially monoclonal antibody, they are to people's quasi-lymphocyte T ₄Proteic genotype antibody has activity, but and to react with the mode of HIV infection and the virus particle of HIV in the donor and in external.In addition, the invention still further relates to immuno active polypeptide useful in vaccine composition, this vaccine composition produces protective response to the infection of HIV.

Following document has been made good summary: Laurence to the epidemiology of the human AIDS cause of disease and the prior art of immunology aspect, " The Immune System in AIDS ", the Scientific Ameri Can 84-93 page or leaf in December, 1985; Gallo, the Scientific American 88-98 page or leaf in " The First Human RetroVirus " in December, 1986; Gallo, the Scientific AmeriCan 47-56 page or leaf in " The AIDS Virus " in January, 1987; 1984 225 phase Science of people such as Levy 840-842 page or leaf; " Mobilizing Against AIDS ", Institute of Medicine, National Academy of Sciences, Harvard University Press(Cambridge, Mass.1986); And 1985 the 3rd phase Ann.Rev.Immunol. of people 477-500 page or leaf such as Lane.The T of human T-lymphocyte in HIV infects ₄The effect of surface glycoprotein (be sometimes referred to as " CD4 albumen or determiner) is studied widely, as 1984 312 phase Nature763-767 of people such as Dalgleish; 1986 312 phase Science767-768 of people such as Klatzman page or leaf; 1984 the 225th phase Science59-62 of people such as Klatzman page or leaf; 1985 the 135th phase J.Immunol.3151-3162 of people such as MCDougal page or leaf; And people's Cell the 47th phase 338-348 page or leaf in 1986 such as Maddon.Also can be referring to people such as Marrack " The T Cell and its Receptor " in February, 1986 SCientific American 36-45 page or leaf; And people's Science the 231st phase 382-385 page or leaf in 1986 such as McDougal.

Recently, the CD4 of soluble form may have the treatment practicality specializing in treatment HIV infects, see people's Nature the 331st phase 76-77 pages or leaves in 1988 such as Fisher; People such as Hussey, Ibid, 78-81 page or leaf; People such as Deen, Ibid, 82-83 page or leaf; And people such as Traunecker, Ibid, the 84-86 page or leaf, all these relate to solubility CD4 in external and HIV infect.The latent defect of plan use solubility CD4 therapeutical agent is CD4 and is present in other immunocyte, the known response that comprises the main histocompatibility complex of II level (" the MHC ") molecule on B cell, scavenger cell, monocyte surface, this means may need to modify (for example, by blocking) before being used for the treatment of with CD4.

In the document existing many reports relate to antibody in vivo with external in and the ability that infects of HIV, particularly relate to the trial of the active immne that carries out in order to form protective immunity.Referring to l.Acad.Sci.(USA) 83 phase 9709-9713 pages or leaves of people such as Matthews Proc.Nat ' in 1986; Norman, " AIDS Therapy:ANew Push For Clinical Trials ", the articles before Science230 phase 1355-1358 page or leaf in 1985 and this are a series of; Newmark, Nature324 phase 304-305 page or leaf in 1986; And appear at the note of in February, 1987 Scientific American 86-88 page or leaf under title " AIDS:HoPe ... And Warnings ", and on December 18th, 1986 New Scientist title " Can protein T Thwart the AIDS Virus ...? " under note.Also can be referring to people such as Mitsuya, Nature325 phase 773-778 page or leaf in 1987; People such as Kennedy Science231 in 1986 phase 1556-1559 page or leaf; People such as Chanh EMBO.Journal 5(11 in 1986) phase 3065-3071 page or leaf; People such as Chanh Eur.J.Immunol.16 in 1986 phase 1465-1468 page or leaf; People such as Putney Science234 in 1986 phase 1392-1395 page or leaf; And the document of people's 1-5 day in June, 1987 such as Matshushita " III International Conference on Acquired Immunodeficiency Syndrome(AIDS) " 106 pages W.3.2.

The result of disclosed anti--genotype immunization research is an important background technology of the present invention.Referring to people such as Kennedy " Anti-Idiotype and Immunity " in July, 1986 Scientific American 48-56 page or leaf; Jerne, " The Immune System ", in July, 1973 Scientific Americal 52-60 page or leaf; Marx, " Making Antibodies Without The Antigens ", Science 228 phase 162-165 page or leaf in 1985; People such as Finberg CRC CritiCal in 1987 Reviews in Immunology the 7th phase 269-284 page or leaf; And people's ScienCe 232 phase 220-223 page or leaf in 1986 such as Kennedy.Also referring to Norman, relevant anti-HIV-immunotherapy of Supra and generation are to potential dependency between the anti-idiotypic antibody of HIV surface protein.

Significant especially background technology of the present invention is the work that people such as MCDougal J.Immunology 137 phase 2937-2944 page or leaf in 1986 is reported, wherein point out: " ... produce anti-four kinds of candidate CD4 monoclonal antibodies (OKT4A; OKT4D, OKT4F and Leu3a(are sometimes referred to as " anti--Leu3a ")) rabbit anti--genotype blood plasma is with the reaction of (HIV) virus or can not suppress virus and join CD4 to ⁺On the T cell.This report should be compared with report (be reported in 1987 year Bio/Technology 5th phase 421-422 page or leaf) and 1987 year June 1-5 day the report on washingtonian three acquired immune deficiency syndrome (AIDS) (AIDS) international conference (see document TH.9.5, and be reported in 1987 year June 11 day New Scientist 26th page) of Ronald C.Kennedy 1-4 day in March, 1987 in the 7th the DNA/ hybrid annual meeting (the 7 th Annual DNA/Hybridoma Congress) in San Francisco.These reports pointed out antagonism anti--development of the polyclonal antiserum of T4 antibody and such antiserum(antisera) identification HIV and in outer body and the ability that infects of HIV.The preparation method of monoclonal anti-genotype antibody has also been mentioned in the report in back, this development people such as Chanh in June, 1987 Proc.Nat ' l.Acad.sci.(USA) the 84th phase 3891-3859 page or leaf also done introduction.

In this technology; really need to be used for to diagnose the HIV particle that biological fluid and tissue samples exist and the novel method and the material of HIV cells infected, and need in the body and the new tool that infects of HIV and development can give the vaccination program of the immunity protection that anti-HIV infects.

The invention provides purified and isolating immuno active polypeptide, be preferably antibody or mosaic or their fragment, monoclonal antibody preferably, they have activity to the proteic genotype antibody of people's quasi-lymphocyte T4 (preferably monoclonal antibody).These products of the present invention have that part of specific immunity bonded ability of carrying out with the HIV virus particle, that part ofly are meant inevitable and T when HIV infects such as the such host cell of human T-lymphocyte and human nervous system's cell ₄The interactional part of surface protein.The bacterial strain that the feature of these products of the present invention especially is them external have in and the unique ability that infects of HIV, be with have approximately from 60000 in the proteic atopy of HIV of 80000 molecular weight (by SDS-PAGE mensuration) and they not reactive to the II level main histocompatibility molecule relevant with the human immunity cell surface.

Therefore, in an example of the present invention, the immunocompetence product generates by " replying " with HIV particle and HIV cells infected surface specific combination, even their generation does not have the direct immunization relation to these surface proteins.

Purification of the present invention and isolated polypeptide are specially adapted to the detection of HIV in the biological fluid and quantitative analytic process, and this testing process is combined into the basis with the immunity between HIV particle and the reactive polypeptide of the present invention (for example antibody).And, antibody, chimeric antibody and their fragment that the present invention forms is optionally to rise immunoreactively with the surface of HIV cells infected, is used for useful reagent when body fluid and tissue samples detect the HIV cells infected and separate the HIV cells infected from comprise the two cell colony of infection and non-infected cell in having provided.Therefore, product of the present invention can be used for diagnosis and therapeutic process, comprises separation and/or optionally eliminates the HIV cells infected.

It is that HIV infected animals, the particularly mankind carry out anti-HIV treatment that the present invention's purification and isolating immunologic active material also are specially adapted to commute.By so a kind of method, use the monoclonal antibody for example of the present invention of immune significant quantity to produce the passive immunity that HIV is infected for the patient who has infected the patient of HIV or be in the HIV risk of infection.According to another kind of way, the cell that infected by HIV for example also can be sent back to patient with the latter in the external cellular segregation that does not infect with patient.

Point out in the following set forth in detail that the polypeptide relevant with antibody of the present invention preferably forms people's lymph T at first ₄The single specific antibody of glycoprotein (CD4 determiner) (preferably monoclonal antibody), then preparation is at this initial T that forms the antibody that forms in the step ₄The antibody in genotype zone (preferably monoclonal antibody).

Fragment, the especially bispecific antibody of chimeric antibody and they also belong to product leech  of the present invention steal forget ⑸  engrave stubborn sole ɡ  entangled know fast   tool brain  dispute  camel Jing pine  clanging or clanking sound go to  ┲ side of body   scratch the solid road of oxime let out the third constellations try to gain  penalize the Buddhist nunnery process the stubborn emerging  a ceremonial jade-ladle, used in libation of cooked food magnesium smile puff try to gain the numerous Suo Yun of  Hou NA sequence and transform or transfection.

As an example, when the configuration data in the genotype zone of grasping relevant antibody of the present invention, just may utilize in substratum, to produce useful antibody fragment (as fab ' and f(ab ') such as the such eukaryotic cells of intestinal bacteria, yeast, insect and mammalian cell ₂Fragment).In addition, prepare chimeric antibody (for example mouse/people's antibody) also within the scope of the present invention with rat bone marrow tumour cell that transforms or hydridization oncocyte (particularly heavy chain deletion mutant cell) as producing the host.People expect that the hydridization oncocyte of hydridization can produce the bispecific antibody with diagnosis and therepic use.Also available recombinant chou method is produced HIV subgroup vaccine material.For example, estimate monoclonal anti physical efficiency of the present invention fabulous be applicable to transform or the tubule of transfection or prokaryotic cell prokaryocyte in filter out the expression product of HIV DNA, the immunity that can isolate the natural generation of having encoded is the DNA of all or part of aminoacid sequence of HIV albumen (comprising glycoprotein) effectively.In suitable host, exist such DNA can high level ground production vaccine substance.

In a kind of preferred scheme, the invention provides and be characterized as monoclonal resisting-mono-clonal-anti-people's lymph T ₄The polypeptide that the antibody of antibody is relevant, preferably monoclonal anti--oKT4 and anti--oKT4A antibody, both all with the 60-80kd HIV albumen activity that responds.At present, preferably monoclonal resisting-okT4A antibody, it has the ability that the very strong HIV at the multiple HIV bacterial strain of external neutralization infects, and participates in complement-intermediary's lysis of HIV cells infected.

In another example, the present invention provides for the first time to people's lymph T ₄Proteic monoclonal antibody is with the hydridization clone of " anti--genotype " monoclonal antibody of antigen/antibody reaction carrying out specific immune reaction.For example, the invention provides new mouse deutero-hydridization clone, JT4C8, JT4C12 and JT4C16, JT1-1F3, JT1-1F3-E5, JT1-1D7 and JT2-N15, they each produce a kind of and anti--T with the supernatant liquor component form of in substratum, growing ₄The monoclonal antibody of genotype specific reaction, and this monoclonal antibody with in vivo with external in and the mode that infects of HIV react with the HIV virion protein.

Hydridization clone JT4C8 is deposited with american agriculture portion mechanism U.S. usual culture collection center on February 18th, 1987, and (Rockville, Maryland), deposit numbers is HB9385.Hydridization clone JT4C12 is deposited with on February 18th, 1987 and locates, and deposit numbers is HB9387.Hydridization clone JT4C16 is deposited with the place on February 18th, 1987, and deposit numbers is HB9386.Hydridization clone JT1-1F3 is deposited with animal cell culture Europe on June 25th, 1987 and deposits center ECACC(Salisbury, Wiltshive, and U.K), deposit numbers is 87062501.Hydridization clone JT1-1F3-E5 also is deposited with animal cell culture Europe on June 25th, 1987 and deposits center ECACC, and deposit numbers is 87062502.Hydridization clone JT1-1D7 is deposited with fermentation research institute (Japanese Ibaragi-Ken) on January 29th, 1988, deposit numbers is FERM BP-1685, hydridization clone JT2-N15 is deposited with Japanese fermentation research institute on January 29th, 1988, and deposit numbers is FEMBP-1684.

In the another one example, the invention provides the method for producing HIV subgroup vaccine substance with well-known affinity purifying, thereby by using the protein component (molecular weight ranges is about 60000 to 80000, especially 65000 to 67000) of isolating HIV by the selectivity immunosorbent of Antibody Preparation of the present invention.

By following detailed description, will come into plain view accompanying drawing 1,2 and 3 expression antibody of the present invention and HIV and the proteic immunity test results of non-infected cells to other purposes of the present invention and advantage to the embodiment of the invention and accompanying drawing; Figure 4 and 5 provide the immune spot detected result relevant with HIV albumen with antibody of the present invention; And Fig. 6 A to 6E provides the photo result who uses the immunofluorescence dyeing of antibody of the present invention to detect to the cell that infects and do not infect.

The following example explanation the present invention produces comprises JT4C8, JT4C12, JT4C16, JT1-1F3, JT1-1F3-E5, therefrom isolates the monoclonal antibody of anti--CD4 and the expansion of such monoclonal antibody and feature thereof at some hydridization clones of JT1-1D7 and JT2-N15.

More particularly, example 1 relates to anti--T that stimulation mouse host produces to be had on the market ₄The antibody of monoclonal antibody, " oKT4 ", splenocyte and myeloma cell's fusion, the screening of hydridization cell, clone also therefrom isolate monoclonal antibody.Example 2 relate to fluoroimmunoassay, analysis and HIV albumen test active Western spot detection method and by external have in and the characteristic of the monoclonal antibody that ability produced that infects of HIV.Example 3 relates to first kind of generation method of the hydridization clone of the monoclonal antibody that the antibody " oKT4A " that has on the market can be provided in growth medium.Example 4 relates to by immunofluorescence analysis, the detection of Werstern spot, external neutralization detection, ELISA detects and the characteristic of the monoclonal antibody that the fluorocyte staining analysis produces.Example 5 relates to the second method that forms hydridization clone, this hydridization clone can provide the monoclonal antibody of the antibody " oKT4A " that has on the market in the medium of their growths, and the characteristic that is detected the monoclonal antibody that produces by the detection of Western spot, external neutralization.

Example 1

By one embodiment of the invention, use such as Oi and Herzenberg at Selected Methods Cell Immunology 351-372(1979) and the standard immunoassay described on the J.Immunol.Meth.39 phase 285-308 page or leaf in 1980 at " Antibody Production By Hybridomas " of Godding learn a skill and produce the hydridization tumor cell line.With monoclonal anti-T ₄The splenocyte of hyperimmune mouse merges with rat bone marrow tumour cell in the presence of polyoxyethylene glycol.Whether the supernatant liquor of each " hydridization knurl " cell culture of check growth exists needed antibody activity.The hydridization oncocyte of selecting is cloned becoming proliferative cell system, this clone in the supernatant liquor of its grown culture, produces have high special anti--resist-T ₄Active antibody.

The A immunization

With monoclonal Anti-Human's lymphocyte T ₄(Rahway N.J) carries out the spleen injection for each BALB/C mouse to antibody for oKT4, ortho Diagnostics.

The freeze dried oKT4 material of 1 microgram is put into 1 ml distilled water, and with 50mM MES damping fluid (Sigma chemical company), PH6.0 dialyses to remove and to replace the organic phosphoric acid salt buffer agent.Then in the FPLC(registered trademark) (Piscataway N.J.08854) carries out high pressure liquid chromatography (HPLC) and separates for Pharmacia, Laboratory Separation Division on the instrument.Separate with the method (wherein the gradient of salt is 0 to 0.5M NaCl) of recommending with a single Q post.The aliquots containig of per 0.5 milliliter of component (20 microlitre) is with the human lymphocyte (10 of fresh collection ⁵Individual cells/ml) analyzes activity.More particularly, with HPLC component and cytomixis and centrifugal, cell is cleaned several times and is suspended in the salt solution (PBS) that phosphate buffered crosses.With 5 micrograms with the rabbit of FITC mark anti--mouse IgG cultivates with this cell, be resuspended in the cell sheet among the PBS after centrifugal and read the result with the fluorocyte counter.Compile the most highly active component of demonstration, analyze on SDS-PAGE, the result shows that purity is higher than 90%.

Four each initial spleen injections of mouse amount to the Inoculant (okT4 of every about 1.5 micrograms of mouse) of about 100 microlitres.Behind fortnight and the 28 sky every mouse is carried out the enhancing injection of 100 microlitre Inoculants.

Strengthen injection the last time and killed these mouse in back four days, sterilely take out spleen, be placed in the PetriShi culture dish (in ice) of the EagleShi medium (Gibco) that fills the DulbecooShi improvement, remove the fat and the reticular tissue of spleen, it is passed through 100 purpose stainless steel sifts, each splenocyte that obtains is by forming the sheet ball down in centrifugal 10 minutes at 1000 rev/mins, clean this cell sheet ball twice with medium (as above), and be suspended in again among the RPMI 1640, counting is measured cell concn in hematimeter under the situation that the blue existence of 0.2% trypanosome is arranged.

The rat bone marrow tumour cell Ns1/1.Ag4.1 that incubation growth is obtained by the Balb/c strain in the RPMI1640 medium of the horse serum that contains 15% heat-inactivation (Pel-Freeze).Descended centrifugal 10 minutes at 1000 rev/mins, make this cell become the sheet ball, with the RPMI1640 washing that does not contain antibiotic.Cell concn is determined by counting in the back in being re-suspended to same media.

With 4: 1 mixed splenocytes and NS1/1 cell, and at 1000 rev/mins times centrifugal 10 minutes.Take out supernatant liquor, at 37 ℃ is that the polyoxyethylene glycol (PEG) 1500 of 500-600 carries out cytogamy with molecular weight down, constantly relaxing under the stirring, in the specified time, add following material: in 1 minute time, add 1 milliliter of RPMI1640 that contains 50%PEG, stirred 1 minute, and in 1 minute, added 1 milliliter of RPMI1640 that contains 15% horse serum; In 1 minute, add 1 milliliter of RPMI1640 that contains 15% horse serum, in 2 minutes, add the RPMI1640 that 8 microlitres contain 15% horse serum.

Under 1000 rev/mins,, be suspended in again and contain 15% horse serum, also contain among the RPMI1640 that penicillin G and Streptomycin sulphate be respectively every milliliter 100 unit and 100 milligrams, use 5%CO in advance centrifugal 10 minutes of the fused cell that obtains ₂On equilibrated 96 plates with 0.1 milliliter in every hole with these cell depositions on orifice plate, this plate is at 37 ℃ down and 5% CO ₂Middle overnight incubation.

Second day, in each hole, add 0.1 milliliter of HAT medium (xanthoglobulin of 13.6 mcg/ml, the thymidine of the aminopterin-induced syndrome of 0.176 mcg/ml and 3.88 mcg/ml) that contains 15% horse serum RPMI1640.This medium optionally allows splenocyte NS1/1 hybrid survival, screen out the NS1/1 cell that do not merge or with other cytogamy the NS1/1 cell.Can not substratum, survive after two days from the original splenocyte that does not melt that adult rats gets.

At the 3rd, 4,6,9 and 12 day, from each hole, take out 0.1 milliliter medium, and add 0.1 milliliter of fresh HAT that contains the RPML1640 of 15% horse serum.At the 15th, 19,23 and 27 day, from each hole, take out 0.1 milliliter medium, and add 0.1 milliliter and contain 15% horse serum and have only the as above xanthoglobulin of same concentration and the RPMI1640 of thymidine.At the 28th day, the culture supernatants in each hole is screened to detect the antibody special to oKT4.

With fluorescence binding immunoassay absorption approach ā Er LISA ") detect to produce the hydridization knurl of oKT4 antibody.Carbonate-the bicarbonate solution that contains the anti--mouse IgGFC of 3.0 mcg/ml with 100 microlitres applies in the hole of 96 orifice plates, spends the night under 37 ℃, with the PBS that contains 0.05% polysorbas20 the hole is cleaned 10 times.These holes were blocked 1 hour under 37 ℃ with 20% horse serum, wash twice, remove blocker with above-mentioned PBS/ polysorbas20.

The aliquots containig of 20 microlitres of the nutrient solution in each hydridization knurl hole and 80 microlitre PBS at room temperature cultivated 1 hour in coated hole, spent the night under 4 ℃ then., and, cultivated 3 hours down these holes washing 10 times with the PBS that contains 0.05% polysorbas20, then 4 ℃ of cultivations 2 hours down at 37 ℃ with mouse serum (8 mcg/ml) blocking-up in 150 microlitres/hole.

In each hole with the okT4(0.3 mcg/ml of the FITC-mark of 100 microlitres) in the dark with 4 ℃ of following overnight incubation, then with above-mentioned PBS/ polysorbas20 solution with hole washing 10 times, add 200 microlitres 5 * 10 in each hole ^-5The N NaoH and 5.6 * 10 of N ^-4The NH of N ₄OH solution.This plate was at room temperature kept 15 minutes, swung then 1 minute.After transferring on Titertek microtitre 1 * 8 plate, fluorometric assay (exciting 490 microns, 530 microns of scatterings) is carried out in these holes with Corona Electric type MTP 100F microplate.

In 2710 screened holes, there are 19 oKT4 reaction presented the significant positive, these 19 male clones are named as JT4C1 to JT4C19.

With the ratio of per 3 about cells in hole with these cell dilution agent in other many holes, formally the hydridization oncocyte that is obtained by these positive holes is cloned.Generally, carry out formal clone by an active hole and produce the formal clone of this same hydridization oncocyte subclone seemingly, in fact, more than the three generations, demonstrate different clonal populations.Subclone from same hole is added a number name (for example the clone who is obtained by hole JT4C7 is marked as JT4C7-1 JT4C7-2 etc.) with its parent's numbering.

Example 2

In order to describe the characteristic of the antibody that produces by example 1 described those positive colonies, carry out some tests to determine monoclonal antibody and original immunogenic relative affinity, this antibody also by the immune response activity of Western spot detection method screening to the HIV virion protein, is gone back in the antagonist and is screened with the ability of HIV virus infection.

A, affine relatively titration

Except the diluent with anti--mouse IgGFc of various rabbits places the test holes, carry out fluorescence in conjunction with detection according to the method for the routine screening antibody that is adopted in the example 1.The results are shown in Table 1 for the fluorescence of the antibody that obtains from clone JT4C1 to JT4C-13 and JT4C-16.The results are shown in Table 2 for the fluorescence of various JT4C7 subclones.

Data in the table 1 show, clone JT4C1, and the antibody of JT4C2 and JT4C4 has higher avidity.The antibody that is obtained by clone JT4C11 and JT4C13 has lower avidity, and the antibody that all the other test clones obtain has medium avidity.

Correspondingly, table 2 shows the subclone of JT4C7, and JT4C7-12 and JT4C7-9 have high and minimum avidity in this test respectively.

B Western spot is analyzed

Differentiate that by all antibody that 19 positive clones obtain measures immune response activity with the HIV virion protein with the analysis of Western spot in example 1.More particularly, use SDS(0.1%) and dithiothreitol (DTT) (0.003M) make the disintegration of HTLV-IIIB particle, and this material is placed in 7.5% polyacrylamide gel, with standard method this gel is carried out electrophoresis, with this substance transfer to the Nitrocellulose filter paper.The initial PBS liquid that contains 20% heat inactivation horse serum of using is cultivated filter paper 1 hour, washs with PBS.Then with each clone's antibody supernatant liquor in room temperature with slightly shake down filter paper was cultivated 3 hours, again 4 ℃ and slightly shake under spend the night.After with the washing of PBS/ polysorbas20, blocked one hour with 20% horse serum as mentioned above, add 10 micrograms

Table 1

Concentration clone numbering

Anti--murine antibody 01234567

(μ g/ml) (blank)

10 -27 2882 2799 2511 2913 2529 2435 1601

3 271 877 803 823 938 723 688 763

1 37 201 200 236 128 178 133 199

0.3 24 31 17 106 37 99 109 142

0.1 205 14 -1 123 23 49 53 103

0.03 392 21 -82 76 68 66 55 120

0.01 301 15 23 105 54 88 46 85

0.003 17 -8 -43 86 -14 116 42 115

Concentration clone numbering

Anti--murine antibody 89 10 11 12 13 16

（μg/ml）

10 2395 2523 2463 1003 2281 1151 2203

3 617 751 716 405 830 497 620

1 84 98 112 92 215 192 272

0.3 38 88 76 101 397 185 263

0.1 19 49 61 16 117 70 157

0.03 43 43 125 76 104 107 202

0.01 -51 40 103 31 80 60 198

0.003 -27 29 126 26 51 65 136

The rabbit of peroxidase labelling resists-mouse IgG, at room temperature cultivates this mixture 4 hours, after PBS/ polysorbas20 washing 10 times, develops the color with standard method.The antibody that is obtained by clone JT4C8, JT4C12 and JT4C16 is that 60000 to 80000 HIV albumen reacts consumingly with molecular weight all.The ability (and not having this ability among their offspring) of these antibody and HIV protein immunization reaction indicated strongly these antibody in vivo with external realization in and the ability of HIV.

Carry out for the above-mentioned antibody of responding property in the Western spot detects that common-size analysis has disclosed JT4C12 and JT4C16 antibody is IgG ₃Homotype antibody.

C, HIV neutralization

HIV neutralising capacity with following method test hydridization knurl culture supernatants.Cultivate 3 days supernatant liquor with 1: 5 ratio with complete developing medium (500 milliliters of RPMI 1640; 6 milliliters of 100 * penicillin/streptomycin; 6 milliliters of 100 * L-Jie acid amides; 100 milliliters of FCS; With 1.2 milliliters of Polybrene stock(1mg/ml)) dilute, get this medium sample of 200 microlitres be added to 24 hole microtiter plates except two holes the institute porose in, two holes only add the medium of equivalent.In the hole of " only adding medium ", add and replenish medium, add the high-titer HIV virus of 200 microlitres in all the other each holes, these plates are sealed in the plastics bag, cultivated 1-1 1/2 hour down, get back to 17 ℃ then and kept 15 minutes at 4 ℃.With the H9 cell with 1 * 10 ⁶The density of cells/ml was cultivated 30 minutes down in 37 ℃ in perfect dielectric, carry out then centrifugal, and with 5 * 10 ⁶The density of cells/ml is suspended in the fresh perfect dielectric, the cell suspending liquid of 200 microlitres is added to (making its cumulative volume is 600 microlitres) in each hole, then these titer plate are incubated 1 hour down in 37 ℃, subsequently 150 microlitres are transferred to every hole and contained on the identical plate of 2.0 milliliters of fresh complete developing mediums.With culture at CO ₂Under 37 ℃, cultivate in the incubator, after 4 days, tell culture, add fresh complete developing medium, at the 7th day that cultivates, preparation was with (seeing people's Nature 316 phase 72-74 pages or leaves in 1985 such as Guroff with the sample of screening in IFA and the reverse transcriptase method; People such as Matthews ProC.Nat ' in 1986 l.Acad.Sci(USA), 83 phases, 9709-9713 page or leaf; With people Proc.Nat ' in 1980 such as Poiesz l.Acad.Sci(USA), 77 phase 7415-7419 pages or leaves).In first kind and in the screening method, its result is negative basically or does not have conclusion, and in the second method that begins simultaneously with first method, 12 antibody that obtain in the JT4C1-19 series among 15 test clones are in IFA or RT test or shown that neutralization is active simultaneously in these two tests, and in the antibody of 6 tested JT4C7 subclones 5 shown in and characteristic.With show in these detect that 100% neutral people AIDS (HTLV-IIIB) patients serum compares, all these neutralizations all are " parts ".

Example 3

With the commercially available monoclonal anti-human lymphocyte T4(Ortho Diagnostics that is called okT4A, Rahway, N.J.) better with Mono S(than Mono Q) the post purification, repeat the formation and the screening procedure of common hydridization knurl in example 1 with its.Some is different a little for specific immune method and example 1, and difference is that initial injection is intraperitoneal injection, and contains the antibody that about 15 micrograms are purified.Abdominal cavity inoculation for the second time is after seven days, and is made up of the purification antibody of about 10 micrograms.After 13 days, apply 5 microgram antibody to strengthen injection for the last time by the spleen injection.In 2090 grooves of screening, 34 are the obvious positive for the reactivity with oKT4A, and wherein 10 demonstrate the activity (to being approximately 200 " background ", fluorescent value is about 100 or higher) of height.These 10 positive colonies are named as JT1-1D11, JT1-1F3, JT1-1G2, JT1-6E12, JT1-6F12, JT2-8E9, JT3-2C4, JT3-5A11, JT3-6D9, and JT3-6E8.

Example 4

The characteristic of the antibody that produces for the described positive colonies of illustrated example 3, carry out and example 2 described essentially identical tests, briefly, in Western spot detection method, the antibody supernatant liquor is to be about 60000 to 80000 HIV albumen with molecular weight to react, this analytical procedure has obviously represented to be about with molecular weight 67000 proteic reactivity, and it is 78000 proteic reactivity that some antibody demonstrate with molecular weight.It should be noted that and the proteic 41kd of HIV chitose or the 120kd component anergy that have been considered to important immunization usually.Show that at these two (JT1-6E12 and JT2-8E9) are that IgM is isostructural in antibody of strong reactivity, and the antibody of clone JT1-1F3 is IgG ₁Isostructural.The results are shown in Table 3 to carry out the fluorescence binding analysis with JT1-1F3 antibody.

Table 3

Anti--contrast of murine antibody concentration

(μ g/ml) blank (HAT) JT1-1F3

20μg/ml 4 754 1093

10 -37 738 1010

5 -43 756 899

2.5 -56 759 908

1.25 -2 454 634

0.625 -38 291 325

0.313 23 315 310

0.157 11 340 331

With example 2(C) the neutrality research carried out simultaneously also represents initial negative neutralization results, in the 2nd test, only has the sign of slight neutralising capacity in rt test.However, select IgG1-secretion JT1-1F3 clone to be used for ascites and amplify, the step of going forward side by side is carried out the antibody ELISA screening with the HIV albumen test.

In the ELISA screening first time, (concentration is respectively 40,20 to coat the HTLV-IIIB albumen of various different concns on some titer plate, 10,5,2.5,1.25,0.625,0.313,0.156,0.078,0.039 and 0.020 μ g/ml), blocking-up (20% horse serum/PBS was blocked 2 hours down at 37 ℃) and dried overnight.Culture supernatants (20 microlitres and 80 microlitre PBS) or the contrast of HAT developing medium are added as first antibody, at room temperature cultivated 1 hour, store down in 4 ℃ and spend the night.Peroxidase-conjugated rabbit anti--mouse IgG(0.3 mcg/ml is in horse serum/PBS of 5%) add as second antibody.After at room temperature cultivating 2 hours, add substrate (0-phenylenediamine, 0.5mg/ml in the McIlvanShi damping fluid, pH5.5; 10 milliliters of 3% hydrogen peroxide).After at room temperature cultivating 20 minutes,, and read optical density going into the 492-610 place with 50 microlitre 0.4M sulfuric acid stopped reaction.Test-results is illustrated among Fig. 1, and the result shows that HIV is detectable on the level of 1.25 micrograms, and when containing 40 micrograms virus, reactive behavior increases gradually.

To being coated with the H that does not infect at the beginning with different concns ₉Cell membrane preparation (10 ⁶, 6 * 10 ⁵, 4 * 10 ⁵, 2 * 10 ⁵, 6 * 10 ⁴, 4 * 10 ⁴, 2 * 10 ⁴, 1 * 10 ⁴, 6 * 10 ³, 4 * 10 ³, 2 * 10 ³Cells/ml) titer plate repeats the ELISA program, and test-results is illustrated among Fig. 2, and the result shows and the preparation anergy that does not infect.

At last, with planting of various same concentration by HTLV-IIIB HIV variation, the Haitian HIV variation of called after AL1212C is planted (by the Massachusetts, Boston, the DaVid Hall doctor of Massachusetts General Hospital obtains) and the proteic ELISA of albumen repetition HIV that obtains of the African HIV variation kind of (by the Massachusetts, the Jerone Groopman doctor of Bostonian New England DeaConess Hospital obtains) of called after 906.As shown in Figure 3, JT1-1F3 antibody can be discerned common epitope of all three kinds of HIV change xenogeneic.Aforesaid RT analytical method and Syncitium induce the such of indication, test the extracorporeal neutralizing activity of the ascites body fluid that is drawn by JT1-1F3 antibody again.In this method, to comparing between the JT1-1F3 antibody (the IgG component of ascites body fluid) of various levels, a kind of negative control (ascites/pristane (Pristane)) and the positive control with a HTLV-IIIB infected patient serum form, according to the method, HIV bacterial strain IIIB, AL1212C and 906 and Mab JR1-1F3, contrast ascites or in and human serum (with 1: 5 extent of dilution in the salt solution that phosphate buffered is crossed) together 4 ℃ of cultivations 90 minutes down.Then each hole is added H9 cell (5 * 10 ⁶), cultivated 1 hour down at 37 ℃.From each hole, take out aliquots containig (150 microlitre) and be added in 2.0 milliliters the fresh culture medium.At the 4th day culture was opened with 1: 1 minute.Active and Syncytium induces in the 4th day and the 7th day record reverse transcriptase.The results are shown in Table 4 in the 7th day for this method, and wherein Syncytium induced representation is as follows relatively: (-), 0/200 cell; (+/-) the 1-5/200 cell; (+),＞10% cell; (++), 25% of＞cell; And (+++), 50% of＞cell.

Result shown in the table 4 clearly illustrated that JT1-1F3 antibody to the HIV variation of all three kinds of tests kind in external neutralization activity, contrast with the patients serum's who does not infect neutralization activity, it is that mutation one is specific fully, and this common epitope that shows available a kind of important kind is discerned.

The molecular weight of JT1-1F3 antibody response kind is measured by immune spot (immunoblo-tting).Analyze purified HTLV-IIIB with SDS-PAGE and Coomasie orchid/silver dyeing (Fig. 4, channel B and C).With some aliquots containigs like the SDS-PAGE analysis classes, transfer on the nitrocellulose paper, and the reactive behavior of analysis and JT1-1F3 antibody.At about 67kd(Fig. 4, passage E and F) locate to detect an independent reaction kind.

The reactive behavior of JT1-1F3 antibody and HTLV-IIIB strain isolated and reactivity to other HIV strain are compared.Obtain and HTLV-IIIB the reactive behavior of the kindred type of AL1212C and 906 strains with the ELISA method.The Western spot analysis of each has disclosed and the antigenic reactive behavior of similar molecular weight (Fig. 5) in JT1-1F3 antibody and the three kinds of HIB strains in addition.These find the surface, can detected relevant or identical antigen-reactive in MAbJT1-1F3 and the various HIV strains.

It also may be useful in the cell that detects the HTV virus infection that above-mentioned discovery to JT1-1F3 antibody has enlightened it.About this point, JT1-1F3 antibody (the IgG component of ascites body fluid) is attached to not the H9 cell that infects with HIV that infects monitors, determine JT1-1F3 bonded degree by anti--mouse IgG of glimmering actinic rabbit.As shown in Figure 6A, if any, have only can detected antibody combining seldom with the H9 cell that does not infect, on the contrary, can detect H9 cell that this antibody and HTLV-IIIB infect concentrated with combine (Fig. 6 B) disperse.With 906 and the AL1212C strain infect the H9 cell time obtain similar conclusion (Fig. 6 C and D).This method has been generalized on AIDS patient's the hematopoietic cell.By Ficoll-Hypaque partition method collecting monocytic cell and check reactive behavior with JT1-1F3 from AIDS patient's peripheral blood.Shown in Fig. 6 E, concentrate and the rabbit epidemic disease fluorescent dye figure of disperse with these cell detection to one, similar to the result that the H9 cell that infects with HIV draws.

JT1-1F3 antibody that it should be noted that screening does not have tangible reactive behavior with the reactive behavior with the important histocompatibility of II level (" MHC ") surface composition (being people's B clone MD1 and normal peripheral blood lymphocytes).

Subclone JT1-1F3-E5 that is selected by the subclone of JT1-1F3 and JT1-1D7 are respectively as producing than the higher levels of IgG1 antibody of JT1-1F3.JT1-1F3, the neutralization analysis data that JT1-1F3-E5 and JT1-1D7 compare mutually are listed in the table below in 5.The reactive behavior of antibody in elisa assay that these three hydridization knurls produce is listed in the table below in 6.

Table 6

The comparison of 3 hydridization knurls in elisa assay

The HIV-IIIB contrast

Viral protein JT1-1F3 JT1-1E5 JT1-1D7 (developing medium)

10 1.337 1.111 1.392 0.031

5 0.914 0.887 1.118 0.023

2.5 0.996 0.831 1.081 0.022

1.25 0.499 0.278 0.523 0.011

0.63 0.221 0.122 0.176 0.020

0.31 0.070 0.075 0.060 0.015

0.16 0.048 0.049 0.036 0.004

0.08 0.027 0.027 0.031 0.012

Example 5

Make hydridization tumorigenesis and screening step that following change comes repetition example 1 and 3 with okT4A and in immunization method, initial immunity comprises the abdominal injection of the okT4A antibody that 10 microgram Mono S purify; Carry out the abdominal injection second time (7 microgram) after 17 days, carry out the abdominal injection of last 7 micrograms after 18 days.After merging and screening, the generation IgG1 male clone who selects a called after JT2-N15 is for further research.Subclone JT1-1F3-E5 and JT1-1D7 as JT1-1F3 and it, clone JT2-N15 produce one in the Western engram analysis with have molecular weight and be about the monoclonal antibody that 65000 to 67000 HIV albumen reacts.The developing medium of JT2-N15 growth is male in elisa assay, and the preliminary neutralization analysis infectious relevant with HIV-IIIB the results are shown in the following table 7.

Table 7

JT2-N15 is to the neutralizing effect of HIV-IIIB

RT activity and %

Neutralizing effect

1, the H9 157/-that does not infect

2、HIV 12194/0

3、HIV+1.5mg/mlAb 343/98.5

4、HIV+750μg/mlAb 568/96.6

In to the active process of doing further screening by the neutralization of anti--idiotype antibody of ascites method breeding, tentatively definite, following scheme can produce the ascites preparation of maximum activity.The mouse in 5 to 8 ages in week is injected 0.5 to 1.0 milliliter pristane first, two week back injections 2 to 8 * 10 ⁶(preferably about 5 * 10 ⁶) the hydridization oncocyte.Begin to collect ascites body fluid after inoculating for two weeks, the body fluid of collecting at first (about 3-5 milliliter) demonstrates the highest neutralization activity.Carry out the collection of ascites body fluid for the second time after three days, obtain 8 to 10 milliliters of low slightly body fluid of activity.Collect from surviving animals for the third time, generally provide the body fluid of 3-5 milliliter, it often has littler activity than the material of the first time or collection for the second time.The neutralizing effect data of expression are based on the ascites of three gleanings compiling in the table 4 and 5, and the JT1-1F3-E5 in table 6 and 7, and the data of JT1-1D7 and JT2-N15 are based on the ascites of only compiling first and second gleanings.

Though the monoclonal antibody formulation okT4 that has on above-mentioned example and the market is relevant with the method for okT4A, but expect the antibody that other is commercially available, as Leu3a(Becton-Dickenson, Immunocytometry System Mountain View, California, 94039) be applicable to too and produce an anti-idiotype antibody of the present invention.The people T4 protein isolate that produces with the human T-cell who expresses T4 glycoprotein, human T lymphocyte and recombinant chou is suitable for equally as the non-commercially available antibody (preferably monoclonal antibody) of original immunogen by known hydridization knurl technology preparation.In addition, the antibody that produces as subclone and JT2-N15 by JT1-1F3 and it is IgG ₁During homotype, can expect that different homotype antibody equally all is useful.For example, IgG ₂Homotype antibody will be more useful in the method that relates to the molten born of the same parents' reaction of complement one intermediary.

People such as Chanh in June, 1987 P.N.A.S(USA) practical feasibility of the report susceptible of proof the inventive method on the 84 phase 3891-3895 pages or leaves has wherein been reported Leu3a(called after HF1.7) monoclonal antibody can be in external and HIV-IIIB infect.Yet the neutralization activity of HF1.7 is characterized by (the seeing 15 pages of 331 phases of Weiss in January, 1988 Nature) of " weak " and does not have proof can expand HIV-IIIB bacterial strain in addition to.In addition, with resisting in the above-mentioned example-okT4 is different with the okT4A monoclonal antibody, people such as people's such as Chanh anti--Leu3a antibody such as Chanh above 3894 pages as shown in Figure 4, do not point out and any responding property of HTV-derived protein except that gP120 (also the relevant polyclone of The Lancet ii 1047-1050 referring to people such as Dalgleish on November 7th, 1987 resists-content of Leu3a antibody).

Though above-mentioned example all relates to small white mouse deutero-hydridization oncocyte, generate and utilize hybrid hydridization knurl (mouse/people and particularly for example use the TIBTECH 147-153 in January, 1986 for example with Borrebaeck; People such as Abrams Methods in 1986 Enzymology 121 phase 107-119 page or leaf; People such as Kozbor Methods in 1986 Enzymology 121 phase 120-140 page or leaf; People such as Suresh Methods in 1986 Enzymology 121 phase 210-228 page or leaf; And people Biochem.﹠amp in 1986 such as Masuho; Biophys.Res.Comm., 135(2) people/people's hydridization knurl of the method for 495-500 page or leaf unanimity preparation all in scope of the present invention.Also referring to Klausner " Single Chain ' Antibodies Become a Reality " Bio/Technology in 1986,4,1041-1042, Klausner, " Stage Set For ' Immunological Star War ' " Bio/Technology in 1987,5,867-868 page or leaf and Marx, " Antibodies Made To Order " Science in 1985,229, the 455-456 page or leaf.

Therefore, be readily appreciated that, above-mentioned production hydridization knurl and discriminating, the concrete grammar that separates monoclonal antibody can not limit practical range of the present invention, the report of " Immunological Techniques; Part I " that the people such as Langone that publish as the many reports among the Methods in Enzy-mology Vo1.121 and New York Academic publishing company in 1986 edit has many alternative methods can reach same result.

In culture, transform or dna sequence dna that transfection protokaryon and eucaryote host cell expression have its coding is formed for immuno active polypeptide in diagnosis of the present invention and the methods for the treatment of also in the scope of the invention.

Anti-HIV methods for the treatment of of the present invention will be understood to include to infected by HIV or be in clothes for patients among the HIV risk of infection with antibody of the present invention or the antibody fragment of effective dose, comprise in vivo with generation in and the passive immunity that infects of HIV. About this point, in order to form immunocompetent anti-HIV therapeutic combination, expect that product of the present invention can be combined with acceptable diluent, assistant and carrier on the immunology.

Belong to anti-HIV methods for the treatment of in the scope of the invention also comprise use to monoclonal anti--active immunization that the biologically active HIV albumen of responding property of monoclonal Anti-Human lymphocyte carries out. These products can be directly by virus formulation by well-known compatibility purifying technique, comprise the immune response mixture that forms earlier between HIV albumen and the antibody of the present invention, then separate desirable proteins and obtain. As an example, in the method for example 4, be that 60000 to 80000 HIV albumen is the initial candidate of being used as vaccine by the molecular weight of JT1-1F3 identification. Can expect equally to express product based on the recombinant with the HIV DNA of antibody mediated immunity of the present invention identification (and/or purification).

Utilize polypeptide products to detect and determine that diagnostic method of the present invention such as the HIV particle weight in the such biological fluid of blood is expected at patient that preliminary examination is benefited from method of passive immunity of the present invention and under monitoring treatment situation of the present invention, consists of a requisite part. Pointed out the purposes of various diagnosis and treatment about the conclusion of antibody of the present invention and the reaction of HIV infection cell surface selectivity, comprise to be the early infection stage of the analytic approach detection on basis with screening antibodies, infect the tissue screening of organizing (for example lymphocyte) and body fluid (for example blood) sample in order to detect HIV. Allow from the cell colony that comprises infection and non-infected cells, to isolate infection cell with antibody recognition infection cell of the present invention. Therefore, can expect that AIDS patient's blood can be processed external with antibody of the present invention, to remove or optionally to kill infected lymphocyte. In addition, realize the lysis of infection cell and carry out processing also within the scope of the present invention in the body with the circulation complement in conjunction with auxiliary with antibody of the present invention. This therapeutic scheme provides preliminary positive findings to the relevant lysis of complement that test JT1-1D7 antibody participates in the H9 cell that HIV infects in the body.

The character of antibody selective reaction activity makes them become drug disposition or is sent to the toxin of infected cell and is used for that development comprises can be for for example good candidate of the bispecific antibody of two determinants of the sub-T cytoactive of building-up effect. Referring to such as the people such as Stearz Proc.Nat ' in 1986 1.Acad.Sci.(USA), 83,1453-1457 page or leaf.

Based on such fact, namely the present invention is based on the immunological characteristic of T4 albumen-it is believed that It is to all variations kind of (AL1212C for example, 906, ARC.LAV, HTLV-IIIRF, HTLV-IIIB) provide common acceptor-rather than any one specific HIV mutation, so, can expect that diagnosis of the present invention and methods for the treatment of will be applicable to that test-and-treat comprises the infection of all HIV mutation. Utilize polypeptide of the present invention to expect to provide the out of Memory that is deep into HIV and host cell interaction as the work of diagnosis and research. As an example, monoclonal antibody of the present invention will be for the form of accurately one-level, secondary and the tertiary structure in the surface protein zone of identification HIV and host cell, these surface proteins the HIV of identification cell infect and relative process in must be interactional. Otherwise, use this information, will make the synthetic polypeptide immunogen with the recombinant generation generate immunologic active material of the present invention.

Above-mentioned detailed description be just in order to be expressly understood invention, rather than in order to limit the present invention, because the professional in this technology can expect various modifications and changes that the present invention is done.

Claims (40)

1, a kind of through purifying and isolating immuno active polypeptide, can carry out specific immune with the part of HIV virus particle combines, said this part HIV virus particle is inevitable when the HIV host cells infected to interact with the T4 surface protein, it is characterized by external have in and the ability that infects of HIV and with the special antibody of human lymphocyte T4 immunity is had the specific immune reactive behavior.

2, a peptide species as claimed in claim 1 is antibody or chimeric antibody or their pieces.

3, a peptide species as claimed in claim 1 is characterized by with oKT4 antibody the specific immune reactive behavior.

4, a peptide species as claimed in claim 1 is characterized by with oKT4A antibody the specific immune reactive behavior.

5, a peptide species as claimed in claim 1 is with the molecular weight of being measured by SDS-PAGE and is about antibody formation, chimeric antibody or their pieces that 60000 to 80000 HIV albumen has the specific immune reactive behavior.

6, a peptide species as claimed in claim 5, it is about 65000 to 67000 HIV albumen with the molecular weight of being measured by SDS-PAGE and has the specific immune reactive behavior.

7, a peptide species as claimed in claim 1, it and II level histocompatibility molecule are unresponsive on immunology.

8, a peptide species as claimed in claim 1 is mouse-derive antibody or antibody fragment form.

9, a peptide species as claimed in claim 1 is people's antibody or antibody fragment form.

10, a peptide species is monoclonal antibody or monoclonal antibody fragment form as claimed in claim 8 or 9.

11, a peptide species as claimed in claim 10 is the form of two determiners, bispecific monoclonal antibody.

12, monoclonal resisting-mono-clonal-Anti-Human's lymphocyte T4 antibody.

13, monoclonal resisting-oKT4 antibody.

14, monoclonal resisting-oKT4 antibody.

15, a kind of hydridization oncocyte system that can in its growth medium, produce a kind of monoclonal antibody of claim 12.

16, a kind of hydridization oncocyte as claimed in claim 15 is to be selected from JT4C8, JT4C12, JT4C16, JT1-1F3, JT1-1F3-E5, JT1-1D7 and JT2-N15.

17, a kind of hydridization oncocyte system that can in its growth medium, produce a peptide species of claim 11.

18, a kind of anti-HIV methods of treatment comprises to being subject to the HIV infected animals and uses the described peptide species of immunity row Я Kang Na ɡ  .

19, a kind of anti-HIV methods of treatment comprises to being subject to the HIV infected animals and uses the described peptide species of claim 12 of immune significant quantity.

20, a kind of being used for infected the described polypeptide of claim 1 that the vaccine composition that produces protective immune response contains immune significant quantity to HIV.

21, a kind of being used for infected the described a kind of antibody of claim 12 that the vaccine composition that produces protective immune response contains immune significant quantity to HIV.

22, a kind of based on HIV and HIV is had immune response between the immuno active polypeptide of specific immune binding ability, be used for detecting and/or definite HIV quantitative analysis method in biological fluid, its improvement comprises adopts the described peptide species of claim 1 as immuno active polypeptide.

23, a kind of based on HIV and HIV is had immune response between the immuno active polypeptide of specific immune binding ability, in biological fluid, be used for detecting and/or definite HIV quantitative analysis method, its improvement comprises adopts the described a kind of antibody of claim 12.

24, a kind of being used for detected and/or determines in body fluid or tissue samples HIV-host cells infected quantitative analysis method, said method comprises: the described peptide species of said sample and claim 1 forms a kind of immune response mixture, and detects the immunity combination that said polypeptide exists the cells infected surface.

25, a kind of being used for detected and/or determines in body fluid or tissue samples HIV-host cells infected quantitative analysis method, said method comprises: the described a kind of antibody of said sample and claim 12 forms a kind of immune response mixture, and detects the immunity combination that said antibody exists the cells infected surface.

26, a kind of analytical procedure as claimed in claim 25, wherein said detection step comprises the detectable mark of determining to be attached on the said antibody.

27, a kind of analytical procedure as claimed in claim 25, wherein said detection step comprises the detectable mark of determining to be attached on the anti-antibody that adds in the said reaction mixture.

28, a kind of analytical procedure as claimed in claim 25, wherein said antibody are selected from anti--oKT4 antibody or anti--oKT4A antibody.

29, a kind of method of in the colony of infection and non-infected cells, separating the HIV cells infected, this method comprises that said cell colony and the described peptide species of claim 1 form a kind of immune response mixture, and according to said polypeptide the selective binding of cells infected is separated cells infected from non-infected cells.

30, a kind of infect with non-infected cells colony in separate the HIV cells infected method, this method comprises that a kind of antibody of said cell colony and claim 12 forms a kind of immune response mixture, and according to said antibody the selective binding of cells infected is separated cells infected from non-infected cells.

31, a kind of proteic method of HIV of from body fluid sample, separating, this method comprises said sample is contacted with a peptide species of claim 1 forming a kind of immune response mixture that comprises said albumen and said polypeptide, and separate said albumen from said reaction mixture.

32, a kind of proteic method of HIV of from body fluid sample, separating, this method comprises said sample is contacted with a kind of antibody of claim 12 forming a kind of immune response mixture that comprises said albumen and said polypeptide, and separate said albumen from said reaction mixture.

33, a kind of through purifying and isolating HIV albumen, it is characterized in that:

(1) determines that by SDS-PAGE its molecular weight is about 60000-80000; And

(2) with monoclonal anti--the specific immune reactive behavior of mono-clonal-Anti-Human's lymphocyte T4 antibody.

34, as claimed in claim 33 a kind of through purifying and isolating HIV albumen, it is characterized in that determining that by SDS-PAGE molecular weight is about 65000-67000.

35, a kind of HIV albumen as claimed in claim 33, its another feature is: can infect HIV in the host of a sensitivity stimulates immunne response in the body that shields.

36, a kind of HIV albumen as claimed in claim 33, its another feature is: adopt to comprise a kind of product of a kind of immunosorbent of monoclonal anti-mono-clonal-Anti-Human's lymphocyte T4 antibody as the affinity method of purification.

37, a kind of being used for comprises acceptable diluent, auxiliary or carrier on the described a kind of HIV albumen of claim 33 of immunocompetence amount and the immunology to the vaccine composition that the HIV virus infection produces protective immune response.

38, a kind of vaccine composition that is used for the HIV virus infection is produced protective immune response; comprise have part and whole be that the DNA of 60000 to 80000 HIV encoding histone transforms or the expression product of the host cell of infection with the SDS-PAGE determining molecular weight, and this product and monoclonal resisting-mono-clonal-anti-human lymphocyte T4 antibody have the specific immune reactive behavior.

39, a kind of usefulness has been encoded, and the dna sequence dna of a peptide species of claim 1 transforms or the microbial host cell of transfection.

40, a kind of usefulness has been encoded, and the dna sequence dna of a peptide species of claim 33 transforms or the microbial host cell of transfection.

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