HRP921484A2 - Vaccine and treatment method of human immunodeficiency virus infection - Google Patents

Description

The present application is a continuation-in-part of U.S. patent application Ser. No. 151,976 filed Feb. 3, 1988, which is in turn a continuation-in-part of U.S. patent application Ser. No. 920,197 filed Oct. 16, 1986 (now Ser. No. 585,266). The mentioned applications and the references mentioned in them have been entered in their entirety in the current application.

Background of the invention

Human immunodeficiency virus type-1 (HIV-1) is a retrovirus that causes a systemic infection with the main pathology in the immune system, and is the etiological agent responsible for acquired immunodeficiency syndrome (AIDS). Barre-Sinoussi et al., Science 220: 868-871 (1983); Popović et al., Science 224: 497-500 (1984). Clinical isolates of HIV-1 are also referred to as lymphadenopathy virus [Feorino et al., Science 225: 69-72 (1984)] and AIDS virus [Levy et al., Science 225: 840-842 (1984)].

AIDS has become a pandemic, and vaccine development has become a priority in global health care. A high percentage of people infected with HIV-1 show a progressive loss of immune function caused by a decrease in the number of T4 lymphocytes. These T4 cells, as well as certain nerve cells, have a molecule called CD4 on their surface. HIV-1 recognizes the CD4 molecule using a receptor located on the envelope of the viral particle, enters these cells and finally replicates and kills the cells. An effective AIDS vaccine should be expected to induce the production of antibodies that would bind to the HIV-1 envelope and thus prevent the virus from infecting T4 lymphocytes and other susceptible cells.

As a rule, vaccines are given to healthy people, before they are exposed to the organism that causes the disease, as an immune prophylaxis. However, it is also reasonable to consider the use of an effective AIDS vaccine in post-exposure immunization as an immunotherapy of the disease. Salk, J., Nature, 327: 473-476 (1987).

The HIV-1 envelope ("env") is widely believed to represent the best candidate for AIDS vaccine development. Francis and Petricciani, New. Eng. J. Med., 1586-1559 (1985); Vogt and Hirsch, Reviews of Infectious Disease, 8: 991-1000 (1986); Fauci, Proc. Natl. Acad. Sci. USA, 83: 9378-9383. The HIV-1 envelope protein was first synthesized as a glycoprotein with a molecular weight of 160,000 (gp160). This precursor is then cleaved into an outer glycoprotein of molecular weight 120,000 (gp120) and a transmembrane glycoprotein of molecular weight 41,000 (gp41). These envelope proteins are the main target antigens for antibodies in AIDS patients. Barin et al., Science, 228: 1094-1096 (1985). Natural gp120 has been shown to be immunogenic and capable of inducing neutralizing antibodies in rodents, goats, rhesus monkeys and chimpanzees. Robey and colleagues, Proc. Natl. Acad. Sci. USA, 83: 7023-7027 (1986).

Considering the very low levels of natural HIV-1 envelope protein in infected cells, as well as the high risk that occurs in obtaining AIDS vaccines from cells infected with HIV-1, to obtain envelope antigens that would be used as AIDS vaccines, recombinant DNA methods. Recombinant DNA technology is so far the best option for the production of "subunit" vaccines against AIDS, considering the possibilities of obtaining large quantities of safe and economical immunogens. The HIV-1 envelope protein was expressed in genetically engineered vaccine virus recombinants. Chakrabarti et al., Nature, 320: 535-537 (1986); Hu et al., Nature, 320: 537-540 (1986); Kieny et al., Biotechnology, 4: 790-795 (1986). The coat protein is also expressed in bacterial cells [Putney et al., Science, 234: 1392-1395 (1986)], mammalian cells [Lasky et al., Science, 23: 209-12 (1986)], and in insect cells. Synthetic peptides derived from amino acid sequences in HIVa-1 gp41 have also been considered as AIDS vaccine candidates. Kennedy et al. (1986). However, no successful AIDS vaccine was obtained using these materials and methods.

The use of a baculovirus-insect cell vector system to produce recombinant HIV-1 envelope proteins represents one aspect of the invention described in US application Ser. No. 920,197 of the same applicant, filed Oct. 16, 1986 (now Ser. No. 585,266). See also Ser. No. 151,976.

It has been shown that baculovirus has a general application in the production of HIVa-1 and other proteins. For example, a baculovirus called Autographae californicae nuclear polyhedrosis virus (AcNPV) was used as a vector to express the entire gp160 and various parts of the HIV-1 envelope gene in infected Spodoptera f'rugiperda cells (Sf9 cells). Earlier applications by the same applicant also described the cleaved gene gp160 (recombinant number Ac3046), the protein obtained from recombinant Ac3046, as well as purification techniques for the Ac3046 gene product, which include lentil lectin affinity chromatography and gel filtration chromatography. The gp160 protein, purified in this way and aggregated into particles, was found to have high immunogenicity in rodents and several primate species.

An ideal AIDS vaccine, in addition to the requirement to be essentially biologically pure and apyrogenic, should provide lifelong protection against HIV-1 infection after one or several injections. This is usually the case with live attenuated vaccines. When killed bacteria or viruses, or substances isolated from them such as toxoids and proteins, are used to obtain vaccines, a weak antibody reaction and only short-term immunity often occur. In order to avoid or minimize these disadvantages, an additional component called an adjuvant may be added. Adjuvants are substances that help stimulate the immune response. Adjuvants commonly used in human vaccines are gels or aluminum salts (aluminum phosphate or aluminum hydroxide), called alum adjuvants. Bomford et al., "Adjuvants", Animal Cell Biotech. Vol. 2: 235-250, Academic Press Inc. (London: 1985).

The present invention provides a vaccine and methods of treating human immunodeficiency virus (HIV), comprising administering to an infected or susceptible individual a recombinant HIV envelope protein. In a preferred embodiment, the envelope protein can be purified, aggregated and combined with an adjuvant (for example with alum) to be used as a vaccine.

Brief description of the drawing

The details of this invention will be listed below with reference to the attached drawings:

Sl. 1 illustrates the cloning strategy used to isolate the HIV-1 envelope (env) gene from E. coli plasmid pNA2. Dark areas are DNA sequences from HIV-1 and open areas are from cloning vectors. The black region in plasmid p1774 was constructed from a synthetic oligonucleotide and inserted as a SmaI-KpnI fragment into the SmaI-KpnI sites in plasmid p1614. The sequence of this synthetic oligonucleotide is also shown.

Sl. 2 illustrates the strategy used to construct the recombinant plasmid vector (p3046), which in turn was used to construct the baculovirus expression vector, Ac3046. Plasmid pMGS3 contains sequences (hatched areas) from the AcNPV baculovirus on either side of the cloning site at position 4.00. This site has unique sites for the restriction endonucleases SmaI, KpnI and Bg1II. The polyhedron promoter of AcNPV is in the 5' direction from position 4.00. The sequence 5'-TAATTAATTAA-3' is in the 3' direction and has a translation stop codon in all three reading frames. Plasmid p1774 and the sequence of the synthetic oligonucleotide region are as described in FIG. 1. Plasmid p3046 contains the entire pMGS3, except for the sequences between the SmaI and Bg1II sites, where the HIV-1 envelope gene from p1774 was inserted.

Sl. 3 shows the DNA nucleotide sequences flanking the gp160 coding sequence from Ac3046. On Fig. 4 is the env sequence of DNA from 3046, from position +1 to +2267.

Sl. 4a-4t show the actual DNA sequences of the env segment of the HIV-1 gene, together with the sequences of the synthetic oligonucleotide at the 5' end of the env gene at Ac3046 (between +1 and +2267). The locations of the restriction endonuclease sites are listed above the DNA sequence, and the predicted amino acid sequences are listed below the DNA sequence. Bases are numbered on the right and on the left.

Sl. 5a-5h compare the DNA sequences in the env gene from Ac3046 with the published env gene sequences from LAV-1. The sequence from LAV-1 is above, and the sequence from Ac3046 is below. The line (1) below the sequence from LAV-1 indicates that the sequence from Ac3046 is the same at that position. The DNA sequence numbering described by Wain-Hobson et al., Cell, 40:9-17 (1985) for LAV-1 was used.

Sl. 6 shows ELISA titers for the highest dilution of HIV-1 antibody-positive human serum (upper graph) and rhesus monkey serum (lower graph) obtained from animals immunized with gp160 (IJ55, KL55) or with gp120 (AB55, CD55, GH55). ELISA titers were measured against highly purified proteins gp120 and gp160. Specific antibody binding was measured with goat anti-human IgG HRP conjugate. The titer represents the highest dilution of the serum that gives a positive reaction in this experiment.

Sl. 7 is a Table summarizing the immune response induced by vaccination with gp160 in seropositive patients.

Sl. 8 (A and B) shows vaccination-induced antibody production responses directed against specific HIV envelope epitopes.

S1. 9 shows the vaccination-induced proliferation of T-cells in response to gp160, in vaccinated seropositive individuals.

Sl. 10 (A-C) shows lymphocyte proliferation reactions related to vaccination.

SI. 11 is a graph showing percent change in CD4 cells over time for vaccine responders and non-responders.

Summary of the invention

Recombinant HIVa envelope protein gp160 ("rgp160"), particularly when adsorbed to an adjuvant such as alum (eg aluminum phosphate), has been found to be particularly useful as an AIDS vaccine. One aspect of the present invention is an AcNPV expression vector, with the coding sequence for a portion of the HIV-1 envelope gene from amino acids equivalent to 1 to 757 of the sequence in Wain-Hobson (ibid), which are amino acids 1-752 in recombinant clone Br. 3046. Another aspect of the invention is to obtain recombinant HIV-1 envelope protein (as well as the protein itself) in insect cells - in particular protein rpg160 which is encoded by the sequence of amino acids 1 to 757 Wain-Hobson ie 03046 having 752 amino acid residues.

Other aspects of the present invention include the purification and construction of recombinant envelope protein particles from a recombinant baculovirus gene product that produces protein 3046, and adsorbing the 3046 particles onto aluminum phosphate aggregates.

The invention also includes prophylactic and/or therapeutic vaccines against AIDS or HIV infection, as well as methods for preventing or treating AIDS or HIV infection.

Detailed description of the invention

The following examples illustrate the invention without limiting its scope.

A recombinant baculovirus, Autographae californicae nuclear polyhedrosis virus (AcNPV) with a spliced HIV-1 gp160 gene encoding amino acids 1-757 of the HIV envelope protein (recombinant Ac3046), is described in US application Ser. No. 920,197 of the same applicant (now Ser. No. 585,260). The cloning steps used to construct a recombinant baculovirus containing genes or portions of genes from HIV-1 are also described in that application and incorporated herein by reference.

The following is a detailed description of the genetic engineering steps used to construct the Ac3046 expression vector. Materials used, including enzymes and immunological reagents, were obtained from commercial sources. Examples are also provided in which the application of the invention is described.

The designation rgp160 is collective and includes other recombinant envelope proteins, including gp120 and gp41. Ac3046 is only one example of an expression vector and recombinant envelope protein according to the invention.

EXAMPLE 1

Construction of recombinant baculovirus Ac3046 with sequences from HIV-1 encoding amino acids 1-757

Cloning and expression of foreign protein coding sequences into a baculovirus vector requires that the coding sequence be aligned with the polyhedrin promoter and upstream sequences on the one hand, and with the baculovirus coding sequences on the other. The alignment is such that homologous recombination with the baculovirus genome results in the transfer of a foreign coding sequence aligned with the polyhedrin promoter and the inactive polyhedrin gene.

Accordingly, various insertion vectors have been developed for use in HIV envelope gene constructs. The MGS3 insertion vector, described below, was constructed to add an ATG codon for translation initiation. Insertion of foreign sequences into this vector must be resolved so that the translational frame, established by the initiation codon, is correctly maintained throughout the foreign sequence.

The MGS3 insertion vector was constructed from a clone of the EcoRI-I restriction fragment in DNA isolated from a plaque-purified AcMNPV (WT-1) isolate. MGS3 was engineered to contain the following structural features: (a) 4000 bp of sequence upstream of the ATG initiation codon in the polyhedrin gene; (b) a polyvalent binding element introduced by site-directed mutagenesis, consisting of an ATG initiation codon in the position of the corresponding polyhedrin codon, restriction sites SmaI, KpnI, Bg1II and a segment of the universal stop codon; and (c) of 1700 bp of sequence extending from the KpnI restriction site (which is within the polyhedrin gene) to the terminal EcoRI restriction site in clone EcoRI-I. See, for example, FIG. 2.

EXAMPLE 2

Construction of recombinant baculoviruses

with sequences coding for LAV env

The recombinant plasmid designated NA2 (Fig. 1) consists of a 21.8 kb segment of the entire HIV-1 provirus inserted into pUC19. This clone is reportedly infectious because it can produce virus after transfection of certain human cells. Adachi et al., J. Virol., 58:284-291 (1986). The complete sequences of the envelope genes contained in NA2 were derived from the LAV strain of HIV. Barre-Sinoussi (1983).

The HIV-1 envelope gene was isolated and genetically engineered as described below and shown in FIG. 1. The envelope gene was first isolated from NA2 as an EcoRI/SacI restriction fragment of 3846 bp, so it was cloned into pUC19 at the EcoRI/SacI restriction site.

The resulting plasmid was designated as p708.

The coat gene was further re-isolated as a 2800 bp KpnI restriction fragment and cloned into pUC18 at the KpnI restriction site. The resulting clone was designated as p1614.

The restriction fragment Kpnl in p1614 contained a slightly truncated part of the HIVa envelope gene, which lacked 121 bp of the corresponding N-terminal sequence. The missing part of the gene, which included the signal peptide sequence, was replaced by inserting a synthetic oligomer with two chains. The inserted oligomer is made from the amino acid sequence of LAV, with the preferred use of the codon of the polyhedrin gene. To facilitate further manipulation, a new SmaI restriction sequence was simultaneously introduced instead of the ATG initiation codon. The ATG initiation codon will be provided from the baculovirus insertion vector. The resulting plasmid was designated as p1774.

As seen in Fig. 2, restriction fragments from p1774, with coding sequences for various HIV-1 envelope regions, were cloned into an MGS insertion vector (for example, MGS3) such that the ATG initiation codon in the insertion vector was in frame with the envelope gene codons . Construct p3046 contained the Smal/BamH1 restriction fragment, isolated from p1774 and inserted into the SmaI/BglII site in the pMGS3 plasmid vector. This clone contains the sequences encoding amino acids 1 to 757 of gp160, and uses a stop codon provided from the MGS3 vector.

EXAMPLE 3

Production and selection of recombinant baculovirus

A plasmid for recombining the HIVa env gene, p3046, was calcium phosphate precipitated with DNA from AcMNPV (WT-1) and added to uninfected Spodopterae frugiperdae cells. The chimeric gene was then inserted into the AcMNPV genome by homologous recombination. Recombinant viruses were identified by occlusion-negative morphology on plaques. Such plaques show an identifiable cytopathic effect but no nuclear occlusion. In order to obtain pure recombinant virus, two additional purifications were performed on plaques. Recombinant viral DNA was analyzed to establish site-specific insertion of HIV env sequences by comparing its restriction and hybridization characteristics with wild-type viral DNA.

EXAMPLE 4

Expression of HIV env from recombinant baculoviruses

in infected insect cells

Expression of HIV env sequences from recombinant viruses in insect cells should result in the synthesis of the primary translation product. This primary product will contain amino acids translated from codons originating from the recombination vector. The resulting kuji protein contains the entire coded amino acids, from the ATG initiation codon from the expression vector downstream of the polyhedrin promoter, all the way to the end-of-translation signal on the expression vector (eg rgp160). The primary translation product of Ac3046 should read Met-Pro-Gly-Arg-Val at the terminus where Arg (position 4) represents the Arg in position 2 of the original LAV clone. Met-Pro-Gly codons were introduced as a result of the cloning strategy.

EXAMPLE 5

Nucleotide sequence of the gp160 insert and flanking DNA

The nucleotide sequence of the gp160 insert and flanking DNA was determined from restriction fragments isolated from the DNA of the viral expression vector Ac3046. The sequencing strategy used included the following steps. The 3.9 kb EcoRV-BamHI fragment was purified by restriction digestion of viral DNA from Ac3046. Viral DNA from Ac3046 was obtained from exogenous virus present in the cell medium used for vaccine production.

As shown in Fig. 2, the 3.9 kg EcoRV-BamHI fragment contains the entire gp160 gene, and 100 bp upstream and about 1000 bp downstream side-linked DNA. From this, the nucleotide sequence of the entire gp160 gene was determined, along with 100 bp of upstream and 100 bp of downstream flanking DNA.

In summary, the sequencing results blame the chimeric construct predicted by the cloning strategy. The sequence of gp160 was essentially the same as reported by WainHobson et al (1985). The sequence of 2256 bases between the putative initiation and translation termination codons predicts 752 amino acid codons and 28 potential non-linked glycosylation sites. The estimated molecular weight of this gp160, including sugar residues, is approximately 145,000.

Sequence analysis of 200 bases of flanking DNA indicated a correct insertion, as illustrated in FIG. 3, 4 and 5.

EXAMPLE 6

Amino acid sequence of gp160

Using standard automated Edman degradation and HPLC procedures, the N-terminal sequence of the first 15 residues in gp160 was determined to be identical to the sequence predicted based on the DNA sequence. The N-terminal methionine is not present in the gp160 protein. This is consistent with the finding that the polyhedrin protein in AcNPV is also produced without the N-terminal methionine. The following is a summary of the actual DNA sequence from gp160 and the N-terminal protein sequence, determined by analyzing DNA from AcNPV 3046 and purified gp160 (Table 1).

TABLE 1

Env gene of LAV in AcNPV 3046 expression vector

Residue

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

Pro Gly Arg Val Lys Glu Lys Tyr Gln His Leu Trp Arg Trp Gly

ATG CCC GGG CGT GTG AAG GAG AAG TAC CAA CAC TGG CGT TGG GGC

These results were compared to the original LAV-1 clone as follows (Table 2).

TABLE 2

Env gon LAV in the original LAV-1 clone

Residue

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

EXAMPLE 7

Purification of recombinant gp160

One aspect of the present invention is the process used to extract and purify the recombinant HIV-1 envelope protein encoded by the Ac3046 expression vector. Recombinant HIV-1 envelope protein, gp160, is produced in S. frugiperda cells 4-5 days after infection with Ac3046. Purification of this rgp160 involves the following

degrees:

1. Cell washing

2. Cell lysis

3. Gel-filtration chromatography

4. Lentil lectin affinity chromatography

5. Dialysis.

This Example describes the purification of recombinant gp160 from approximately 2 x 109 cells infected with Ac3046.

1. Cell washing Infected cells were washed in a buffer containing 50 mM Tris buffer (pH 7.5), 1 mM EDTA and 1% Triton X-100. Cells were resuspended in this buffer, homogenized using standard methods, and centrifuged for 20 minutes at 5000 rpm. The process was repeated three times.

2. Cell lysis Washed cells were lysed by exposure to ultrasound in SOmM Tris buffer (pH 8.0 - 8.5), 4% deoxycholate and 1% β-Swrcaptoethanol. Exposure to ultrasound is performed using standard methods. After that, only the remnants of the nuclear membrane are intact and they are removed by centrifugation for 30 minutes at 5000 rpm. The supernatant containing the extracted gp160 is free from whole cells, which was confirmed by observation through an optical microscope.

3. Gel filtration Gel filtration is performed in a Pharmacia 5.0 x 50 cm glass column filled with Sephacryl resin (Pharmacia). The total volume of the layer is about 1750 ml. In order to depyrogenate and sanitize the column and compounds, at least 6 liters of 0.1 N NaOH were passed through the column for 24 hours. The effluent from the column was connected to a flow-through ultraviolet cell, monitor and recorder (Pharmacia), and the column was then equilibrated with 4 liters of Gel filtration buffer. Crude gp160 was loaded onto the column and developed with Gel Filtration Buffer.

The column separates the crude mixture into three main fractions that absorb UV light. The first maximum comes from the column between about 500 and 700 ml, the second between 700 and 1400 ml, and the third between 1400 and 1900 ml of buffer. The same profile was observed on small analytical columns, from which it was concluded that the first maximum gives material with a molecular weight of ≥ 2,000,000.

This maximum is partially transparent, due to the concentration of lipids and lipid complexes of high molecular weight. This peak also contains from 10 to 20% of gp160 extracted from infected cells. This fraction of gp160 appears to be complexed with itself or with other cellular components to form high molecular weight aggregates.

The second, broad peak contains most of gp160 and proteins with molecular weights between about 18,000 and 200,000.

The third maximum contains little protein, and most of the absorption of UV light is the result of the presence of β-mercaptoethanol in the sample.

When the second maximum is first detected by monitoring the UV absorbance, the effluent from the column is fed directly into the lentil lectin column. When the second peak exits the column, the effluent is excluded from the lentil lectin column and discarded.

4. Lentil lectin Lentil lectin affinity gel medium (Lentil Lectin-Sepharose 4B) was purchased in bulk from Pharmacia. Lentil lectin was isolated by affinity chromatography on Sephadex to a purity of more than 98%, after which it was immobilized by coupling to Sepharose 4B using cyanogen bromide. The matrix contains about 2 mg of ligand per ml of gel. The lentil lectin column is a 5.0 x 30 cm glass column (Pharmacia) containing 125 ml of lentil lectin-Sepharose 4B gel. The affinity matrix can be reused after extensive washing and regeneration using the procedure recommended by the supplier. When not in use, the gel is stored in the column, in a solution of 0.9% NaCl, 1 mM MnCl2, 1 mM CaCl2, and 0.01% thimerosal. The column is washed and equilibrated with 250 ml of the lentil lectin buffer described above, before each use.

Crude gp160 is applied to the column directly, as eluted from the gel filtration column, as described above. When crude gp160 is bound to the column, it is washed with 800 ml of lentil lectin buffer containing 0.1% deoxycholate. Under these conditions, all gp160 binds to the column. Lentil lectin buffer plus 0.3M α-methyl monoside is used to elute bound glycoproteins, which is controlled through a UV monitor at a wavelength of 280 nm.

5. Dialysis Sugars and deoxycholates are removed by conventional dialysis.

Purification of gp160 from 1 liter of infected cells can be summarized in the following table (Table 3).

In the following embodiment, conventional ion exchange chromatography can be used instead of gel filtration. Similarly, the order of the steps is not critical: for example, gel filtration or ion exchange chromatography may follow the lentil lectin purification step. Other reagents can also be used according to the invention. For example, instead of deoxycholate, other detergents can be used to purify the recombinant protein. These include nonionic detergents such as Tween 20 (polysorbate 20), Tween 80, Lubrol and Triton X-100.

TABLE 3 - Summary of purification

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1 Total protein was estimated from the absorbance at 280 nm.

EXAMPLE 8

A. Assembly of gp160 particles

As one aspect of the present invention, it has been discovered that the gp160 antigen can be assembled into particles having a molecular weight of ≥ 2,000,000 during purification. The gp160 protein was extracted from cells as a mixture of 80-90% monomer (molecular weight 160,000) and 10-20% polymer (particulate form). Running gel filtration removes the aggregated form of gp160. Attempts to purify gp160 from this fraction (the first peak coming off the gel filtration column) indicate that it is complexed with other proteins from the cell, and possibly even with membrane fragments. However, the gp160 antigen in the second peak coming off the gel filtration column has a molecular weight of about 160,000 - 300,000, and is therefore predominantly monomeric or dimeric.

The construction of aggregates or polymers of gp160 takes place during the development of the lentil lectin column. It was determined that the antigen forms aggregates regardless of whether it is eluted from the lectin column in 0.5% deoxycholate, which is about 0.2% of the critical micelle concentration (CMC) for deoxycholate, or gp160 is eluted from the column in 0.1% deoxycholate.

Aggregate size is measured on a Superose 12 high-resolution FPLC column (Pharmacia). Samples from representative batches of purified gp160 are of a size substantially equal to or greater than 2,000,000 molecular weights of the blue dextran size standard.

A cross-linking study by Schwaller et al. (1989) demonstrates that gp160 produced in insect cells is a tetramer of identical units. This study also showed that gp160 is a tetramer in both HIV-infected cells and virus particles. Therefore, recombinant gp16U particles may have tertiary and quaternary structures similar to those found in native HIV gp160.

The correct three-dimensional structure could be important for shaping epitopes that require proper folding of gp160. It is likely, considering that non-glycosylated proteins are removed from the connection with the gp160 antigen during binding and washing on the lentil lectin column, that the hydrophobic parts of gp160 begin to form intermolecular associations. Deoxycholate is probably not bound to gp160, given that the concentration can be kept above the CMC and the antigen still forms complexes. Assembly of this antigen into aggregates is, apparently, an innate property of this protein when it is purified in accordance with the invention. It is possible that the highly hydrophobic N-terminal sequence present on the gp160 protein contributes to the natural ability of this protein to build particles. After purification, gp160 complexes can be sterile filtered through a 0.2 μm cellulose-acetate filter without significant protein loss.

B. Analysis of particle formation

Analysis of purified gp160 particles by electron microscopy shows that they are spherical particles with a protein appearance, size from 30 to 100 nm.

To further test the presence of particles, purified gp160 was analyzed by gel filtration. About 100 μg of gp160 was loaded onto a FPLC gel filtration column Superose 12, HR 10/30 (Pharmacia, Inc.). This column was first calibrated with protein standards of molecular weights. The protein profile from this column is highly reproducible; the elution volume is inversely proportional to the molecular weight of the protein standard. The column separates monomeric gp160 from polymeric forms and excludes globular proteins of molecular weight ≥ 2 x 106. When developed on this column, virtually all purified gp160 is eluted in the blank

by volume and, accordingly, has a molecular weight ≥ 2 x 106 (2,000,000).

EXAMPLE 9

A. Adsorption of gp160 on alum

The effectiveness of insoluble aluminum compounds as immune adjuvants depends on the complete adsorption of the antigen to the solid phase. As part of the present invention, it has been discovered that compositions can be obtained with alum that will effectively adsorb pg160 at pH values at which the potency of the gp160-alum complex as an immunogen will not be reduced. The factors that were controlled during the formulation of this alum composition (aluminum phosphate gel) were as follows:

1. The optimal pH for adsorbing antigens on alum is around 5.0. However, gp160 was found to lose immunogenicity at pH 6.5 compared to pH 7.5, so alum with a pH of 7.1 ± 0.1 was prepared. It was found that at this pH, practically 100% of the gp160 will still be adsorbed onto the alum.

2. The ionic strength originating from the NaCl present is relatively low and amounts to less than 0.15 M.

3. A molar excess of aluminum chloride was used in relation to sodium phosphate, in order to ensure that there are no free phosphate ions in the supernatant.

4. Antigen gp160 was added to freshly prepared alum to stop crystal growth and minimize particle size.

Below is the procedure for obtaining 200 ml of alum and adsorbing the purified gp160 on the alum so that the antigen concentration is 40 μg/ml.

B. Preparation of reagents

(total volume of formulation 200 ml)

The following solutions are prepared in sterile, pyrogen-free vials or 100 ml glasses. Salts for Solution 1 and Solution 2 and sodium hydroxide are prepared, then filtered through a 0.2 μm cellulose-acetate filter into sterile, pyrogenic 100 ml bottles.

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Treat the solutions for 30 minutes in an autoclave, with gentle draining. Cool to room temperature.

C. Molding of alum

1. Add Solution 1 (aluminum chloride-sodium acetate) to the formulation container, using sterile 25 ml disposable pipettes. Record the volume of Solution 1 and start mixing the solution.

2. Add Solution 2 (sodium phosphate) to the container, using sterile 25 ml single-use pipettes, and continue mixing while the precipitate forms and record the volume of Solution 2.

3. Add 3 ml of Solution 3 (sodium hydroxide) and continue mixing for another 5 minutes. Take a 0.5 ml sample and measure the pH. If the pH is less than 7.0, add another 0.5 ml of sodium hydroxide, mix for another 5 minutes and measure the pH again. Continue until the pH is between 7.0 and 7.2.

4. Determine the total volume added to the formulation container (Solution 1 + Solution 2 + Solution 3), then add sterile VZI to a volume of 100 ml.

5. Immediately add 8,000 μg of purified gp160 in 100 ml of 1 mM Tris, pH 7.5, directly to the formulation vessel.

6. Continue mixing for at least 20 minutes, and then pour the formulated vaccine into sterile vials.

EXAMPLE 10

Immunogenicity of gp160 absorbed on alum

(reaction of specific antibodies)

The accepted method for determining the immunogenicity of an antigen preparation (vaccine) is to measure the reaction of specific antibodies in groups of mice that were given one dose of the antigen. At the end of the 4th week, the mice were bled and serum levels of antibodies against the designated antigen (usually the antigen used to immunize the animals) were measured by a standard antibody test, for example ELISA (enzyme-linked immunosorbent assay).

Immunogenicity in mice, for purified gp160 without adjuvant at pH 6.0 and adsorbed on alum (as described in Example 9) at pH 7.5, or mixed with Freund's complete adjuvant, is shown collectively in Table 4.

TABLE 4

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Mice immunized with a single dose of 1.0 μg of gp160 antigen without any added adjuvant show an antibody response against gp160 (see table above). However, a significantly stronger antibody reaction was observed in the group of mice immunized with 1.0 μg of gp160 absorbed on alum adjuvant. A single dose of less than 0.1 μg of gp160 mixed with complete Freund's adjuvant, or formulated with alum, will seroconvert ≥ 50% of immunized mice. Although to a lesser extent, the gp160 antigen was immunogenic in mice both as unformulated antigen at pH 7.5 and at pH 6.0, but a loss of immunogenicity was observed at lower pH.

EXAMPLE 11

Immunogenicity of gp160 absorbed on alum (ELISA test of serum)

The ability of a vaccine candidate formulation to elicit an immune response is a very important biological property. The following tests were performed to determine whether the gp160 vaccine formulated with alum is immunogenic in animals, and whether the alum adjuvant increases this immunogenicity.

On day zero, mice (groups of 10) were injected with one dose (0.5 μg, 1.0 μg, or 5.0 μg) of gp160 alone, gp160 adsorbed on alum, or gp160 in complete Freund's adjuvant (CFA). On day 28, the mice were bled, and the sera were tested in an ELISA test (dilution 1:10) to determine the presence of antibodies against gp160.

The results for the sera collected on 28 days are shown collectively in Table 5. In all groups, more than 50% of mice showed seroconversion. At all doses, the number of seroconversions and the average serum absorbance (OD450nm at 1:10 dilution in the ELISA test) were higher for gp160 adsorbed on alum, compared to the values obtained in mice immunized with gp160 alone.

The results indicate that the alum adjuvant significantly increases the immunogenicity of the gp160 antigen.

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2Mice were bled 28 days after immunization and sera were tested at a 1:10 dilution in an ELISA assay against gel-purified gp160. Similar results were obtained using a commercial ELISA (Genetic Systems, Inc.; EIATM ELISA) against native HIV-1 proteins at a serum dilution of 1:400.

3 Number of seroconverted mice (P) according to the total number tested (N).

TABLE 5 - 28 days after injection

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4Number of mice that seroconverted (P) compared to the total number tested (N) at 28 days after immunization with 0.5 μg, 1 μg, or 5 μg of HIV-1 VaxSynTM.

5Mean absorbance (OD450) for seroconverted mice measured by sponsor's ELISA against gp160, at 1:10 serum dilution.

EXAMPLE 12

Neutralization data

HIV-1 neutralization assays are an accepted method for determining whether an antibody preparation will inhibit HIV-1 infection of a culture of susceptible human lymphocyte cells. Antisera from animals immunized with gp160 were analyzed in the HIV-1 neutralization test, and the results are collectively presented in Table 6.

TABLE 6

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6μg of gp160 or gp120 given at the time of the first/second/third immunization.

7 The highest dilution of antiserum that will inhibit infection by 50%, compared to cells infected with HIV-1 that were exposed to the serum of non-immunized animals.

Guinea pigs, rabbits and rhesus monkeys were also immunized with gp160 (using alum or Freund's adjuvant). In general, immunization of these animals produced a good antibody response against HIV-1 envelope proteins.

EXAMPLE 13

Immunogenicity in chimpanzees

Genetically speaking, the chimpanzee is man's closest relative and is currently the only animal model for HIV-1 infection. In a safety/immunogenicity study in three chimpanzees, two chimpanzees were immunized with 40 μg or 80 μg of gp160 in an alum-formulated vaccine. Each was reimmunized after 4 weeks with 40 μg and 80 μg of gp160. A control animal was inoculated with 1 ml of saline at the same time. Serum samples from each animal were analyzed weekly for the presence of antibodies against gp160 and HIV-1 viral antigens, using three immunoassays - an ELISA against purified gp160, developed at MicroGeneSys, Inc., a Western Blot assay, and a commercial ELISA for HIV-1. The results of these analyzes are described below.

A. ELISA (MGSerch HIV 160)

MGSearch HIV 160 ELISA test, where MGSearch is a registered trademark of MicroGeneSys, Inc. of Meriden, Connecticut, U.S.A., presents an immunosorbent assay against gp160 and is described in U.S. Patent Application Ser.No. 920,197 (now 585,266) of the same applicant.

Serum samples taken before immunization and weekly for 11 weeks after primary immunization were diluted from 1:10 to 1:100,000 and then incubated with nitrocellulose strips containing 100 μg of purified gp160 at one point. The endpoint titer of the dilution represents the highest dilution at which the test was positive for antibodies against gp160, determined using a conjugate of goat anti-human IgG and alkaline phosphatase.

Serum samples from control animals and pre-immune sera from immunized animals were negative. A chimpanzee receiving a dose of 80 μg was positive at a 1:10 dilution by week 4. Anti-gp160 antibody titers continued to rise until week 5, at which time the end-point dilution titer was approximately 1:100,000 and 1:2,000,000, respectively. The antibody titer decreased slightly in both animals during days 6-11. week.

This type of reaction is similar, both quantitatively and qualitatively, to the antibody reaction that is usually obtained in chimpanzees vaccinated with the human hepatitis B virus vaccine.

B. Commercial ELISA test

Both MGSearch HIV 160 ELISA and Western Blot tests of sera from chimpanzees immunized with the VaxSyn8 vaccine showed that the animals were seroconverted and possessed antibodies against recombinant gp160. To determine whether the animals produced anti-HIV antibodies that recognized the envelope proteins of the native virus, pre-immunization sera and sera collected from weeks 1 to 11 were tested with a recognized commercial ELISA kit, the LAV EIATM kit from Genetic System Corporation, Seattle, Washington, U.S.A. An animal immunized with 80 μg of gp160 was positive at a dilution of 1:100 by week 2 and continued to show increasing antibody levels until week 6. An animal immunized with 40 μg was positive at a 1:100 dilution by week 6.

EXAMPLE 14

Distribution of antibodies between gp120 and gp41

It is important to determine whether the antibody response against gp160 in vaccinated animals is directed against gp41, gp120, or both. Multiple immunological methods were used to detect and measure the distribution of antibodies against different regions of the HIV-1 envelope protein, including radioimmunoprecipitation (RIP), immunofluorescence (IF), Western Blot (WB) analysis, and quantitative ELISA against three different recombinant envelope antigens.

Sl. 6 collectively shows the immunoreactivity of three different recombinant antigens: [ART] [TAB] (1) gd120-delta (truncated recombinant gp120 from HIV-1 missing about 40 amino acids from the C-terminus of the molecule); [ART] [TAB] (2) gp120 (complete recombinant gp120 from HIV-1); and [ART] [TAB] (3) gp160.

Human sera obtained from 50 HIV-1 antibody-positive individuals and 3 pooled human sera were highly reactive with gp160 and moderately reactive with gp120, while little or no antibody reacted with truncated gp120. It is likely that the truncated gp120, which represents more than 90% of the outer glycoprotein of HIV-1, contains protective determinants. The observation that AIDS-positive human sera have few antibodies against this region of the envelope is consistent with the fact that the immune response to viral infection is not fully protective and that human positive sera usually show a low level of inhibitory activity in vitro.

In contrast, rhesus monkeys immunized with either the gp160 immunogen or the truncated gp120 have antibodies that strongly react with the truncated gp120 portion of the HIV-1 envelope. This difference in the distribution of sites along the virus envelope, which is recognized by the antibody, and the higher titers observed in monkeys, may explain the fact that monkey sera have high neutralizing titers.

Quantitative evaluation of the immunoreactivity of these three recombinant envelope antigens with human sera and with rhesus immune sera is presented in FIG. 7. All tested monkey sera have a high titer of antibodies against the truncated gp120 antigen (gp120-delta), including the sera of animals immunized with gp160.

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8VaxSyn is a trademark of MicroGeneSys, Inc., for the AIDS vaccine described herein.

These results show that recombinant gp160 in rhesus monkeys elicits an antibody response that is different from that often seen during natural infection. In the gp 120-delta region of the gp160 protein, there are epitopes that are effectively recognized in immunized monkeys, but which are not recognized by the human immune system during infection. These new epitopes may be important for protection against HIV-1, and may represent an important feature of recombinant gp160 in the prevention and treatment of HIV infection.

EXAMPLE 15

Therapeutic administration of vaccines

To determine the effects of vaccination with cloned HIV gp160 (obtained in the baculovirus system as described above) on HIV-infected individuals, a clinical trial was conducted with 30 human HIV-seropositive patients.

Vaccination with recombinant gp160 led to an increase in humoral and cellular immune responses, specific to HIVa gp160, in 19 to 30 (63%) HIV-seropositive volunteers. Fourteen of 15 (93%) volunteers who received 6 doses of the vaccine showed an increase in the total level of antibodies against gp160. Accordingly, recombinant HIVa proteins (ie, rgp41, rgp120, rgp160, and mixtures thereof) can be conveniently administered in a method for treating human patients infected with HIV.

Effective amounts of HIV protein used in this embodiment of the invention can be determined according to techniques well known in the art, such as the techniques illustrated below. In general, such effective amounts may range from about 1 μg to about 100 μg per kilogram of patient body weight. The frequency of administration can also be determined by known means. In a preferred embodiment, administration will be by the parenteral route, i.e., intravenously, intraperitoneally, intramuscularly, intradermally, etc., as will be well known to those skilled in the art.

A. Selection of volunteers

Thirty volunteers with HIV infection were recruited. Only seropositive volunteers with an early stage of infection, defined as Walter Reed stage 1 or 2 (CD4 cell count for at least 3 months not less than 400, with or without lymphadenopathy) were eligible. (Redfield et al., New Engl. J. Med. 314: 131-132 (1986). Additional criteria narrowed the volunteers to adults 18 to 50 years of age, with a normal complete blood count, who showed no signs of end-organ disease, who had not abused alcohol or drugs in the past 12 months and had not used antiretroviral or immunomodulating drugs. All patients underwent a two-month baseline assessment and were then randomized to treatment groups. No patient received any antiretroviral or immunomodulating drugs during treatment. meds.

Twenty-six of the 30 volunteers were men; 4 were women. Fourteen were Caucasian (white), 13 black and 3 Hispanic. The median age was 29 years (range, 18 to 49). At entry, 8 volunteers were classified as Walter Reed stage 1, and 22 volunteers were staged as Walter Reed stage 2. The median baseline CD4 cell count was 668 (range, 388 to 1639). The median time between initial diagnosis and access to trials was 24 months (range, 3 to 49 months).

B. Vaccines and vaccination program

As described, the test vaccines contained a non-infectious "subunit" glycoprotein derived from gp160, as a recombinant protein expressed by baculovirus. The immunogenic protein was obtained in Lepidoptera-insect cells, biochemically purified and adsorbed on aluminum phosphate to obtain the final vaccine formulation.

Formulations with three doses of gp160 were used: 40 μg/ml, 160 μg/ml and 320 μg/ml. The injection volume for the 40 μg and 160 μg doses was 1 ml; for injection with 640 μg of gp160, 2 ml of the 320 μg/ml formulation was used.

Thirty volunteers were divided into six groups of five people each. Two immunization programs were examined: Program A, with vaccination on days 0, 30 and 120; and Program B, with vaccination on day 0, 30, 60, 120, 150 and 180. For each Program (A or B) there were three groups that received different doses of vaccine (Table 7 below). All vaccinations were performed by intramuscular injections into the deltoid muscle. The duration of the experiment was 10 months: 2 months of baseline assessment and 8 months of evaluation after the first vaccination.

TABLE 7 - Immunization program

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C. Safety and Toxicity Assessment

Each volunteer was interviewed and examined at 0, 1, 2, 3, 15 and 30 days after each injection. Volunteers were examined for fever, chills, nausea, vomiting, arthralgia (joint pain, myalgia (muscle pain), malaise, urticaria (hives), intestinal cramping, fainting, or headache. Examination to assess local reaction at the injection site they included redness, swelling, itching, pain and sensitivity, skin discoloration, skin cracking, changes in regional lymphadenopathy, changes in the functioning of the injected limb, and the formation of a subcutaneous nodule at the injection site. Also, once a month, a complete blood count, serum chemistry, coagulation profile and urinalysis.

Cellular immune function was assessed by T-cell phonotyping (cellular phenotypes of total lymphocytes, CD4 and CD8 cells) as described by Rikman et al., Clinical Immuno. 52: 85-95, 1989; Birx et al., J. Acquir. Immune Deficiency. Syndr. 4: 188-196, 1991). The proliferative reaction of T-cells to mitogens (Phytolaccae berries or Con A) and to control antigens (Candida alchicans and tetanus) was also evaluated. Birx et al., see above. In vivo cellular immune function was assessed by delayed skin hypersensitivity testing to control antigens (ie, mumps, tetanus toxoid, Candia albicans, and trichophyton).

Quantitative viral cultures in peripheral blood mononuclear cells (PBMC) and plasma were determined as described by Burke et al., J. Acquir. Immune Deficiency. Syndr. 3: 1159-1167, 1991. DNA polymerase chain reaction (Wages et al., J. Med. Vir-ol. 33: 58-63, 1991) and serum p24 antigen levels were determined to control the amount of HIV virus in vivo. .

No evidence of systemic toxicity was observed, but local reactogenicity was observed in 87% of subjects (13 in each vaccination group). Local reactions included hardness, tenderness, and the formation of a transient subcutaneous nodule at the injection site; an increase in regional adenopathy was rarely observed. No person refused revaccination. There were no differences in local reactions during the first immunization and revaccination, nor depending on the dose.

Data on adverse effects on the immune system were not obtained either by measuring in vitro, via the proliferative reaction to mitogen or antigen-specific proliferative reactions, nor by measuring in vivo, via reactions in delayed skin hypersensitivity testing, nor by measuring the acceleration of the quantitative decrease in the number of CD4 cells. The baseline median CD4 cell count was 716 for responders and 605 for vaccine non-responders. Median CD4 cell counts on days 180-240 were 714 for responders and 561 for vaccine non-responders. During the 240-day trial, the net change in median CD4 cell count was -0.2% for responders and -7.3% for vaccine non-responders (Fig. 11). HIV-immunogenicity induced by the vaccine was not associated with indications of an accelerated decrease in the number of CD4 cells in any individual during the entire duration of the experiment.

In order to examine the possibility of increased HIV replication and an increased number of viruses in certain individuals, as a consequence of vaccination, the activity of the virus in vivo was measured on quantitative cultures of the virus in plasma and in PBMC, via DNA polymerase chain reaction and via the p24 antigen level in serum. Quantitative culture tests and DNA polymerase chain reaction tests did not show any changes during the experiment. Antigen p24 could not be detected in the tested persons.

D. Immunogenicity test

Antibodies directed against HIV proteins were measured using recombinantly obtained viral gene products go160, p66 and p24, as well as the entire viral lysate of the prototype MN strain of HIV. Dot Blot and Western Blot techniques were used, as described by Toubin et al., Proc. Natl. Acad. Sci. USA 76: 4350-4354 (1979). Antibody reactions to specific envelope epitopes were also measured (see Fig. 7).

Epitopes 88 (amino acids 88-98 in gp120) and 448C (amino acids 448-514 in gp120) in FIG. 7 were chosen because antibodies directed against these regions of gp120 have been reported to correlate with early stages of HIV infection.

Epitopes 106 (amino acids 106-121 in gp120), 241 (amino acids 241-272), 254, (amino acids 254-272), 300 (amino acids 300-340), 308 (amino acids 308-322), 422 (amino acids 422-454) and 735 (amino acids 735-752) were chosen for their putative functional importance. Epitopes 106 and 422 are implicated in CD4-binding; epitopes 241, 254 and 735 are implicated in group-specific neutralization; both epitopes 300 and 308 are implicated in type-specific neutralization.

Epitope 582 (amino acids 582-602) was chosen as a control, since it represents the immunodominant envelope domain in natural HIV infection. Additional epitopes investigated include 49 (amino acids 49-128); and 342 (amino acids 342-405).

On Fig. 7, hatched boxes indicate a documented change in the immune response directed at the HIV envelope. Hatched fields with (=) indicate primary humoral reaction; hatched fields with (+) indicate secondary humoral reaction; (-) indicates an antibody negative to a specific epitope before and after immunization; and (+) indicates antibody positive for specific epitope before and after immunization, but without quantitative change. Hatched fields with (•) indicate a new T-cell proliferation response to gp16O after immunization. Only (•) indicates no cellular response to gp160; hb stands for "high base value" (cannot be interpreted); and nd means "not done".

Neutralizing activity was measured against three prototype isolates (HIV-IIIB, RF and MN) in the syncytium inhibition assay described by Nara, Nuture, 333: 469-470 (1988). HIV-specific cellular responses were measured by a known lymphocyte proliferation assay technique, using gp160, p24 and the control protein of the baculovirus expression system (Birx, see above).

E. Vaccine responders and non-responders

Individuals were classified as vaccine responders only if both cellular and humoral immune responses were associated with the vaccination series (see S1. 7). Vaccine-induced humoral immunity was defined as seroconversion to specific HIV envelope epitopes and/or as an immune response to envelope-specific epitopes after secondary revaccination. Vaccine-induced cellular immunity is defined as the development of a new, reproducible, vaccine-associated proliferative response to gp160. Persons in whom neither a humoral nor cellular immune reaction developed, as well as persons in whom only a humoral or only a cellular proliferative reaction to epitopes in gp160 or to the HIV envelope developed, were classified as non-responders to the vaccine.

F. Vaccine-induced humoral reaction

As can be seen in Fig. 7, 19 of 30 individuals (63%) showed a vaccine-induced increase in both humoral and cellular immune responses specific to HIV gp160. These 19 are classified as “responders to

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This definition of people who react to the vaccine is highly restrictive in light of the scientific goals of this experiment, which consist in assessing the possibility of immunization after infection.

vaccine". 6 of the 11 "vaccine non-responders" showed only a humoral or only cellular immune reaction. All 7 people who did not show any noticeable reaction to the vaccine received only three doses each (Program A). They did not no changes observed in antibody binding to HIV polymerase (p66), structural gene product (p24), or non-HIV checkpoint antigen, tetanus No individuals developed anti-baculovirus antibodies against the Lepidoptera cell checkpoint protein.

An increase in the number of antibodies against the envelope (gp160) was observed in 13 individuals in a Western Blot analysis in which the entire lysate of the HIV-MN virus was used. The changes were related to the immunization program. Three out of 15 people (20%) in Program A and 10 out of 15 people (67%) in Program B developed an increase in the number of antibodies against the envelope protein (P=0.025 in two-fold Fischer's exact test). All 13 individuals also seroconverted to specific envelope epitopes.

Conversely, of the 10 individuals who did not seroconvert to any specific envelope epitope, none showed an increase in the number of antibodies directed against the envelope in the Western Blot test. Of the remaining 7 persons who seroconverted in relation to specific epitopes of the envelope, not one of them showed a change in the number of antibodies directed against the complete envelope of the virus in the Western Blot test. No changes in the number of antibodies directed against non-enveloped proteins of HIV were observed in any individual.

Fourteen of 15 subjects (93%) in Program B (6 doses) showed an increase in the total number of anti-gp160 antibodies, in contrast to only 7 of 15 subjects (47%) in Program A (3 doses) (P=0.01 in two-tailed Fischer test). (Fig. 7).

As illustrated in FIG. 8, the representation of each of the gp160-specific epitopes, before and after immunization, was as follows: epitope 49 (27 to 70%), epitope 88 (28 to 52%), epitope 106 (50 to 87%), epitope 214 (0 to 14%), epitope 254 (0 to 13%), epitope 300 (47 to 77%), epitope 308 (42 to 69%), epitope 342 (0 to 27%), epitope 422 (3 to 10%), epitope 448C (73 on 87%) and epitope 735 (17 on 33%). Vaccine-induced seroconversion was observed for all specific epitopes except 58? (Fig. 7). Antibodies (seroconversion) directed against epitopes 241, 254 or 342 were observed only after vaccination.

Secondary immune reactions were detected against the following epitopes: 88, 106, 300, 448C and 582. The representation of antibody directed against epitope 582 was 100% before vaccination, and only one person (3%) showed a secondary immune reaction.

The pattern of vaccine-induced anti-HIV antibodies directed against envelope epitopes was variable (SI. 7). Primary antibody reaction (seroconversion) in relation to at least one epitope was observed in 20 persons; 14 out of 15 people in Program B and 6 out of 15 people assigned to Program A (P=0.005 in two-tailed and Fischer's test). Individuals from Program A seroconverted against only 15 of 110 (14%) potential epitopes

against which they did not have antibodies before immunization. Individuals from Program B seroconverted versus 60 of 129 (47%) (P<0.0001 in two-tailed Fischer test). Seroconversion in relation to three or more envelope epitopes occurred in 9 persons (60%) assigned to Program B, but in only 2 persons (13%) assigned to Program A (P=0.02 in double Fischer test).

Neutralizing activity in serum against three different types (HIV-IIIB, MN and RF) was determined on days 0, 90 and 195 in 7 individuals. Four out of 5 vaccine responders showed an increase in neutralizing activity against one or more isolates. Vaccine responders also, relative to non-vaccine responders, showed an increased ability to inhibit syncytial formation.

G. Vaccine-induced cellular reactions

Changes in cellular immune responses were based on comparison of pre-vaccination (baseline) and post-vaccination mean lymphocyte stimulation indices (LSIs) using the Wilcoxon rank sum test.

Twenty-one out of 30 persons (70%) developed a new T-cell proliferation reaction after immunization (Fig. 7).

Sl. 9 illustrates the time-dependent proliferative responses to gp160, p24, and baculovirus checkpoint protein in four typical vaccine responders. In all subjects, gp160-induced proliferation increased from a baseline mean LSI of 3 to an LSI of 10 (calculated using the mean of 4 values obtained after the last immunization). In contrast, no change was observed for the proliferative reaction against the HIV-protein p24 or the baculovirus control protein.

Vaccine-induced changes in the mean value of LSI for all persons, for persons classified into sub-groups depending on the reaction to the vaccine, and for persons classified into groups according to the immunization program, are illustrated in Fig. 10.

The change in proliferative response to gp160 was significantly different between responders and non-responders (<0.001, two-tailed Wilcoxon test). Proliferative responses to gp160 induced in Program B (6 doses) were greater than those induced in Program A (3 doses) (P < 0.10, two-fold Wilcoxon test).

Nineteen of the 21 people who developed a proliferative reaction to gp160 also developed a humoral reaction (vaccine responders). The maximum mean lymphocyte stimulation index (LSI) to gp160 observed for all vaccine responders was 50.1. However, the response of each individual responding to the vaccine was different (maximum values ranged from 3 to 171) (Fig. 7), as was the dependence of the magnitude and duration of cellular responses to gp160 on the time of vaccination (Fig. 9). .

H. Discussion of results

Despite the limited sample size in this trial, several factors have been shown to be associated with vaccine immunogenicity. Six of 15 individuals (40%) in Program A versus 13 of 15 individuals (87%) in Program B were vaccine responders (P=0.02, two-tailed Fischer test) (SI. 7). Of the 16 subjects with a mean baseline CD4 cell count greater than 600 cells/ml, 13 (81%) were vaccine responders, in contrast to 6 of 14 (43%) subjects whose mean baseline CD4 cell count was less than 600 cells/ml (P=0.07, double Fischer test). As collectively illustrated in Table 8, multiple immunizations improved immunogenicity even in patients with baseline CD4 cell counts less than 600 cells/ml. For example, 5 of 6 individuals in Program B (6 injections) were vaccine responders, compared with only 1 of 8 individuals in Program A (3 injections) (P=0.03, two-tailed Fischer test) (Table 8). .

TABLE 8

Baseline-dependent immune reactivity to gp160

CD4 cell count and immunization program

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The therapeutic use of vaccines was first started by Pasteur in the 19th century, to treat acute rabies infection. The suitability of such an approach in the treatment of other infections has not been widely studied. Although there are other examples of post-infectious modification of virus-specific immunity (for example after exposure to the hepatitis A or B virus), there is a lack of well-documented human studies demonstrating the possibility of using this approach in developed or chronic viral infections.

The present invention provides modification of virus-specific immunity, by active immunization after infection. In particular, a vaccine with gp160, derived from the HIV envelope gene, increased the human virus-specific humoral and cellular response in 19 of 30 people infected with an early form of HIV.

This study qualitatively and quantitatively measured different antibody responses to specific epitopes of HIV in natural infection, compared to responses after post-infectious immunization. In this way, vaccine-induced humoral immunogenicity was documented in 70% of already infected persons. For example, twenty individuals (19 responders and 1 vaccine non-responder) seroconverted against specific envelope epitopes. Seroconversion associated exclusively with vaccination (epitopes 241, 254 and 342) occurred in 10 persons.

In addition, variations in the humoral response to this vaccine, which is characterized through epitope mapping, will enable analysis of the causes and consequences of specific antibody reactions, and provide a unique opportunity to characterize potential immunoregulatory mechanisms that are not triggered during natural infection.

Although the significance of neutralizing activity in serum in vivo is currently unknown, the observed increase in neutralizing activity against different types of HIV (IIIB, RF, MN) in 4 of 5 vaccine responders indicates that post-infection immunization causes changes in functional antibodies. The tested vaccine induced an increase in neutralizing capacity in serum against different types of HIV, and will potentially be helpful in defining group-specific neutralizing epitopes.

A proliferative reaction against the HIV envelope protein rarely occurs in natural HIV infection. However, after immunization with gp160, a proliferative response of specific T-cells was documented in 21 individuals (70%). The reason for this difference is not clear. One possibility is that the new proliferative response may be directed against one or more envelope epitopes that are unique to the vaccine (as a result of vaccine production technology or alternative antigen processing in vivo). Another possibility is that the protein used in the proliferation study may not stimulate the primary proliferative response of T-cells against the homologous "wild-type" envelopes of the native virus. However, additional evidence was obtained that vaccination enhances the cellular immune response in the host: selected vaccine responders showed, after repeated immunization, a response to produce HIV-IIlB-specific cytotoxic T-cells.

The factors responsible for vaccine immunoreactivity in HIV-infected persons are still not elucidated. Even at an early stage of infection, individual individuals respond suboptimally to various vaccines compared to an appropriate control. Hyporesponsiveness has been attributed to early B-cell dysregulation and T-cell dysfunction. Here, vaccine immunoreactivity is associated with baseline CD4 cell count, which is consistent with the hypothesis that host immune status is an important determinant of vaccine immunoreactivity. however, immunization schedule also influenced vaccine reactivity: Schedule B (6 injections) was superior in individuals within the same range of T-cell counts. Indeed, the reduced reactivity observed in individuals with lower CD4 cell counts can be ameliorated by increasing the number of vaccinations, indicating that host immunoreactivity can be further improved by further modifying dosages, regimens, adjuvants, or formulations.

Although doubts have been raised about the safety of actively immunizing HIV-infected persons with HIV-specific vaccine products, there is no evidence of immuno-specific toxicity. Quantitative cultures, DNA polymerase chain reaction assays, and serum antigen assays show increased levels of HIV in vivo. A factor that perfectly reflects the replication of HIV in vivo, the rate of decrease in the number of CD4 cells, showed positive changes in the tested persons, especially in persons who respond to the vaccine. The change in mean CD4 cell count was -0.2% for responders and -7.3% for non-responders. These data show that post-infectious immune reactivity is not associated with increased CD4 cell depletion, and suggests an association with reduced HIV replication in vivo.

Vaccination results in this study were also compared to a database of ten infected, untreated individuals matched for age, ethnicity, and baseline CD4 cell count. The mean CD4 cell count decreased by 8.7% in this reference group, decreased by 7.2% in individuals assigned to Program A, and increased by 0.6% in individuals assigned to Program B. These results indicate that post-infection vaccination with

by recombinant HIV envelope proteins possible and, moreover, provide encouragement in terms of prophylactic applications of these vaccines.

VACCINES AND VIRUS TREATMENT METHODS

HUMAN IMMUNITY DEFICIENCY

DNA NUCLEOTIDE SEQUENCES LATERALLY LINKED TO CODING]IN THE SEQUENCE OF gp160 FROM Ac3046

TGCTGATATC ATGGAGATAA TTAAAATGAT AACCATCTCG CAAATAAATA

-100

AGTATTTTAC TGTTTTCGTA ACAGTTTTGT AATAAAAAAA CCTATAAATA

-50

ATG -----/3046/----- TAATTAATTAA GT ACC GAC TCT GCT GAA GAG

+ 1 +2256

GAG GAA ATT CTC CTT GAA GTT TCC CTG GTG TTC AAA GTA AAG GAG

+2288

TTT GCA CCA GAC GCA CCT CTG TTC ACT GGT CCG GCG TAT TAA

+2333 +2374

3.

VACCINES AND VIRUS TREATMENT METHODS

HUMAN IMMUNITY DEFICIENCY

DNA NUCLEOTIDE SEQUENCES AND PRESUMED SEQUENCES

AMINO ACID AT THE END OF THE GENE IN Ac3046

FIG. 4a

With engines:

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HUMAN IMMUNITY DEFICIENCY

FIG. 4b

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FIG. 4c

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FIG. 4s

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Cleaving enzymes:

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Non-cleaving enzymes:

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FIG. 4t

OF NUMBERED BASES OF ENV GENAA IN Ac3046: 2256

NUMBER OF AMINO ACID CODONS: = 2256 ÷ 3 = 752

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Total weight estimate

of non-glycosylated polypeptides: = 84,895.5

Total number of glycosylation sites:

28 x 2100 (per oligosaccharide)

Total estimated molecular weight of gp160 = 84,895.5 + 58800

= 143,695.5

VACCINES AND METHODS OF TREATMENT OF HUMAN IMMUNE DEFICIENCY VIRUS

COMPARISON OF LAV-1 SEQUENCE AND RECOMBINANT Ac3046 pg160 SEQUENCE

The sequence and the corresponding codons at the top of the reeds are those it predicts

FIG. 5a processing and Wain-Hobson et al. (1985). The sequence at the bottom of each

sequence is the one determined for Ac33046 from recombinant DNA virus.

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FIG. 5b

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FIG. 5d

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FIG. 5 years

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FIG. 5 a.m

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Claims (36)

1. A method of treating persons infected with the human immunodeficiency virus (HIV), indicated by the fact that it consists in giving the infected person a recombinant HIV envelope protein. 2. The method according to claim 1, characterized in that the recombinant protein is given in a dose of about 1 to 100 μg per kilogram of body weight. 3. The method according to claim 1, characterized in that the recombinant protein is administered in a dose of about 10 μg to about 4000 μg. 4. The method according to claim 1, characterized in that the recombinant protein is given in a dose of about 40 μg to about 1280 μg. 5. The method according to claim 3, characterized in that at least three doses are administered. 6. The method according to claim 4, characterized in that at least six doses are administered. 7. The method according to claim 5, characterized in that individual doses are given at intervals of about 30 to 60 days. 8. The method according to claim 6, characterized in that individual doses are given at intervals of about 30 to 60 days. 9. A method of treating persons infected with the human immunodeficiency virus (HIV), indicated by the fact that it consists in giving the infected person a recombinant HIV envelope protein, in an amount sufficient to cause an enhanced HIV-specific cellular or humoral reaction. 10. The method according to claim 1, characterized in that the recombinant protein is produced in the baculovirus-insect cell expression system. 11. The method according to claim 3, characterized in that the recombinant protein is produced in the baculovirus-insect cell expression system. 12. The method according to claim 5, characterized in that the recombinant protein is produced in the baculovirus-insect cell expression system. 13. The method according to claim 1, characterized in that the recombinant protein has a molecular weight of approximately 145,000. 14. The method according to claim 3, characterized in that the recombinant protein has a molecular weight of approximately 145,000. 15. The method according to claim 5, characterized in that the recombinant protein has a molecular weight of approximately 145,000. 16. The method according to claim 1, characterized in that the HIVa envelope protein represents at least one of gp 160, gp120 and gp41. 17. The method according to claim 3, characterized in that the HIV envelope protein represents at least one of gp160, gp120 and gp41. 18. The method according to claim 5, characterized in that the HIV envelope protein represents at least one of gp160, gp120 and gp41. 19. The method according to claim 1, characterized in that the recombinant protein is expressed by a baculovirus vector for insect cells, Ac 3046. 20. The method according to claim 3, characterized in that the recombinant protein is expressed by a baculovirus vector for insect cells, Ac3046. 21. The method according to claim 5, characterized in that the recombinant protein is expressed by a baculovirus vector for insect cells, Ac3046. 22. The method according to claim 1, characterized in that the recombinant protein is agglomerated into particles with a molecular weight of at least about 2,000,000. 23. The method according to claim 3, characterized in that the recombinant protein is agglomerated into particles with a molecular weight of at least about 2,000,000. 24. The method according to claim 5, characterized in that the recombinant protein is agglomerated into particles with a molecular weight of at least 2,000,000. 25. The method according to claim 1, characterized in that the recombinant protein is combined with an adjuvant. 26. The method according to claim 3, characterized in that the recombinant protein is combined with an adjuvant. 27. The method according to claim 5, characterized in that the recombinant protein is combined with an adjuvant. 28. A method for treating persons infected with the human immunodeficiency virus (HIV), characterized in that the infected person is given a preparation consisting of a recombinant HIV envelope protein and an alum adjuvant, wherein the recombinant protein is formed into particles with a molecular weight of at least approx. 2,000,000. 29. The method according to claim 28, characterized in that the recombinant protein is produced in the baculovirus-insect cell expression system. 30. The method according to claim 28, characterized in that the recombinant protein is selected from the group comprising recombinant gp160, recombinant gp120, recombinant gp41, recombinant HIVa envelope protein with a molecular weight of about 145,000, and recombinant protein expressed by the Ac3046 vector. 31. The method according to claim 28, characterized in that the recombinant protein contains about 757 consecutive amino acids from gp160, and essentially does not contain about 40 consecutive amino acids from gp160. 32. The method according to claim 28, characterized in that the recombinant protein is administered in a dose of about 10 μg to about 4000 μg. 33. A therapeutic vaccine preparation against HIV, characterized in that it contains a recombinant HIV envelope protein and an alum adjuvant, wherein the recombinant protein is formed into particles with a molecular weight of at least about 2,000,000. 34. The preparation according to claim 33, characterized in that the recombinant HIV envelope protein is provided in an amount of about 10 μg to about 4000 μg per dose. 35. The preparation according to claim 34, characterized in that the recombinant protein is produced in the baculovirus-insect cell expression system. 36. The preparation according to claim 34, characterized in that the recombinant protein contains about 757 consecutive amino acids from gp160, one gp160 peptide shortened approximately at position 757 and substantially excludes about 40 bordering amino acids from gp160.