CN1068266A - Human immunodeficiency virus's vaccine and Therapeutic Method thereof - Google Patents

Abstract

In the baculovirus insect cell carrier system, produce acquired immune deficiency syndrome (AIDS) (acquired immune deficiency syndrome (AIDS)) vaccine that contains people's defective immunity virus, 1 type (HIV-1) envelope protein by HIV-1 env gene of cloning.Recombinant HIV-1 albumen is purified and is assembled into granule and is adsorbed on the aluminum phosphate adjuvant.This AIDS vaccine is induced body fluid and the cell immune response that makes new advances in the individuality that HIV infects, and can be used as that the vaccine therapy agent is used for postponing or pre-anti-virus to immune destruction.

Description

Human immunodeficiency virus's vaccine and Therapeutic Method thereof

The application is the part continuation application of the U.S. Patent application 151,976 submitted on February 3rd, 1988, and the latter is the part continuation application (present series number is 585,266) of the U.S. Patent application 920,197 submitted on October 16th, 1986.These applications and the document of quoting are herein all listed in this description with the list of references form.

1-type human immunodeficiency virus (HIV-1) is a kind of retroviral that causes systemic infection, its main diseases Neo-Confucianism is immune system, and be the pathogen (Barre-Sinoussi that causes acquired immune deficiency syndrome (AIDS) (acquired immune deficiency syndrome (AIDS)), et al, Science, 220:868-871(1983); Popovic et al.Science，224：497-500（1984）〕。The clinical isolates of HIV-1 also is known as Lymphadenopathy-associated virus (Feorino, et al.Science, 225:69-72(1984)) and AIDS related virus (Levy et al., Science 225:840-842(1984)).

It is more and more general that acquired immune deficiency syndrome (AIDS) has become, and its developing vaccines has become a major subjects in publilc health field, the world.The patient that major part has infected HIV-1 causes the forfeiture gradually of immunologic function because of the lymphocytic minimizing of T4.On the surface of these T4 cells and some neurocyte, has the molecule that is called CD4.HIV-1 enters these cells and also finally duplicates and cell killing by being positioned at the receptor identification CD4 molecule on the virion district film.Effectively AIDS vaccine is expected to cause and produces and the bonded antibody of HIV-1 peplos, thereby prevents that it from infecting T4 lymphocyte or other permissive cell.

Vaccine normally gives healthy individual with the form of preventive before the ill organism of contact.But, also can consider effective AIDS vaccine is used (Salk, J., Nature, 327473-476(1987)) as the immunotherapy agent to this disease in the immunity after contact.

In the exploitation of AIDS vaccine, generally believe HIV-1 peplos (" env ") be most promising candidate (Francis and Petricciani.New Eng.J.Med., 1586-1559(1985); Vogt and Hirsh, Reviews of Infeetious Disease, 8:991-1000(1986); Fauci, Proc.Natl.Acad.Sci.USA, 83:9278-9283).Initial synthetic HIV-1 envelope protein is that molecular weight is 160,000 glycoprotein (gp 160), to be cut into molecular weight be that 120,000 outside glycoprotein (gp 120) and molecular weight are 41,000 transmembrane glycoprotein (gp 41) to these gp 160 precursors then.These envelope proteins are main target antigens (Barin, et al., Science, 228:1094-1096(1985)) of HIV sufferers internal antibody.Proved natural HIV-1 gp 120 be exempt from disease originality and can in rodent, goat, Rhesus Macacus and chimpanzee, induce and produce neutralizing antibody (Robey, et al., Proc.Natl.Acad.Sci.USA83:7023-7027(1986)).

Because the level of natural HIV-1 envelope protein is extremely low in the infected cell, and exist and the relevant danger of cell preparation AIDS vaccine, so adopted recombinant DNA method to produce HIV-1 envelope antigen as AIDS vaccine by the HIV-1 infection.Because recombinant DNA technology can produce a large amount of safe and economic immunogens, so it is the optimum selection of production acquired immune deficiency syndrome (AIDS) subunit vaccine.Expressed in the vaccinia virus recombinant that in heredity, has changed the HIV-1 envelope protein (Chakrabarti, et al., Nature, 320:535-537(1986); Huet al., Nature, 320:537-540(1986) Kieny, et al., Biotechnology, 4:790-795(1986)).In addition, also bacterial cell (Patney, et al., Science, 234:1392-1395(1986), mammalian cell (Lasky, et al, Science, 23:209-12(1986)) and expressed in insect cells envelope protein.The candidate (Kenneely, et al., (1986)) that also is considered to AIDS vaccine by the deutero-synthetic peptide of HIV-1 gp41 aminoacid sequence.But, utilize these materials and method successfully not to produce AIDS vaccine.

Utilizing baculovirus-insect cell carrier system production Recombinant HIV-1 envelope protein is the U.S. Patent Application Serial 920 of the common unsettled and common transfer submitted on October 16th, 1986, (the application's series number is 585 in disclosed invention aspect in 197,266) (see series number 151,976 again).

Proved that rhabdovirus system has versatility in producing HIV-1 albumen and other albumen.For example, baculovirus autographa california (Autographa Californica) nucleopolyhedrosis virus (AcNPV) is used as carrier, in infected autumn mythimna separata (Spodoptera frugiperela, fall armyworm) cell (sfg cell), express the total length gp160 and the various piece thereof of HIV-1 env gene.Protein that in above-mentioned common pending application application, also discloses the gp160 gene (recombinant Ac3046) that blocks, produced by recombinant Ac3046 and the Ac3046 gene outcome purification technique that comprises Lens culinaris lectins affinity chromatograph and gel permeation chromatography.Found with this mode purification and be gathered into particulate gp160 albumen in rodent and primate, to have very high immunogenicity.

Ideal AIDS vaccine also should can provide the lifelong protection of anti-HIV-1 infection after once or several times injecting except satisfying the requirement that is essentially biological pure and mild non-pyrogen.The viral vaccine of living is like this usually.When antibacterial that kills when utilization or virus or its separator (as toxoid or protein) preparation vaccine, usually produce the very weak antibody response and the immunity of short-term.In order to overcome or reduce these defectives of vaccine as far as possible, can use the supplementary element that is called adjuvant.Adjuvant is the material that helps immune stimulatory to reply, adjuvant commonly used in people's vaccine is the gel of aluminum salt (aluminum phosphate or aluminium hydroxide), be commonly referred to adsorbed onto alum adjuvant (Bomford, et al., " Adjuvants ", Animal Cell Biotech.Vol.2:235-250, Academic Press Inc.(London:1985)).

The invention provides the vaccine and the Therapeutic Method thereof of HIV (human immunodeficiency virus) (HIV), it comprises to infected individuality or susceptible individual takes the Recombinant HIV envelope protein.In a preferred embodiment, can combine with envelope protein purification, gathering and with adjuvant (as Alumen) with as vaccine.

Below in conjunction with description of drawings particular content of the present invention.

Fig. 1 illustrates clone's scheme of separating HIV-1 env gene (env) in escherichia coli plasmid pNA2.Shaded area is the HIV-1 DNA sequence.The open zone is from cloning vehicle.Black area among the plasmid p1774 is made up by synthetic oligonucleotide, and is introduced in as the SmaI-KpnI fragment in the SmaI-KpnI site of plasmid p1614.The sequence of this synthetic oligonucleotide is listed.

Fig. 2 has illustrated the scheme of construction of recombinant plasmid vector (p3046), and this carrier is used to make up rhabdovirus expression vector Ac3046 again.The sequence (cross-hatched area) that derives from baculovirus AcNPV is contained in the both sides of plasmid PMGS3 4.00 place's cloning sites in the position.This site is the specific limited Cobra venom endonuclease point of contact of SmaI, KpnI and Bgl II.AcNPV polyhedral body promoter is positioned at 5 ' direction of 4.00 positions, sequence 5 '-TAATTAATTAA-3 ' is positioned at 3 ' direction, and reads that at all three rotaring inter-transtating terminating cipher is all arranged in the sign indicating number.The sequence in plasmid p1774 and synthetic oligonucleotide zone as shown in Figure 1.The sequence between SmaI and Bgl II site (the HIV-1 env gene of p1774 inserts at this), plasmid p3046 contains all PMGS3.

Fig. 3 shows the dna nucleotide sequence of flank Ac3046 gp160 coded sequence.+ 1 and+3046env DNA sequence between 2264 is shown among Fig. 4.

Fig. 4 a-4k shows that Ac3046(+1 is between+2264) in the real DNA sequence of HIV-1 enV genetic fragment and the sequence synthetic oligonucleotide of enV gene 5 ' end.List the location in restriction endonuclease site above the DNA sequence, listed the aminoacid sequence of inferring below the DNA sequence.Indicate the numbering of base on left and right side.

Fig. 5 a-5d has compared from the DNA sequence of the env gene of Ac3046 and the disclosed env gene order from LAV-1.Being the LAV-1 sequence above, is the sequence of Ac3046 below.Sequence among line below the LAV-1 sequence (1) the expression Ac3046 is identical in this position.Employed DNA sequence numbering is (Cell, 40:9-17(1985)) described numberings such as Wain-Hobson.

Fig. 6 shows the people HIV-1 antibody positive serum (last figure) and the gp160(IJ55 that must use by oneself, KL55) or gp120(AB55, and CD55, GF55) the ELISA terminal point dilution titer of Mian Yi animal Rhesus Macacus serum (figure below).The ELISA titre records by highly purified gp120 and gp160 albumen.The specificity binding antibody is measured with goat anti-human igg HRp junctional complex.The highest serum dilution factor that produces positive reaction in the test is titre.

Fig. 7 has summed up in seropositivity patient's body of inoculating vaccine by the vaccine-induced immunoreation of gp160 with the form of table.

Fig. 8 (A and B) shows the antibody response of vaccine-induced anti-specificity HIV peplos surface antigen determinant.

Fig. 9 is presented in the seropositivity individuality of inoculating and is reacted by the T-cell proliferation of vaccine-induced anti-gp160.

Figure 10 (A-C) shows the lymphopoiesis reaction relevant with inoculation.

Figure 11 is percent and the time relation figure that cd4 cell changes in respondent and non-responder.

Found Recombinant HIV-1 gp160 envelope protein (" rgp 160 "), be particularly suited in the time of particularly on it is adsorbed in as Alumen adjuvants such as (as aluminum phosphate) as AIDS vaccine.One aspect of the present invention is the Ac NPV expression vector with section H IV-1 env gene coded sequence, and said part comprises the amino acid/11-757 that sees in the recombinant clone 3046.Another aspect of the present invention is to produce HIV-1 envelope protein (and protein itself)-especially by aminoacid sequence 1-757(promptly 03046 in insect cell) the rgp160 albumen of encoding.

The present invention also comprises by purification in the gene outcome that produces 3046 proteic recombinant baculovirus and forms the recombinant envelope protein granule and 3046 plasmids are adsorbed on the aluminum phosphate aggregation.

The present invention also relates to prevent and/or treat the vaccine of acquired immune deficiency syndrome (AIDS) or HIV infection and the method for prevention or treatment acquired immune deficiency syndrome (AIDS) or HIV infection.

Following examples illustrate the present invention, but do not limit its scope.

U.S. Patent Application Serial 920 at common unsettled and common transfer, the present series number of 197(is 585,260) baculovirus of having narrated reorganization in is autographa californica nuclear polyhedrosis virus (AcNPV), and it contains the HIV-1 gp160 gene (recombinant Ac3046) of the truncate of coding HIV envelope protein 1-757 amino acids.Wherein also disclose the clone's step that is used to make up the recombinant baculovirus that contains HIV-1 gene or its part, it also classifies this paper list of references as.

It below is detailed description to the genetic engineering step that is used to make up the Ac3046 expression vector.Employed material comprises that enzyme and immunoreagent all obtain by commercial sources.And provide and how to have finished and utilized embodiments of the invention.

The present invention also considers other recombinant envelope protein that is referred to as rgp160, comprises reorganization gp120 and gp41 albumen.Ac3046 only is an example of expression vector of the present invention and recombinant envelope protein.

Embodiment 1

Carry the structure of the baculovirus recombinant Ac3046 of HIV-1 amino acid/11-757 coded sequence

Clone and express the exogenous proteins coded sequence and need at one end in baculovirus vector with this coded sequence and polyhedral body promoter and upstream sequence arrangement, and the baculovirus coded sequence is arranged in the other end.Arrangement will make the transfer of outside the genomic homologous recombination of baculovirus can cause arranging with polyhedral body promoter non-activity polyhedrosis gene source coding sequence like this.

Therefore, designed the insertion carrier that a large amount of being used to makes up the HIV env gene.Design insertion carrier MGS3 described below to be used to providing ATG translation initiation codon.Exogenous array is inserted this carrier must make the sign indicating number of being set up by start codon of translating correctly keep in whole exogenous array.

Insertion carrier MGS3 is cloned by the EcoRI-I restriction fragment of AcMNPV separator (WT-1) separated DNA of plaque purification to make up.MGS3 is designed to be made up of following structure: (a) the 4000bp sequence of the ATG upstream from start codon of polyhedrosis gene; (b) the poly connexon of introducing by direct mutagenesis, it is made up of ATG start codon and restriction enzyme site Sma I, KPn I, Bgl II and the general termination codon fragment at corresponding polyhedral body codon position place; (c) extend to the 1700bp sequence (see figure 2) of ECORI-I clone's terminal ECORI restriction site from Kpn I restriction site (being positioned at polyhedrosis gene).

Embodiment 2

Structure carries the baculovirus recombinant of LAV env coded sequence

The recombiant plasmid of naming to NA2 (Fig. 1) is made up of the proviral 21.8Kb fragment of complete HIV-1 that is inserted among the pUC18.Because this can produce virus after being cloned in some people's cell of transfection, so there is report to think that it is to have infective (Adachi, et al., J.Virol.59:284-291(1986)).The complete packet membrane gene sequence that contains among the NA2 derives from the LAV Strain (Barre-Sinoussi(1983) of HIV).

Separate and operation HIV-1 env gene by the method shown in the following stated and Fig. 1.Env gene is the EcoRI/SacI restriction site of separating and be cloned into PUC19 with the form of 3846bp EcoRI/SacI restricted fragment in NA2 at first.The plasmid that is produced is called p708.

Separate env gene once more as 2800bp Kpn I restriction fragment then and be cloned in the Kpn I restriction site of PUC18.The plasmid called after p1614 that is produced.

Kpn I restriction fragment among the p1614 contains the fraction HIV env gene of truncate a little, makes the terminal corresponding sequence of its N-lack 121bp.This disappearance part (having comprised signal peptide sequence) in the gene is substituted by inserting double-stranded synthetic oligomer.The oligomer that is inserted into utilizes preferred polyhedrosis gene codon to design according to the LAV aminoacid sequence.For ease of further operation, a new SmaI restriction sequence is introduced simultaneously, to replace the ATG start codon.The ATG start codon will insert carrier by baculovirus and provide.The plasmid that is produced is named and is p1774.

With reference to Fig. 2, the restriction fragment that derives from p1774 and contain each regional coded sequence of HIV-1 peplos is cloned into MGS and is inserted in the carrier (as MGS3), thereby makes the ATG start codon that inserts carrier and the codon of env gene be arranged in the same sign indicating number of reading.Construction p3046 is made up of the isolating SmaI/BamHI restriction fragment of p1774 in the SmaI/Bgl II site that is inserted into plasmid vector pMGS3 certainly.This clone contain gp160 amino acid/11-757 coded sequence and the termination codon that provides by the MGS3 carrier has been provided.

Embodiment 3

Preparation and screening recombinant baculovirus

With HIV env gene recombination plasmid p3046 and AcMNPVDNA(WT-1) one reinstate calcium phosphate precipitation, and be added in the Spodoptera frugiperda cell that does not infect.By homologous recombination mosaic gene is inserted in the AcMNPV genome then.According to the negative plaque identification of morphology of closure recombinant virus.This class plaque shows identifiable cytopathic effect, but seedless closure.Carry out twice other continuous plaque purification to obtain recombinant virus.By relatively the restricted cutting of recombinant virus dna and wild-type virus DNA and the locus specificity that hybridization characteristics is analyzed HIV env sequence insert.

Embodiment 4

Derive from the HIV env of recombinant baculovirus in the expressed in insect cells that infects

The HIV env sequence that derives from recombinant virus in expressed in insect cells will cause the synthetic of elementary translational product.This elementary product will be made up of the aminoacid that the codon that recombinant vector provided is translated.Its result obtains a kind of protein, and it contains from the ATG start codon of polyhedral body promoter downstream expression vector and translates all coded aminoacid of codon the termination signal to the expression vector (as rgp160).The end of level translational product should be Met-Pro-Gly-Arg-Val at the beginning of the Ac3046, and wherein the Arg on 4 is the Arg on 2 of the original LAV clones.The Met-Pro-Gly codon is that the result as this clone's scheme provides.

Embodiment 5

The nucleotide sequence of gp160 insertion and flanking DNA thereof

Separating the nucleotide sequence that in the restriction fragment of virus expression carrier Ac3046 DNA, determines gp160 insertion and flanking DNA.Sequence analysis method may further comprise the steps.Restrictive diges-tion purification 3.9Kb EcoRI-BamHI fragment by the Ac3046 viral DNA.From be present in the extracellular virus that is used in the cell culture medium of mass production vaccine, make the Ac3046 viral DNA.

As shown in Figure 2,3.9Kb EcoRV-BamHI fragment is made up of with about 1000bp downstream flanking DNA complete gp160 gene and 100bp upstream.Wherein, the nucleotide sequence of complete gp160 gene is determined, and it comprises 100bp upstream and 100bp downstream flanking DNA.

Briefly, the result of sequence analysis discloses the chimeric construct thing as clone's scheme is desired.The sequence of gp160 basically with (1985) such as Wain-Hobson reported identical.Be speculated as the glycosylation site that 751 amino acid whose codons are connected with 28 potential N-at the rotaring intertranslating start of left and right sides and the sequence of 2253 bases between the termination codon.The molecular weight of estimated this rgp160 that comprises saccharide residue is about 145,000.

The sequence analysis that flanking DNA carried out of 200 bases has been shown correct insertion shown in Fig. 3,4 and 5.

Embodiment 6

The aminoacid sequence of gp160

Utilize the N-end sequence of the automatic edman degradation method of standard and preceding 15 residues that the HPLC method records gp160 identical with the sequence of inferring by DNA sequence.The terminal methionine of N-does not appear on the gp160 albumen.This conforms to the observed result that also produces the AcNPV polyhedrin when the terminal methionine of no N-.As the gp160 that passes through analysis AcNPU3046 DNA and purification is determined, now true gp160 DNA and N-terminal protein matter sequence are summarized as follows (table 1).

Table 1

LAV env gene residue in Ac NPV 3046 expression vectors

Original LAV-1 clone shown in these results and following (table 2) is compared.

Table 2

LAV env gene residue among the original LAV-1 clone

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Met?Arg?Val?Lys?Glu?Lys?Tyr?Gln?His?Leu?Trp?Arg?Trp?Gly

ATG?AGA?GTG?AAG?GAG?AAG?TAT?CAG?CAC?TTG?TGG?AGA?TGG?GGG

Embodiment 7

The purification of recombinant gp160

An aspect involved in the present invention be extract and purification Ac3046 expression vector in the method for coded Recombinant HIV-1 envelope protein.Recombinant HIV-1 envelope protein gp160 produces in Spodoptera frugiperda cell when infecting 4-5 days with Ac3046.The proteic purification of this rgp160 comprises the steps:

1. washed cell

2. lysis

3. gel permeation chromatography

4. Lens culinaris lectins affinity chromatograph

5. dialysis

This embodiment has narrated from about 2 * 10 ⁹Purification of Recombinant gp160 in the cell that individual Ac3046 infects.

1. washed cell

In the buffer that contains 50mM Tris buffer (PH7.5), 1mM EDTA and 1% Triton X-100, wash infected cells.Cell is suspended in this buffer again, uses the conventional method homogenate, and with 5000rpm centrifugal 20 minutes.With this method triplicate.

2. lysis

In 50mM Tris buffer (PH8.0-8.5), 4% dexycholate and 1% beta-mercaptoethanol, pass through the washed cell of supersound process cracking.Ultrasonic Treatment is carried out according to a conventional method.It is complete having only the nuclear membrane residue after handling, and is removed in centrifugal 30 minutes with 5000rpm.Determine to contain by observation by light microscope in the supernatant of gp160 of extraction and do not have intact cell.

3. gel filtration

On Pharmacia 5.0 * 50cm glass column of having filled Sephacryl resin (Pharmacia), carry out gel filtration.Total bed volume is about 1750ml.For removing pyrogen and cleaning post and connection tube, wash post with 6 liters of 0.1N NaOH at least, total time is 24 hours.To in post, be connected on UV flow cell, monitor and the chart recording instrument (Pharmacia) by effusive liquid, use 4 liters of gel filtration buffer balances then.Thick gp160 sample on capital is also opened up layer with the gel filtration buffer.

This post is separated into three main uv absorption parts with crude mixture.The 1st peak flows out between about 500-700ml buffer, and second peak flows out between the 700-1400ml buffer, and the 3rd peak flows out between the 1400-1900ml buffer.Observe same washing curve on small-sized analytical column, it has recorded first peak is that molecular weight is 〉=2,000,000 material.

Owing to concentrating of high molecular lipid and lipid complex makes that this peak is translucent.This peak also contains from the gp160 of the 10%-20% of infected cell extraction obviously, and this part gp160 is compound with itself or other cell component, has formed high molecular weight aggregates.

Second broad peak contains most of gp160 and the protein of molecular weight between about 18,000 to 200,000.

The 3rd peak contains protein seldom, and most of uv absorption is because due to the beta-mercaptoethanol that exists in the sample.

When at first detecting second peak, be that effluent directly is added on the Lens culinaris lectins post by the uv absorption spike.In case second peak washes out from post, remove effluent from Lens culinaris lectins post immediately, and import in the solution.

4. Lens culinaris lectins

Lens culinaris lectins affinity gel substrate (Lentil Lectin-Sepharose 4B) is buied from Pharmacia with bulk form.Reach purity more than 98% separating the Lens culinaris lectins by affinity chromatograph on the Sephadex, utilize Bromine cyanide. that it is coupled on the Sepharose 4B and immobilization then.Every milliliter of gel contains the 2mg part of having an appointment in this substrate.Lens culinaris lectins post is the 510 * 30cm glass column (Pharmacia) that contains 125ml Lens culinaris lectins-Sepharose 4B gel.Affinity substrate can use repeatedly through after thorough washing and pressing the method regeneration of manufacturer's recommendation.When not using this gel is added in by 0.9%NaCl, 1mM MnCl ₂, 1mM CaCl ₂In post, preserve in the solution of forming with 0.01% ethyl record thiosalicylic acid sodium.Wash and this post of balance with the above-mentioned Lens culinaris lectins of 250ml buffer before each the use.

When behind eluting on the above-mentioned solvent resistant column, with the direct upper prop of thick gp160.In a single day thick gp160 combines with post, promptly wash post with the 800ml Lens culinaris lectins buffer that contains 0.1% dexycholate.Under these conditions, all gp160 all combine with post.With Lens culinaris lectins and the bonded glycoprotein of 0.3M Alpha-Methyl mannoside eluting, and by the ultraviolet light monitor in wavelength 280nm place's monitoring.

5. dialysis

Remove desaccharide and dexycholate with the conventional dialysis method.

In the table 3 below the method for the cell purification gp160 of 1 liter of infection can be summarized in.

In another embodiment, the ion-exchange chromatography of available routine (anionic or cationic) replaces gel filtration.Equally, the order of each step is not strict: for example, can carry out gel filtration or ion-exchange chromatography after Lens culinaris lectins purification step.According to the present invention, also can use other reagent.For example, available other detergent replaces the deoxycholic acid purification of recombinant proteins.This class detergent comprises nonionic detergent, as Tween 20(multi-sorbitol ester 20), Tween 80, Lubrol and Triton x-100.

¹Total protein is to estimate according to the absorptance at 280nm place.

Embodiment 8

A。The particulate assembling of gp160

As one aspect of the present invention, found to be purified in the process gp160 antigen and can be assembled into molecular weight and be equal to or greater than 2,000,000 granule.The gp160 albumen that extracts from cell is the mixture of being made up of 80-90% monomer (molecular weight 160,000) and 10-20% polymer (particle form).To the trial prompting of purification gp160 in this part (effusive first peak in the solvent resistant column) it and other cell protein even compound with the film fragment.But the gp160 molecular weight in effusive second peak in the gel Filter column is about 160,000-300, and 000, so it mainly is monomer or dimeric forms.

The formation of gp160 aggregation or polymer occurs in the Lens culinaris lectins post exhibition layer process.Clear and definite no matter be in 0.5% dexycholate (it is about 0.2% critical capsule concentration (CMC) of dexycholate) from lectins post eluting, still in 0.1% dexycholate from the post eluting, this antigen all forms aggregation.

Go up the size of measuring aggregation at high-resolution FPLC Superose 12 posts (Pharmacia).The gp160 sample of the representational purification of different lot numbers has the molecular weight (2,000,000) that is equal to or greater than blue dextran standard mostly.

By crosslinked studies confirm that (1989) such as Schwaller are carried out, the gp160 that produces in the insect cell is by the tetramer that equates that subunit is formed.This research shows that also cell and the gp160 in the virion that HIV infects are the tetramer.Therefore, reorganization gp160 granule may have three grade and the quarternary structure similar to natural HIV gp160.

In order to form the correctly folding required antigenic determinant of gp160, suitable three dimensional structure may be very important.Be likely since non-glycosylated protein combine with Lens culinaris lectins post and washing process in lost with gp160 is antigenic and got in touch, the hydrophobic part of gp160 begins to form the intramolecularly contact.Because its concentration can remain on more than the CMC, dexycholate does not combine with gp160 probably, and antigen still can form complex.Its civilian dress is made into polymer when according to this antigen of method purification of the present invention, seemingly this proteinic intrinsic property.This may be that the very hydrophobic N-end sequence that is present on the gp160 albumen causes this protein to have the particulate natural ability of formation.After the purification, can under the proteinic situation of not obvious forfeiture, carry out aseptic filtration to the gp160 complex by the cellulose acetate filter membrane by 0.2 μ m.

B. the formation of analysing particulates

With the gp160 granule of electron microscope observation purification, prove that they are spheroidal particle of the 30-100 μ m of analogous protein.

Identify that another test that granule exists is the gp160 that analyzes purification with gel filtration.About 100 μ g gp160 are added to Superose 12, FPLC gel filtration HR 10/30 post (Pharmacia, Inc.) on.This post is at first proofreaied and correct with the protein molecular weight standard product.The protein scattergram that obtains from this post has very high repeatability; The molecular weight of elution volume and protein standard substance is inversely proportional to.This post is separated monomer gp 160 and get rid of molecular weight with polymer and is equal to or greater than 2 * 10 ⁶Globular protein.On this post when exhibition layer the equal eluting in the water capacity outside of gp160 of all purification basically, so its molecular weight size is equal to or greater than 2 * 10 ⁶(2,000,000).

Embodiment 9

A。Gp160 is adsorbed onto on the Alumen

Insoluble aluminium compound depends on the degree fully that antigen adsorbs as the effectiveness of immunological adjuvant on solid phase surface.As a part of the present invention, to have found to make such Alumen compositions, they can adsorb gp160 effectively not reducing under the PH condition of gp160-Alumen complex as the effectiveness of immunogen.The factor that will control in Alumen (Fosfalugel (Yamanouchi)) compositions forming process has:

1. the best PH that is adsorbed on the Alumen of antigen is about 5.0.But, found to compare with PH7.5, therefore gp160 forfeiture immunogenicity will prepare Alumen under PH7.1 ± 0.1 condition when PH6.5.Found that when this pH value 100% gp160 will be attracted on the Alumen basically.

2. relatively low from the ionic strength of NaCl, and less than 0.15M.

3. the aluminum chloride that has molar excess with respect to sodium phosphate is to guarantee not having the free phosphoric acid radical ion in the supernatant.

4. gp160 antigen is added in the Alumen of firm preparation, to stop crystalline growth and to reduce granular size as far as possible.

As described below, prepare 200ml Alumen and method that purification gp160 is adsorbed onto on the Alumen should guarantee that antigenic ultimate density is 40 μ g/ml.

3. the preparation of reagent (preparing 200ml altogether)

The following solution of preparation in the aseptic apyrogeneity bottle of 100ml.The salt of mixed solution 1 and solution 2 and sodium hydroxide, and the cellulose acetate membrane filtration by 0.2 μ m is to the aseptic apyrogeneity bottle of 100ml.

Solution 1 AlCl ₃6H ₂O 0.895g

NaHAc·3H ₂O 0.136g

Be dissolved in the 40ml water for injection (WFI) 0.2 μ m filter membrane

Filter

Solution 2 Na ₃PO ₄12H ₂O 1.234g

Be dissolved in 40ml WFI, 0.2 μ m membrane filtration

Solution 3 NaOH 2.0g

Be dissolved in 100ml WFI, 0.2 μ m membrane filtration

Solution 4 Tris 1.25g

Be dissolved in 100ml WFI, add 1ml to 90ml WFI,

Regulate pH to 7.5 with 0.5N HLL, and add with WFI

To 100ml

With solution autoclaving 30 minutes; Slowly find time.Be cooled to room temperature.

C. the generation of Alumen

With the aseptic Dispette of 25ml with solution 1(aluminum chloride-sodium acetate) be added in the preparation container, the volume of recording solution 1 also begins agitating solution.

With the aseptic Dispette of 25ml with solution 2(sodium phosphate) add in the solution, continue to be stirred to and form precipitation, the volume of recording solution 2.

3. add 3ml solution 3(sodium hydroxide) also continue stirring 5 minutes, get 0.5ml sample determination pH value.If PH is lower than 7.0, add the 0.5ml sodium hydroxide again, continue to stir after 5 minutes and measure PH once more.Continue this process to PH be 7.0-7.2.

4. measure the cumulative volume (solution 1+ solution 2+ solution 3) that adds in the preparation container, add aseptic WFI then volume is supplied 100ml.

5. will be dissolved in 100ml 1mM Tris(PH7.5 rapidly) in the gp160 of 8,000 μ g purification directly be added in the preparation container.

6. continue at least to stir 20 minutes, then the vaccine branch for preparing is filled in the sterile vials.

Embodiment 10

The immunogenicity (specificity Ab reaction) of the gp160 of Alumen absorption

The immunogenic method of mensuration antigen preparation (vaccine) that is adopted is to measure one group of specific antibody reaction that has given the antigenic mice of single dose.Give the mice blood-letting 4 weekends, and measure the antibody horizontal of anti-specific antigen (antigen that normally is used for immune animal) in the serum by conventional antibody test (as ELISA, elisa).

Table 4 summed up under PH6.0 and PH7.5 condition, adsorbed adsorbed onto alum adjuvant (embodiment 9) or with the gp160 of the blended purification of Freund's complete adjuvant in the intravital immunogenicity of mice.

²Mice is in immunity blood-letting in back 28 days, and detects the antibody titer of the gp160 of the anti-gel purification in the serum (dilution in 1: 10) with the ELISA detection method.Utilize commodity ELISA medicine box (Genetic Systems Inc.; EIA ^TmELISA) the anti-proteic antibody of natural HIV-1 also obtains analog result in the detection serum (dilution in 1: 400).

³Seroconversion number of mice (P) is than tested total mice (N).

The mice immunized of gp160 antigen that does not add any adjuvant with 1.0 μ g will induce the antibody response (seeing the above table) at gp160.But, in the gp160 mice immunized group that is adsorbed in 1.0 μ g on the adsorbed onto alum adjuvant, observe stronger antibody response.Mix with complete Freund's adjuvant or will make by immune mouse and produce seroconversion more than 50% with the single dose gp160 less than 0.1 μ g of adsorbed onto alum adjuvant preparation.Although the antigen of not preparing when gp160 is as PH7.5 and PH6.0 when being lower than this extreme value still has immunogenicity in mice, immunogenicity will be lost to some extent when lower pH value.

Embodiment 11

The immunogenicity (research of ELISA serum) of the gp160 of Alumen absorption

It is a very important biological characteristics that candidate vaccine brings out immunoreactive ability.For confirm gp160 vaccine with adsorbed onto alum adjuvant preparation in animal immunogenicity and confirm that adsorbed onto alum adjuvant can strengthen this immunogenicity, carries out following experiment.

In 0 day, use single dose (0.5 μ g, 1.0 μ g or 5.0 μ g) gp160 respectively, be adsorbed on the adsorbed onto alum adjuvant or complete Freund's adjuvant (CFA) on gp160 immune mouse (10 groups).Give the mice blood-letting on the 28th day, and detect the existence of anti-gp160 antibody in the serum (dilution in 1: 10) with the ELISA method.

Extracted the results are summarized in the following table (table 5) that serum obtained from the 28th day.In all each groups, all there is the mice more than 50% to show seroconversion.Under all dosage, all be higher than the numerical value that in only with the gp160 mice immunized, is obtained obtaining seroconversion number and average serum absorption value (OD450nm value) with the gp160 mice immunized that is adsorbed in adsorbed onto alum adjuvant to dilute at 1: 10 in ELISA.

This result proves that adsorbed onto alum adjuvant has strengthened the gp160 immunogenicity of antigens significantly.

⁴With 0.5 μ g, 1 μ g or 5 μ g Vaxsyn ^TmThe number of mice (P) of generation seroconversion in the 28th day and the ratio of test mice sum (N) after the HIV-1 immunity.

⁵Use Sponsor ' s ELISA detection method to detect 1: 10 dilution factor serum with definite mean absorbance (OD450) that the mice serum of seroconversion takes place at gp160.

Embodiment 12

In and data

The HIV-1 neutralization test is to be used to measure the method whether antibody preparation will suppress the susceptible human lymphocyte of HIV-1 viral infection cultivation.In the HIV-1 neutralization test, the antiserum with the animal of gp160 immunity is detected, it the results are summarized in the following table 6.

⁶Throw the gp120 that gives or the micrograms of gp160 in the first/the second/for the third time when immunity.

⁷HIV-1 infection cell with respect to contact nonimmune animal serum produces the 50% the highest antiserum dilution factor that suppresses.

Embodiment 13

Immunogenicity among chimpanzee

The most approaching from hereditary angle, chimpanzee and people, and it is unique animal model that HIV-1 infects at present.In the safety of among three chimpanzees, the carrying out/immunogenicity test, with 40 μ g in the vaccine of Alumen assistant preparation or two chimpanzees of 80 μ g gp160 immunity, with 40 μ g or 80 μ g gp160 two chimpanzees are carried out booster immunization respectively in the 4th week.Simultaneously with 1ml saline solution inoculation control animal.Gather blood serum sample weekly, detect three kinds of immunodetections of medicine box with the ELISA detection method at pure gp160, Western engram analysis method and the commodity in use ELISA of MccroGeneSys company exploitation anti-gp160 antibody and the anti-HIV-1 virus antigen-antibody of three chimpanzees are analyzed.Analysis result is as described below.

A.ELISA（MGSearch HIV 160）

ELISA detection method (MGSearch HIV 160, MGSearch is the (Meriden of MceroGenesys company, Conneeticut U.S.A) trade mark of product) is aimed at the immuno-sorbent assay of gp160, and in U.S. Patent Application Serial 920 at common unsettled common transfer, the present system of 197(number is 585,266) in be described.

With the blood serum sample gathered in the 11st week before the immunity and behind the initial immunity 1: 10-1: dilution in 100,000 the scope, the celluloid bar that contains the gp160 of 100 μ g purification then on every is incubated.The terminal point dilution titer is high dilution, and wherein this test antagonism gp160 antibody is positive when detecting with monoclonal antibody human IgG one alkali phosphatase junctional complex.

The blood serum sample of preimmune serum that derives from control animal and immune animal is negative.The chimpanzee to the of accepting 80 μ g dosage is positive at 1: 100 dilution factor during 2 weeks, and chimpanzee to the 4 Zhou Shicai that accept 40 μ g dosage present the positive at 1: 10 dilution factor place.Antibody titer one cover of anti-gp160 increased to for the 5th week, and so far the terminal point dilution titer is about 1: 100 respectively, and 000 and 1: 2,000,000.The antibody titer of two animals all descends a little in 6-11 week.

The immunoreation of this type no matter be on qualitative or quantitatively all among the chimpanzee of B-mode liver viroid vaccination of choosing viewed antibody response similar.

B. commercial ELISA test

From trade mark to the AIDS vaccine of VaxSyn(MicrogeneSys company) can know MGSearch HIV 160 ELISA that carry out of the chimpanzee of immunity and the Western engram analysis and find out that they have produced seroconversion and have had the antibody of day anti-subgroup gp160.Whether also producing the anti-HIV antibody of identification natural viral envelope protein in order to measure them, is Genetic System Corporation(Seattle at licensed commercially available ELISA test medicine box, Washington) the LAV EIA of company ^TmThe test medicine box in to preimmune serum and 1-11 the serum after week test.2 weeks of animal to the with 80 μ g gp160 immunity begin the positive to occur at 1: 100 dilution factor place, and its antibody horizontal shows as increase up to the 6th week always.Animal with 40 μ g gp160 immunity just presents the positive at 1: 100 dilution factor place up to the 6th week.

Embodiment 14

The distribution of antibody between gp120 and gp41

Also anti-gp41gp120 or the two are very important to measure the anti-gp160 antibody response that produces in the inoculation animal.Employing comprises that (the various immunological methods of WB and quantitative ELISA method are analyzed to detect and to measure the distribution of antibody between the various piece of HIV-1 envelope protein three kinds of different recombinant envelope antigens for the radioimmunoassay sedimentation method (RIP), fluorescent immune method (IF), Western engram analysis method.

Fig. 6 has summed up the immunoreactivity of following three kinds of different recombinant antigens: (ART) (TAB) (1) gp120-δ (its for the C-terminal deletion of molecule Recombinant HIV-1 gp120 of about 40 amino acid whose truncates); (ART (TAB) (2) gp120(total length Recombinant HIV-1gp120) and (ART) (TAB) (3) gp160.

With gp160 immunoreactivity is arranged from the individual human serum that obtains of 50 HIV-1 antibody positives and 3 parts of blended human serums, middle isoreactivity is arranged, do not have to react or only have very weak reactivity with the gp120 of truncate with gp120.This is likely because represented the truncate gp120 of the outside glycoprotein of 90% above HIV-1 to contain the protectiveness determinant.The antibody that anti-this part peplos is seldom only arranged in the acquired immune deficiency syndrome (AIDS) positive human serum, this observed result and immunoreation do not have protection fully and positive human serum to viral infection and demonstrate low-level neutralization activity usually this is consistent really external.

In contrast, with the Rhesus Macacus of the gp120 immunity of gp160 immunogen or truncate have on the antibody recognition site distributes in this peplos of antibody with the gp120 part kickback of HIV-1 peplos truncate difference may since monkey serum have higher in and titre.

Quantitatively value of commenting that personnel selection and immune Rhesus Macacus serum are done these three kinds of antigenic immunoreactivities of recombinant envelope is shown among Fig. 7.The antibody titer of the gp120 antigen (gp120-δ) of the anti-truncate that the equal tool of all tested monkey serums of serum of animal of gp160 immunity of comprising using by oneself is very high.

These results prove that reorganization gp160 has brought out and are different from the natural infection process immunoreation that usually takes place in Rhesus Macacus.Have the antigenic determinant that can effectively be discerned in immune monkey in the gp120-of gp-160 δ zone, it is not found by the human immune system in course of infection.The epitope that this class is new may be very important to the anti-HIV-1 protective effect, and may be the critical nature that is used to prevent and treat the reorganization gp160 of HIV infection.

Embodiment 15

The use of treatment vaccine

Carrying out clinical trial with 30 HIV seropositivity patients produces in aforesaid rhabdovirus system with definite HIVgp160(with the clone) the back influence of inoculation to the HIV infected individuals.

With causing having among 30 HIV seropositivity volunteers 19 people's (63%) gp160HIV one specificity humoral and cell immune response to strengthen after the reorganization gp160 inoculation.There are 14 people (93%) to demonstrate the increase of its total gp160 antibody among the volunteer of 15 acceptance 6 dosage vaccines.Therefore, can use Recombinant HIV albumen (being rgp41, rgp120, rgp160 and composition thereof) to treat the patient who is infected by HIV by advantageous manner.

Can determine the proteic effective dose of the HIV that is used for the embodiment of the present invention according to the technology of knowing in the field.In general, the scope of this effective dose is the about 1-100 μ of every kg of patient body weight g.Also can determine to take number of times by known method.In a preferred embodiment, be by the outer administration of gastrointestinal tract such as intravenous well known to those of ordinary skill in the art, intraperitoneal intramuscular, intradermal.

A。The screening of volunteers

Absorb 30 volunteers that infected by HIV by HIV, only suffering from early stage HIV infects one and is called Waltar Keed first phase or second phase (the cd4 cell counting is no less than 400 in during more than 3 months, have or do not have a lymphadenopathy) one the just qualified selected (Kedfield of seropositivity volunteers, etal, New Engl J.Med.314; 131-132(1986)).Other selected condition comprises that volunteers are defined as the 18-50 adult in year, it has normal complete blood count, endless last organ disease symptom, pro-be not abuse of alcohol or drugs in 12 months time, and do not accept degeneration-resistant viral medicine or immunoregulation medicament.Before being divided into some treatment groups at random, all patients have all been carried out 2 months baseline estimate.All volunteers all do not accept any degeneration-resistant virus drugs or immunoregulation medicament in process of the test.

Have 26 to be the male in 30 volunteers, 4 is the women.14 white people, 13 Black people, 3 Spaniards.Its mean age is 29(18-49).8 volunteers are Walter Keed first phase when selected, and 22 is the Walter Keed second phase.The average CD4 counting of baseline is 668 (scope is 388-1639).Making a definite diagnosis the average time that enters between the research at first is 24 months (from 3 months to 49 months).

B. vaccine product and immunization protocol

As described herein, experimental vaccine comprises the non-infectious subunit glycoprotein of the gp160 of the recombinant protein that derives from baculovirus expression.This immunogen protein produces in lepidopteran insect cell, through the biochemistry purification and be adsorbed on the aluminum phosphate with as final bacterin preparation.

Used the gp160 preparation of following three kinds of dosage; 40 μ g/ml, 160 μ g/ml and 320 μ g/ml.The volume injected of 40 μ g and 160 μ g dosage is 1ml; Use 2ml injection (320 μ g/ml) to carry the vaccine of 640 μ g dosage.

30 volunteers are divided into 6 groups, every group 5 people.Two kinds of immunization protocols are studied: option A, inoculation in the 0th, 30 and 120 day; Option b was inoculation in the 030th, 60,120,150 and 180 day.In every kind of immunization protocol (A or B), all there are three groups of volunteers to accept the vaccine (table 7) of various dose.All inoculums all are expelled in the triangular muscle by the intramuscular approach.Experimental period, is 10 months 2 months baseline estimate and begins to inoculate back 8 months assessment of following up a case by regular visits to.

Table 7-immunization protocol

The dosage of gp160 (μ g)

They are 0 30 60 120 150 180 years old

Option A

The 1st group 40 40 40

The 3rd group 160 160 160

The 5th group 640 640 640

Option b

The 2nd group 40 40 40 160 160 160

The 4th group 160 160 160 640 640 640

The 6th group 640 640 640 640 640 640

C. safety and toxic evaluation

Inquiring and inspection are carried out to each volunteers in after per injection the 0th, 1,2,3,15 and 30 day, understand that it has or not heating, cold, feels sick, symptoms such as vomiting, arthralgia, myalgia, discomfort, urticaria, asthma, dizziness or headache be inspection that local response that evaluation takes place in the injection site is carried out comprise observe erythema, swelling, itch, pain and tenderness, dyschromasia, skin injury, the variation of regional nodes's disease, the changes of function of injection limbs and the formation of injection site subcutaneous nodule.Result to every month full blood count, serum chemistry, coagulation curve and urinalysis also evaluates.

According to people such as Richman [Clinical Lmmuno S2:85-95,1989] people [J.Acguir.lmmue Defic, Syndr, 4:188-196 such as Birx, 1991] described method, also estimated [total lymphocyte, CD4 and cd8 cell phenotype] by the detection of T-cell phenotype and do not evaluated the cell in vitro immunologic function.Also estimated the T-cell proliferative reaction [Birx etc., the same] of mitogen [phytolacca american and ConA] and contrast antigen [Candidealbicans and tetanus] has been assessed the cells in vivo immunologic function by the tardy property hypersensitivity tuerculoderma of contrast antigen [being mumps, tetanus toxoid, Candida albicans and trichophyton].

According to the described method of people such as Burke [J.Acguir.lmmnuke DeficSyndr3:1159-1167,1991] the quantitative viral cultures of peripheral blood lymphocytes [PBMC] and blood plasma is assessed.Assessment archaeal dna polymerase chain reaction [Wages, etal ,] J.Med.Virol.33:58-63,1991) and serum p24 antigen levels with the viral capacity of HIV in the monitoring body.

Do not observe general toxicity, but in 87% object (in every group 13), find to have local response originality.Local response comprises the injection site sclerosis, touches a tender spot and forms of short duration subcutaneous nodule.Seldom find the increase of local adenopathy.Nobody's refusal is accepted booster injection.Viewed local response number of times does not have difference when initial immunity, booster injection or dosed administration.By external mitogen and antigenic specificity propagation reply or body in the super quick test of tardy property skin or confirm that by the acceleration that quantitative cd4 cell is removed it is free from side effects to immune system.For vaccine reaction person and non-responder, the average CD of baseline ₄Cell counting is respectively 716 and 605.For vaccine reaction person and non-responder, 180-240 days the average cd4 cell counting that begins in research is respectively 714 and 561.In the process of the test of lasting 240 days, what average cd4 cell was counted among the vaccine reaction person changes into-0.2% only, and average cd4 cell is counted the 7.3%(Figure 11 that descended in the non-responder of vaccine).In any individuality in entire test, vaccine-induced HIV immunogenicity does not have related with the acceleration of CD4 decline.

In order to assess probability, measure body inner virus activity by quantitative blood plasma and PBMC viral cultures, PBMC archaeal dna polymerase chain reaction and the antigenic serum levels of P24 as inoculation result's HIV breeding and the load increase of virus in individuality.Quantitative culture thing and archaeal dna polymerase chain reaction detection method proof do not change in this process of the test.In individuality, do not detect blood-serum P 24 antigens.

D. immunogenicity assessment

Utilize the totivirus lysate of viral gene product gp160, p66, p24 and the prototype HIV Strain MN of reorganization generation to measure the anti-proteic antibody of whole HIV.Use people (Proc.Natl.Acad.Sci.USA 76:4350-4354(1979) such as Toubin) described dot blotting and Western engram technology.Detect antibody response (see figure 7) simultaneously to specified packet membrane antigen determinant.

Selected among Fig. 7 is 88-98 amino acids among the antigenic determinant 88(gp120) and 448c(gp120 in the 448-514 amino acids), because it is reported that these regional antibody of anti-gp120 infect relevant with early stage HIV.

Why select the 106-121 amino acids among the antigenic determinant 106(gp120), 241(aminoacid 241-272), 254(aminoacid 254-272), 300(aminoacid 300-340), 308(aminoacid 308-322), 422(aminoacid 422-454) and 735(aminoacid 735-752) be because they have the functional importance of supposition. Antigenic determinant 106 and 422 influences the CD4 combination; Antigenic determinant 241,254 and 735 has influence on the neutralization of cohort specificity; Antigenic determinant 300 and 308 has influence on the type specific neutralization.

Why select antigenic determinant 582(aminoacid 582-602) be because it has represented the natural HIV immunodominance peplos zone in infecting in contrast.Other antigenic determinant that is studied comprises 49(aminoacid 49-128) and 342(aminoacid 342-405).

Among Fig. 7, dash box has been represented the immunoreactive variation of HIV peplos specific aim that the document record is arranged.The dash box that has (=) is represented elementary humoral response; The dash box that has (+) is represented secondary humoral response; (-) expression to before the specific antigenic determinant immunity and immunity after negative antibody; (+) expression does not change but have quantitatively before the specific antigenic determinant immunity and immunity back antibody positive.The dash box that has () is represented the new T-cell proliferative response to the gp160 follow-up immunization.Independent () expression is to the acellular reaction of gp160; Hb represents " high background " (can not explain); Nd represents " not doing ".

Use Nara(Nature, 333:469-470(1988)) described syncytium suppresses detection method and detected neutralization activity to three prototype separators (HIV-III BRF and MN).According to known lymphopoiesis detection technique, use gp160, p24 and baculovirus expression system reference protein (Birx, the same) detect the HIV specific cell immunoreaction.

E. vaccine reaction person and non-responder

For tested personnel as long as occur among the repeatably selectivity increase (Fig. 7) the relevant person that just it classified as the vaccine reaction with inoculation series at the cell of HIV peplos specific antigen determinant and humoral immune reaction.Vaccine-induced humoral immunity is defined as at the seroconversion of HIV peplos specific antigen determinant and/or at the reaction of the secondary booster immunization of peplos specific antigen determinant.Vaccine-induced cellular immunity be defined as at gp160 form new, repeatably, the cell proliferative response of vaccine dependency (this vaccine reaction person's definition is the science purpose of this test relatively, is strict as the suitability of assessment premunition).Gp160 antigenic determinant or HIV peplos are neither produced the individuality that humoral response do not produce the individual of cell proliferative response yet or only produce humoral response or cell proliferative response be decided to be non-responder.

F. vaccine-induced humoral response

Referring to Fig. 7, there are 19 (63%) to demonstrate the vaccine-induced gp160 HIV specificity humoral and the enhancing of cell immune response among 30 examinees.These 19 patients are decided to be " vaccine reaction person ".There are 4 people body fluid or cell immune response only to occur in 11 " non-responders ".All 7 do not show that the patient of any vaccine-induced reaction that detects has only accepted the vaccine (option A) of 3 dosage.Do not record the variation of the binding ability of antibody and HIV polymerase (p66), structural (p24) gene outcome or non-HIV contrast antigen (tetanus).In any individuality, all do not form the antibody that anti-baculovirus squama sticks up order insect cell reference protein.

Utilize totivirus pyrolysis product HIV-MN, record by the Western blotting and occur peplos antibody (gp160) in 13 individualities and increase.The anti-envelope protein antibody of 10 (67%) among 3 (20%) among 15 patients of option A and 15 patients of option b increases (recording p=0.025 by the FisherShi accuracy test, the two ends hangover).All 13 individualities have also produced the seroconversion at specified packet membrane antigen determinant.

In contrast, in 10 individualities not, do not find that by the Western engram analysis peplos antibody of any individuality increases to any peplos specific antigen determinant generation seroconversion.Do not find in remaining 7 individualities, to occur the variation of whole peplos antibody by the Western trace.In any individuality, do not observe the variation of anti-non-peplos HIV protein antibodies.

There are 14 (93%) to show the increase of total gp160 antibody in 15 individualities of option b (6 dosage), and in 15 individualities of option A (3 dosage), have only 7 to demonstrate antibody increase (P=0.01) (Fig. 7).

As shown in Figure 8, each gp160 specific antigen determinant is respectively from the preceding extremely postvaccinal advantage of immunity: antigenic determinant 49(27-70%), antigenic determinant 88(28-52%), antigenic determinant 106(50-87%), antigenic determinant 214(0-14%), antigenic determinant 254(0-13%), antigenic determinant 300(47-77%), antigenic determinant 308(42-69%), antigenic determinant 342(0-27%), antigenic determinant 422(3-10%), antigenic determinant 448C(73-87%) and antigenic determinant 735(17-33%).Observe the vaccine-induced seroconversion (Fig. 7) of anti-all specific antigen determinants except that 582.Anti-epitope 241,254 or 342 antibody (seroconversion) only detect when follow-up immunization.

Detection is to the secondary immune response of following antigenic determinant: 88,106, and 300448C and 582.Antibody advantage 100% to antigenic determinant 582 occurs before immunity, and only proves in a patient (3%) that secondary immune response is arranged.

The response curve of vaccine-induced anti-envelope antigen determinant HIV antibody is variable (Fig. 7).In 20 individualities, produced primary antibody reaction at least one antigenic determinant; Being respectively in 15 people's the group has 14 people to accept option b, and 6 people accept scheme A(P=0.005, Fisher's, two tails).The patient who adopts option A only potentially produces seroconversion to its 15 (14%) that does not have in the antigenic determinant of pre-immune antibody to 110.Adopt the patient of option b that 60 (47%) in 129 antigenic determinants is produced seroconversion.(P＜0.0001, Fisher's, two tails).Adopt 9 patients (60%) of option b to three or more envelope antigen determinant generation seroconversion at randomization, but option A group only 2 patients (13%) produces seroconversion (P=0.02 Fisher's, two tails) to three or more envelope antigen determinant.

In detecting in the 0th, 90 and 195 day 7 individualities for the serum of three different virus strains (HIV-III B, MN and RH) in and active.There are 4 to show among 5 vaccine reaction persons among one or more separators and active enhancing.Compare with non-responder, the ability that the vaccine reaction person suppresses syncytium formation also increases.

G。Vaccine-induced cell effect

The average lymphocyte stimulation indices (LSI) of (baseline) and inoculation back was definite before the variation of cell immune response was based on and utilizes Wilcoxon ranks summation tests (rank sum test) relatively to inoculate.

Immunity has in back 30 individualities 21 people (70%) to produce new T cell proliferative response (Fig. 7) at gp160.

Fig. 9 has illustrated the cell proliferative response at gp160, p24 and baculovirus reference protein that occurs in different time among four typical vaccine reaction persons.For all individualities, the inductive propagation of gp160 is 3 to increase to 10(and utilize the mean of back 4 numerical value of last immunity to calculate from the average LSI of baseline).On the contrary, being directed to the proteic breeder reaction of HIV p24 albumen or control baculovirus does not then change.

Figure 10 illustrated all individualities, according to the variation of the vaccine-induced average LSI value that is occurred in the individual of vaccine reaction grouping and the individuality according to the immunization protocol grouping.

Vaccine reaction person and non-responder are aspect the variation of the breeder reaction of gp160 obviously different (＜0.0001, Wilcoxon' one side is trailed).Be higher than by the inductive gp160 breeder reaction of option A (3 dosage) (P＜0.10, the hangover of Wilcoxon' one side by the inductive gp160 breeder reaction of option b (6 dosage).

Gp160 is produced among 21 patients of breeder reaction, have 19 people also to develop humoral response (vaccine reaction person).For all vaccine reaction persons, viewed maximum average lymphocyte stimulation indices (LSI) to gp160 is 50.1 still, each vaccine reaction person's reaction all be variable (the peak value scope is 3 to 171 from LSI) (Fig. 7), as to the cell effect of gp160 and inoculum concentration and the transitory phase mutual relation between the reaction period (Fig. 9).

H. discussion of results

Although the sample number that should test is limited, but confirmed that Several Factors is relevant with vaccine immunogenicity, for option A, there are 6 (40%) to be the vaccine reaction person among 15 patients, and in 15 patients of option b, have 13 (87%) to be the vaccine reaction person) (P=0.02, Fisher's, two hangovers) (Fig. 7).There are 13 (81%) to be the vaccine reaction person at average baselining CD4 counting in greater than 16 patients of every milliliter 600, and only have 6 (43%) to be the vaccine reaction person on average entering 14 patients of CD4 counting less than every milliliter of 600 cells.As summing up in the table 8, repeatedly immunity can improve immunogenicity, even the patient who counts less than every milliliter of 600 cells for its baseline CD4 also is like this.For example, and only there is 1 for the vaccine reaction person compares among 8 patients that accept 3 injections (option A), has 5 to be vaccine reaction person (P=0.03, Fisher's, two hangovers) (table 8) among 6 patients of option b (6 injections)

Table 8

According to baseline CD4 number and the definite GP160 vaccine immunity reactivity of immunization protocol

CD4 counts N responder number (%) non-responder number (%)

Option A

>600 7 5(71%) 2(29%)

500-600 5 1(20%) 4(80%)

<500 3 0(%) 3(100%)

Subtotal 15 6 (40%) 9 (60%)

Option b

>600 9 8(89%) 1(11%)

500-600 2 2(100%) 0(0%)

<500 4 3(75%) 1(25%)

Subtotal 15 13 (87%) 2 (13%)

Amount to 30 19 (63%) 11 (37%)

Just vaccine being applied to treat acute rabies at the 19th-century Pasteur infects.But deeply do not inquire into the practicality of this treatment means in tackling other infection.Although some examples that improve to infect back virus-specific immunity (behind contact first type or hepatitis B virus) are arranged, do not have ample evidence prove this method in human body to forming or the feasibility of chronic viral infection.

Therefore, the invention provides the method for improving the virus-specific immunity by metainfective active immunity.Specifically, for 19 among 30 patients of early stage HIV infection, the gp160 vaccine that is derived from the HIV env gene has strengthened human body host specific aim virus-specific body fluid and cell immune response.

This research is qualitative and measured the different antibodies reaction that is directed to specific HIV antigenic determinant in the natural infection immunity relative with premunition quantitatively.In this respect, in 70% individuality, accurately measure vaccine and in the individuality that has infected, induced humoral immunization originality.For example, 20 individualities (19 vaccine reaction persons and 1 vaccine nonresponder) are to specific envelope antigen determinant generation seroconversion.Only (antigenic determinant 241,254 is relevant with 342 with the vaccination that 10 patients are carried out in seroconversion.

Therefore, to the change of being identified by antigenic determinant mapping at the humoral response of this vaccine, to make expection cause and effect analysis become possibility, and the chance of identifying the potential immune regulation mechanism uniqueness of not brought out in the natural infection process is provided the specific antibodies reaction.

Although also do not know in the serum at present and active interior dependency, observe the neutralization activity that in 5 vaccine reaction persons, has 4 to improve anti-different HIV Strain (MB, RF, MN), the prompting premunition has been induced the variation of functional antibodies.Experimental vaccine has been induced the raising to specific HIV strain serum neutralising capacity.And may will help to limit in the cohort specificity and antigenic determinant.

Rare in natural HIV infects to the breeder reaction of HIV envelope protein.But after with the gp160 immunity, in 21 patients (70%), confirmed specificity T-cell proliferative response.The reason of this difference is not clear.A kind of may be to have caused a kind of new breeder reaction at the distinctive envelope antigen determinant of vaccine (as the result of immunogenic production process or the processing of body endoantigen).Perhaps, the protein that is used to breed detection may not stimulate the elementary T-cell proliferative response of homology " wild type " peplos at natural viral.But, obtained vaccination and can improve immunoreactive other evidence of host cell: after having confirmed in selected vaccine reaction person that HIV-III Type B specificity cellular toxicity T-cell effect results from booster immunization.

The reactive factor of vaccine immunity of being responsible for hiv infected patient is still waiting clarification.Even under the situation that HIV infects, compare with corresponding contrast in early days, individuality all can produce comparatively ideal immunoreation to various vaccines.This hyperergy is relevant with T-cellular machine dysfunction with early stage B cell insufficiency of accommodation.Therefore, vaccine immune response and baseline cd4 cell enumeration correlation, and this is being that this hypothesis of important determiner of vaccine reaction is consistent with host's immune state just.But the immunization protocol in specificity T-cell counting interval also has influence on vaccine reaction: option b (6 injections) is more superior.In fact, by increasing inoculation times the reduction of observed vaccine reaction in the patient of low cd4 cell counting is improved, this prompting can further improve host immune response by changing dosage, administering mode, adjuvant or prescription.

Although the patient who HIV is infected with HIV specificity vaccine product carries out the safety of active immunity and caused concern, do not produce the toxic evidence of immunologic opsonin at present.Quantitative culture thing, archaeal dna polymerase chain reaction detect and serum antigen detects the increase that demonstrates HIV load in the body.Among the patient, among the person's that especially is categorized as the vaccine reaction the patient, the fabulous body internal labeling-cd4 cell rate of descent that HIV duplicates-be subjected to advantageously influencing.The average CD4 change in count of responder is-0.2%, and the nonresponder then is-7.3%.This data declaration premunition reactivity is irrelevant with the destructive increase of CD4, and prompting is interior restricted relevant with the HIV body that descends.

In the research, also immunity inoculation result and 10 are infected and do not treat basic parameters such as year the making of patient, race and baseline cd4 cell counting comparing.Average CD4 counting has descended 8.7% in this reference group, accepts this counting of option A patient and has descended 7.2%, and the patient who accepts option b has then increased by 0.6%.It is feasible that these presentation of results carry out premunition inoculation with the Recombinant HIV envelope protein, and also is challenging with regard to this result of prophylactic applications of this vaccine.

Claims (36)

1, the method that the individuality that infected by human immunodeficiency virus (HIV) is treated, it comprises to the infected individual Recombinant HIV envelope protein that uses.

2, method according to claim 1, wherein recombiant protein is the dosage use with about 1-100 μ g/ kg body weight.

3, method according to claim 1, wherein recombiant protein is the dosage use with about 10 μ g-4000 μ g.

4, method according to claim 1, wherein recombiant protein is the dosage use with about 40 μ g-1280 μ g.

5, method according to claim 3 is wherein used 3 dosage at least.

6, method according to claim 4 is wherein used 6 dosage at least.

7, method according to claim 5, wherein each dosage uses with about 30-60 days interval.

8, method according to claim 6, wherein each dosage uses with about 30-60 days interval.

9, treatment is subjected to the method for the individuality of human immunodeficiency virus (HIV) infection, and it comprises:

To be enough to improving the amount of HIV specific cell or humoral immune reaction to the infected individual Recombinant HIV envelope protein that uses.

10, method according to claim 1, wherein recombiant protein is produced by shape virus-insect cell expressioning system.

11, method according to claim 3, wherein recombiant protein is produced by baculovirus insect cell expression system.

12, method according to claim 5, wherein recombiant protein is produced by baculovirus insect cell expression system.

13, method according to claim 1, wherein the molecular weight of recombiant protein is about 145,000.

14, method according to claim 3, wherein the molecular weight of recombiant protein is about 145,000.

15, method according to claim 5, wherein the molecular weight of recombiant protein is about 145,000.

16, method according to claim 1, wherein HIV peplos is at least a among gp160, gp120 and the gp41.

17, method according to claim 3, wherein HIV peplos is at least a among gp160, gp120 and the gp41.

18, method according to claim 5, wherein HIV peplos is at least a among gp160, gp120 and the gp41.

19, method according to claim 1, wherein recombiant protein is expressed by baculovirus insect cell carrier A c3046.

20, method according to claim 3, wherein recombiant protein is expressed by baculovirus insect cell carrier A c3046.

21, method according to claim 5, wherein recombiant protein is expressed by baculovirus insect cell carrier A c3046.

22, method according to claim 1, wherein recombiant protein is gathered into molecular weight and is at least about 2,000,000 granule.

23, method according to claim 3, wherein recombiant protein is gathered into molecular weight and is at least about 2,000,000 granule.

24, method according to claim 5, wherein recombiant protein is gathered into molecular weight and is at least about 2,000,000 granule.

25, method according to claim 1, wherein recombiant protein combines with adjuvant.

26, method according to claim 3, wherein recombiant protein combines with adjuvant.

27, method according to claim 5, wherein recombiant protein combines with adjuvant.

28, treatment is subjected to the method for the individuality of human immunodeficiency virus (HIV) infection, and it comprises that wherein recombiant protein has formed molecular weight and has been at least about 2,000,000 granule to the infected individual compositions of being made up of Recombinant HIV envelope protein and adsorbed onto alum adjuvant of using.

29, method according to claim 28, wherein recombiant protein is produced by baculovirus insect cell expression system.

30, method according to claim 28, wherein recombiant protein is selected from the Recombinant HIV envelope protein of reorganization gp160, reorganization gp120, reorganization gp41, molecular weight about 145,000 and by the recombiant protein of Ac3046 vector expression.

31, method according to claim 28, wherein recombiant protein comprises 757 continuous amino acids of gp160 and does not comprise 40 of gp160 terminal continuously aminoacid basically.

32, method according to claim 28, wherein recombiant protein uses with the dosage of about 10 μ g-4000 μ g.

33, therapeutic HIV vaccine combination, it comprises Recombinant HIV envelope protein and adsorbed onto alum adjuvant, wherein recombiant protein has formed molecular weight and has been at least about 2,000,000 granule.

34, compositions according to claim 33, wherein the Recombinant HIV envelope protein is that amount with every dose of about 10 μ g-4000 μ g provides.

35, method according to claim 34, wherein recombiant protein is produced by baculovirus insect cell expression system.

36, method according to claim 34, wherein recombiant protein comprises the about 757 successive aminoacid of gp160 and does not comprise the aminoacid of 40 ends of gp160 basically.

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