LT3365B - Vaccine for human immunodeficiency virus - Google Patents

Abstract

An Acquired Immunodeficiency Syndrome (AIDS) vaccine containing the Human Immunodeficiency Virus, Type-1 (HIV-1) envelope proteins is produced from cloned HIV-1 envelope genes in a baculovirus-insect cell vector system. The recombinant HIV-1 proteins are purified, assembled into particles and then adsorbed on an aluminum phosphate adjuvant. The resulting adsorbed recombinant HIV-1 virus envelope protein formulation (AIDS vaccine) is highly immunogenic in animals and elicits antibodies which bind to the HIV-1 virus envelope and neutralize the infectivity of the virus in in vitro tests. The above AIDS vaccine induces new humoral and cellular immune responses in HIV-infected patients and is useful as a form of vaccine therapy to delay or prevent the destruction of the immune system.

Description

This application is a U.S. Patent Application Serial No. 151,976 completed in 1988. February 3, cont. Application no. 151,976 are U.S. Patent Application Serial Nos. 920,197 completed in 1986. October 3 (now Serial No. 585,266), cont. These applications and cited works are included in this patent, although they are quoted only.

Human Immunodeficiency Virus type 1 (HIV-1) is a retrovirus that causes pathogenesis in the immune system. Human immunodeficiency virus is a causative agent of acquired human immunodeficiency syndrome (AIDS). BarreSinoussi, et al., Science, 220: 868-871 (1983); Popovic et al., Science, 224: 497-500 (1984); Clinical isolates of HIV1 were further classified as associated with lymphadenopathy virus (Feorino et al., Science 225: 69-72 (1984) and AIDS-related virus (Levy et al., Science 225: 840-842 (1984))).

As AIDS has become pandemic, the development of a vaccine against AIDS has become one of the major health challenges. A high percentage of people infected with HIV-1 rapidly lose their immune function due to a decrease in T4 lymphocytes. These T4 cells, like certain nerve cells, have a molecule called CD4 on their surface. HIV-1 recognizes the CD4 molecule through a receptor on the envelope of viral particles, enters the cell, replicates and eventually kills it. An effective vaccine against AIDS is expected to induce antibody binding to the HIV-1 virus envelope, thereby protecting T4 lymphocytes and other sensitive cells from HIV-1.

As a general rule, vaccines are used as immunoprophylaxis in healthy individuals who have not yet developed the disease. However, it is completely acceptable to use an effective vaccine against AIDS as an immunotherapeutic agent after infection. Salk, J., Nature, 327: 473476 (1987).

It is now widely believed that the HIV1 envelope (env) is the most appropriate candidate for developing an AIDS vaccine. Francis and Petricciani. New Eng. J. Med., 1586-1559 (1985); Vogt and Hirsh, Rewiews of Infectious Disease, 8: 991-1000 (1986); Fauci, Proc.Natl.Acad.Sci.USA, 83: 9278-9283. Initially, the HIV1 envelope protein is synthesized as a 160,000 molecular weight glycoprotein (gp160). The gp160 precursor is then cleaved into 120,000 molecular weight outer glycoprotein (gp120) and 41,000 molecular weight transmembrane glycoprotein (gp41). These envelope proteins are the most important antibody targeting antigens in patients with AIDS. Barin, et al., Science, 228: 1094-1096 (1985). Native HIV-1 gp120 has been shown to be immunogenic and capable of inducing neutralizing antibodies in rodents, goats, rhesus monkeys and chimpanzees. Robey, et al., Proc.Natl.Acad.Sci.USA 83: 7023-7027 (1986).

Because of the very low levels of native HIV-1 envelope protein in infected cells and the risks associated with preparing the AIDS vaccine from HIV-1 infected cells, recombinant DNA techniques have been introduced for HIV1 envelope antigens for the production of anti-AIDS vaccine. Recombinant DNA technology has proven to offer the best potential for the production of subunits of the AIDS virus vaccine by enabling the safe and cheap production of large quantities of immunogens. The HIV1 envelope protein gene was expressed in genetically engineered recombinant vaccine viruses. Chakrabarti, et al., Nature 320: 535-537 (1986); Hu, et al., Nature, 320: 537-540 (1986); Kieny, et al.,

Biotechnology, 4: 790-795 (1986). In addition, envelope protein genes were expressed in bacterial cells (Putney et al., Science, 234: 1392-1395 (1986)), in mammalian cells (Lasky, et al., Science, 23: 209-12 (1986)), and in insect cells. Synthetic peptides corresponding to the HIV-1 gp41 amino acid sequence were also considered suitable for the production of anti-AIDS vaccines. Kennedy, et al. (1986). Unfortunately, such methods and materials have failed to develop an effective vaccine against AIDS.

One aspect of the invention is the use of a baculovirus, an insect cell vector system, for the production of recombinant HIV-1 envelope protein, described in U.S. Patent Application Serial No. 4,672,124, filed and signed. 920,197 completed in 1986. October 16, (now Serial No. 585, 266). See also serial no. 151.976.

The baculovirus system has been shown to be generally useful in the production of HIV-1 and other proteins. For example, baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV), was used as a vector for expression of full-length gp160 and individual portions of the HIV-1 envelope gene in infected Spodoptera frugiperda cells (Sf9 cells). Further, previous patent applications describe a truncated gp160 gene (recombinant number Ac3046); protein produced by recombinant Ac3046; and purification methods of the Ac3046 gene product using lens lentin affinity chromatography and geofiltration chromatography. The gp160 protein purified and aggregated in this manner was found to be highly immunogenic in rodents and primates.

The ideal vaccine against AIDS should not only be biologically pure and apyrogenic, but one or more of its injections should protect against HIV-1 infection for life. This is typical of live attenuated vaccines. When killed bacteria or viruses or substances isolated from them, such as toxoids, proteins, are used in the production of vaccines, a weak immune response is usually induced and only transient immunity is acquired. To overcome or reduce these vaccine deficiencies, an additional substance called an adjuvant is usually included in the vaccine. Adjuvants are substances that help trigger an immune response. In general, adjuvants used in vaccines for human use are aluminum salt gels (aluminum phosphate or aluminum hydroxide), commonly referred to as aluminum adjuvants. Bomford, et al., Adjuvant, Animal Cell Biotech. Vol. 2: 235-250, Academic Press Inc. (London: 1985).

The present invention is a vaccine against human immunodeficiency virus (HIV). In a preferred embodiment, the envelope protein is aggregated and mixed with an adjuvant (e.g., alum) for vaccine preparation,

Details of the present invention and references to the accompanying drawings are given below:

FIG. 1 illustrates the cloning strategy used to isolate the HIV-1 envelope gene (env) from the E, coli plasmid pNA2. Blank portions represent the HIV1 sequence, unblocked the DNA sequence of the cloning vector. The dark region of plasmid pl774 is constructed from synthetic oligonucleotides and inserted into the Smal-KpnI site of plasmid 01614 as a Smal-KpnI fragment. The sequence of this synthetic oligonucleotide is shown.

FIG. 2 illustrates a strategy for constructing a recombinant plasmid vector (p3046), which in turn has been used to construct the baculovirus expression vector Ac3046.

vector

Plasmid pMGS3 contains baculovirus AcNPV sequences (cross-hatched) on each side of the cloning site at position 4.00. This region has unique sites for the restriction endonucleases SmaI, KpnI and BglII. The AcNPV polyhedrin promoter is located 5 'to the 4.00 position. The sequence 5'-TAATTAATTAA-3 'is 3'-directional and has a broadcast termination codon in all three reading frames. The plasmid pl774 and the sequence of the synthetic oligonucleotide region are shown in Figs. 1. Plasmid p3046 contains the entire pMGS3, except for the sequences between the SmaI and BglII sites, where the PL774 HIV1 envelope gene is inserted.

FIG. Figure 3 shows the nucleotide sequences of the DNA surrounding the coding sequences for Ac3046 gp160.

FIG. 4 is shown in 3046 env DNA sequence between +1 and +2267. FIG. 4a-4t shows Ac3046 HIV env gene segment primary DNA sequence together with of a synthetic oligonucleotide

sequence located at the end of the 5 'env gene (between +1 and +2267). The restriction endonuclease links are listed above the DNA sequence, and the predicted amino acid sequence is below the DNA sequence. The bases are numbered left and right.

FIG. 5a-5h compare the DNA sequence of the Ac3046 env gene and the published LAV-1 env gene. The LAV-1 sequence is at the top and the Ac3046 is at the bottom. Line (1) following the LAV-1 sequence indicates that the Ac3046 sequence at this position is the same. The DNA sequences were numbered in the same manner as LAV-1 Wain-Hobson et al., Cell, 40: 9-17 (1985).

FIG. 6 shows antibody titers against HIV-1 as determined by immunoassay assay (IFA) in positive human sera (upper graph) and rhesus monkeys immunized with gp160 (IJ55, KL55) or gp120 (AB55, CD55, GH55) (lower graph). Serum titer against well-purified proteins gp120 and gp160 was measured by IFA. Specific binding antibodies were raised using goat anti-human IgG conjugated to horseradish peroxidase. For this test, the titre is the highest dilution of serum giving a positive response.

FIG. Table 7 summarizes the immune responses elicited by the gp160 vaccine in patients with positive sera.

FIG. 8 (A and B) show vaccine-induced immune responses to specific HIV-1 envelope epitopes.

FIG. 9 shows a vaccine-induced T cell proliferative response in gp160-vaccinated patients with positive sera.

FIG. 10 shows an increase in lymphocyte counts following gp160 vaccination.

FIG. 11 is a graph showing the percentage change in CD4 cell count over time in vaccine-sensitive and non-vaccine individuals.

It has been found that recombinant HIV-1 gp160 envelope protein (rgp160), particularly adsorbed on an adjuvant such as aluminum (e.g. aluminum phosphate), is very suitable for the AIDS vaccine.

One aspect of the present invention is an AcNPV expression vector comprising a portion of an HIV-1 envelope gene coding sequence that is equivalent to recombinant clone no. 3046 1-757 to the Wain-Hobson (ibid) sequence and corresponds to amino acids 1-752.

Another aspect of the invention is the production of this recombinant HIV1 envelope protein (and the protein itself) in insect cells, particularly the rgp160 protein encoded by the amino acid 1-757 Wain-Hobson sequence (i.e., 03046 having 752 amino acid residues).

Other aspects of the present invention include purification of the envelope protein and particle formation from the recombinant baculovirus producing protein 3046, and adsorption of particles of that product onto aluminum phosphate aggregates.

The invention also encompasses vaccines and agents for the prophylaxis and / or therapy of AIDS and HIV infections.

The following examples illustrate the invention without limiting its scope.

Recombinant baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV), having the truncated HIV-1 gp160 gene encoding amino acids 1-757 of the HIV-envelope protein, is described in U.S. Patent Application Ser. 920,197 (now Serial No. 585,260). The cloning steps used to construct recombinant baculovirus containing HIV-1 genes or portions of HIV-1 genes are also described in and incorporated herein by reference.

The following is a detailed description of the gene engineering steps used to construct the Ac3046 expression vector. The materials used, including enzymes and immunological reagents, were obtained from commercial sources. The following examples illustrate how to carry out and apply the invention.

Also contemplated are other recombinant envelope proteins, all of which are classified as rgp160, including recombinant gp120 and gp41 proteins. Ac3046 is an example of such an expression vector and recombinant envelope protein according to the invention.

EXAMPLE

Construction of the recombinant baculovirus Ac3046 having a portion of the HIV1 coding sequence corresponding to amino acids 1-757

Cloning and expression of the foreign protein coding sequences in the baculovirus vector requires that the coding sequence with the polyhedrin promoter and the sequences upstream of it be aligned and the baculovirus coding sequences on the other. In such an arrangement, foreign coding sequences are transferred together with the polyhedrin promoter and the inactive polyhedrin gene during homologous recombination with the baculovirus genome.

Therefore, various insertion vectors have been developed for the construction of the HIV envelope gene. The insertion vector MGS3, described below, was constructed to supply the ATG translation initiation codon. The insertion of foreign sequences into this vector should be such that the broadcast frame starting with the initiation codon will exactly match the foreign sequences.

The insertion vector MGS3 was constructed from an Eco RII restriction fragment clone whose DNA was isolated from a purified AcMNPV isolate (WT-1). MGS3 was constructed to have the following structural properties: (a) 4000 b.p. the sequence upstream of the polyhedrin promoter initiation codon ATG; (b) a polylinker inserted by site-specific mutagenesis that has an initiation codon at the ATG position corresponding to the codon position and KpnI, BglII, as well as polyhedrin restriction initiation sites at SmaI, a universal stop codon segment; (c) 1700 b.p. a sequence stretching from the Kpn restriction site (which is in the polyhedrin gene) to the EcoRI-I terminal EcoRI-I clone. See also: e.g.: fig. 2.

EXAMPLE

Construction of baculovirus recombinants containing LAV env coding sequences

The recombinant plasmid labeled NA2 (Figure 1) consists of a total HIV-1 provirus 21.8 k.b. of a segment inserted into pUC18. This clone has been reported to be infectious because it can produce a virus capable of infecting certain human cells. Adachi, et al., J. Virol. 59: 284-291 (1986). All envelope gene sequences present in NA2 are derived from the HIV LAV strain. Barre-Sinoussi (1983).

The HIV-1 envelope gene was isolated by a genetic engineering procedure described below and shown in Fig. 1. Pi NA2 was isolated at 3846 b.p. EcoRI / SacI fragment containing the envelope gene and EcoRI // SacI restriction site pUC19. a plasmid named p708.

and with it as most cloned from restriction into Constructed

The envelope gene was re-isolated as 2800 b.p. KpnI restriction fragment and cloned into pUC18 KpnI restriction site. The constructed plasmid was named pi 614.

The KpnI restriction fragment at pl614 contained a slightly truncated HIV envelope gene lacking 121 b.p. sequences corresponding to the N-terminus of length. This missing part of the gene encoding the signal peptide sequences was altered by the insertion of a double-stranded synthetic oligomer. The inserted oligomer was synthesized from the amino acid sequence of LAV using codons specific to the polyhedrin gene. To facilitate further manipulations, a new Smal restriction sequence was inserted in place of the ATG initiation codon. The ATG initiation codon is derived from the baculovirus insertion vector. The constructed plasmid was named pl774.

Based on FIG. 2., restriction fragments of pl774 containing sequences encoding various HIV envelope domains were cloned into the MGS insertion vector (e.g., MGS3) such that the ATG initiation codon for the insertion vector was in the reading frame along with the codons for the envelope gene. Constructed p3046 consists of a Smal / BglII restriction fragment isolated from p1774 inserted into the Smal / BamHI site of plasmid vector pMGS3. This clone contains sequences encoding the 1775 amino acids of the gp160 protein and the termination codon of the MGS3 vector.

EXAMPLE

Preparation and selection of recombinant baculovirus

The recombinant plasmid p3046 of the HIV env gene was precipitated with calcium phosphate with AcMNPV DNA (WT-1) and inserted into uninfected cells of Spodoptera fruglperda. The chimeric gene was then inserted into the AcMNP genome by homologous recombination. Recombinant viruses were morphologically indexed by negative plaque occlusion. Such plaques show a cytopathic effect but without nuclear occlusion. In order to obtain a pure recombinant one, one after the purification procedure.

virus, two other sequential, plaque comparisons of restriction and hybridization characteristics with that of wild-type viral DNA were performed to determine whether site-specific insertion of HIV env sequences occurred in recombinant viral DNA.

EXAMPLE

Expression of HIV env from recombinant baculovirus 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 parent product will be composed of amino acids translated from the codons of the recombinant vector. In this way, a protein is obtained which contains all the amino acids encoded in the region from the expression vector initiation codon ATG outside the polyhedrin promoter to the translation termination signal located in the expression vector (e.g., rgp160). The parent Ac3046 streaming product should read Met-Pro-Gly-Arg-Val, where Arg at position 4 in the original LAV clone should be at position 2. Met-Pro-Gly codons are a consequence of the cloning strategy.

EXAMPLE gp60 insert and surrounding DNA nucleotide sequence The gp160 insert and flanking DNA nucleotide sequence was determined by the restriction fragment isolated from the DNA of the viral expression vector Ac3046. The sequences were sequenced as follows: The 3.9 kb EcoRV-BamHI fragment was purified by restriction endonuclease digestion of Ac3046 viral DNA. Ac3046 viral DNA was prepared from extracellular virus in the medium of cells used to make the vaccine series.

As shown in FIG. The 2.9 kb EcoRV-BamHI fragment consists of the entire gp160 gene and 100 b.p. DNA located upstream of the gene and 1000 b.p. DNA outside the gene.

Thus, the sequencing results indicated that a chimeric construct resulting from the cloning strategy was obtained. The gp160 sequence was essentially the same as that reported by Wain-Hobson, et al. (1985). The sequence of 2256 base pairs between putative translation initiation and termination codons leads to a codon of 752 amino acids and 28 potential N-glycosylation sites. The molecular weight of this rgp160 (together with the carbohydrate moieties) is approximately 145,000.

As can be seen in Figs. Sequence analysis of 3,4 and 5,200 bases belonging to the surrounding DNA showed accurate insertion.

EXAMPLE gp! 60 amino acid sequence

Using standard automated Edman degradation and HPLC, the N-terminal sequence of the first 15 gp160 residues was found to be identical to the DNA predicted sequence. The gp160 protein has no N-terminal methionine. This is consistent with the observation that the AcNPV polyhedrin protein is also synthesized in the absence of the N-terminal methionine. Analysis of AcNPV 3046 DNA and purified gp160 revealed that the primary gp160 DNA and N-terminal protein sequences were as follows (Table 1):

TABLE

LAV env in AcNPV 3046 expression vector

Remain

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

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

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

The following results are compared with the original LAV-1 clone (Table 2):

TABLE

LAV env gene AcNPV in original LAV-1 clone

Remain

1 2 3 4 5 6th 7th 8th 9th 10th 11th 12th 13th 14th Mat Arg Or 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

Purification of recombinant gp! 60

One aspect of the present invention is a procedure used for the extraction and isolation of a recombinant HIV-1 envelope protein encoded in an Ac3046 expression vector. The recombinant HIV-1 envelope protein gp160 is synthesized in S. frugiperda cells 45 days after Ac3046 challenge. Purification of this rgpl60 protein proceeds as follows:

1. Cell washing

2. Cell disruption

3. Gel filtration chromatography

4. Lens lentine affinity chromatography

5. Dialysis

This example describes the purification of recombinant gp160 from approximately 2x10 cells infected with Ac3046.

1. Cell washing. Infected cells were washed in buffer containing 50mM Tris buffer (pH 7.5), 1mM EDTA and 1% Triton Χ-100. In this buffer, cells are resuspended, homogenized by standard methods, and centrifuged for 20 minutes at 5000 rpm. This process is repeated 3 times.

2. Cell disruption. The washed cells are sonicated in a mixture of 50 mM Tris buffer (pH 8.0-8.5), 4% deoxycholate and 1% beta-mercaptoethanol. Ultrasonic destruction is performed using standard methods. After sonication, only nuclear membrane particles remain which are removed by centrifugation at 5000 rpm for 30 minutes. Observations under the light microscope indicate that the supernatant containing the gp160 extract does not contain any disrupted cells.

3. Gel filtration. Gel filtration is performed on 5.0x50 cm glass columns (Pharmacia) filled with Sefacryl (Pharmacia). The total volume of sorbent is approximately 1750 ml. The column and hose connections are depyrogenated and disinfected by passing at least 6 liters of 0.1 N NaOH per column for 24 hours. The eluate exiting the column passes through a UV cuvette coupled to a monitor and recorder (Pharmacia), and the column is equilibrated with 4 liters of gel filtration buffer. Crude gp160 is loaded onto the column and passed through a gel filtration buffer.

In the column, the crude mixture breaks down into three large UV-absorbing fractions. The first peak is drawn between approximately 500 and 700 ml, the second peak between 700 and 1400 ml and the third peak between 1400 and 1900 ml of buffer. The same elution profile is observed in small analytical columns which have been found to yield first peak material with molecular weight> 2,000,000.

This peak fraction is poorly absorbed due to its high molecular weight lipid and lipid complexes. In addition, this peak fraction contains 10% to 20% gp160 extracted from infected cells. Undoubtedly, this fraction of gp160 complexes with itself and with other cellular components, forming high molecular weight aggregates.

The second, broad, peak fraction contains most of the gp160 and proteins with an approximate molecular weight of 18,000 to 200,000.

The third peak fraction is low in protein and much of the UV absorption is due to the beta-mercaptoethanol present in the sample.

As soon as the second peak is recorded with the UV detector, the eluate exiting the column is directed directly to the column with the lens plate. When the second peak leaves the column, the eluate is disconnected from the column with the lens plate and deflected.

4. Lens Plate. A large amount of lentil Lectin Sepharose 4B lentine affinity medium was purchased from Pharmacia. The lentil lithin was purified to affinity chromatography on Sephadex to 98% purity and then immobilized on Sepharose 4B using bromocyan. The amount of ligand in the matrix is approximately 2 mg / ml of gel. Lens sputum column is a 5.0x30 cm glass column (Pharmacia) containing 125 ml of Leptin spinach Sepharose 4B gel. After thorough washing and regeneration according to the manufacturer's recommendations, the affine matrix can be reused. When not in use, the gel is stored in a column filled with 0.9% NaCl, 1mM MnCl ₂ , 1mM CaCl _2, and 0.01% mertiolate. Prior to each use, the column is washed and weighed with 250 ml of the lentil buffer described above.

Crude gp160 is passed directly through the column as it is eluted from the gel filtration column as described above. When crude gp160 binds to lentil volatile buffer,

Under these conditions, columns. After connecting the columns, it is washed with 800 ml of 0.1% deoxycholate. all gp160 binds to a glycoprotein detected by UV detector at 280 nm, using an elution lens of leptin buffer with 0.3 M alpha-methyl-manozide.

5. Dialysis. Carbohydrates and deoxycholate are removed by conventional dialysis.

The procedure for purification of gpl60 from 1 liter of infected cells can be summarized in the following table (Table 3). Alternatively, conventional ion exchange chromatography (anion and cation) may be used instead of gel filtration. The workflow is not decisive either: for example, gel filtration or ion-exchange chromatography can be performed after lens lectin chromatography. Other reagents may be used according to the invention. For example, other detergents may be used instead of deoxycholate for the purification of recombinant protein, i. y. nonionic detergents such as Tween 20 (polysorbate 20), Tween 80, Lubrol and Triton Χ-100.

TABLE - Summary of purification

Purification General gp160 protein General Removed stage protein mass (mg) ¹ gp160 (mg) impurity% Cell sediment 1-2000 20th 1-2 Growth medium 1,2,3 250 15th 6th Serum albumin, washing most nucleic acids, soluble cellular proteins Gel Filter 120 12th 12th Lipids, nucleic cation acids, high mol. weight aggregates Lenses 14th 10th 70 Not glycosylated

lentin proteins affinity chromatography ¹ Total protein was determined by absorbance at 280 nm.

EXAMPLE

A. gp! 60 particle assembly.

One aspect of the present invention was that the gp160 antigen can be assembled into particles of> 2,000,000 molecular weight during purification. A mixture of gp160 proteins containing 80-90% monomers (160,000 molecular weight) and 10-20% polymers (in the form of particles) is extracted from the cells. In the gel filtration step, aggregated forms of gp160 are removed. Failure in the membrane fraction isolated from gp160 from this fraction (first peak after gel filtration) suggests that it forms complexes with other cellular proteins and possibly even fragments. However, gp160 antigen presenting a second peak after gel filtration has an approximate molecular weight of 160000-300000. This indicates that monomeric and dimeric forms predominate.

Aggregates or polymers of the gp160 protein are formed in a column with a lenticular lens. The antigen was found to aggregate both by eluting 0.5% deoxycholate from the column with lenticular lentil at a critical micelle concentration (KMC) of about 0.2% deoxycholate and by eluting gp160 from the column with 0.1% deoxycholate.

Aggregate size is measured in a high resolution FPLC Superose 12 column (Pharmacy). Typical examples of the purified gp160 series are typically 20,000,000 molecular weight or more compared to the Dekstran Blue standard.

Schwaller, et al. (1989) show that gp160 is synthesized in insect cells, which are tetrameric particles composed of identical subunits. In addition, these studies indicate that gp160 is tetrameric in HIV-infected cells and viral particles. Thus, particles of recombinant gp160 may have tertiary and quaternary structures similar to native HIV gp160.

An appropriate three-dimensional structure may be important for the formation of epitopes that require precise gp160 spatial distribution. As non-glycosylated proteins are separated from gp160 antigen lenses by lithin chromatography, hydrophobic portions of gp160 appear to begin to form intermolecular assemblies. Deoxycholate does not appear to bind to gp160, as its concentration can be maintained above KMC and antigen continues to form complexes. It appears that such antigen aggregation is an intrinsic property of this protein when purified according to the present invention. It is possible that the hydrophobic N-terminal sequence present in the gp160 protein contributes to the natural ability of this protein to form particles. After purification, the gp160 complexes can be sterile filtered through a 0.2 micron diameter cellulose acetate filter without losing much protein during this procedure.

B. Analysis of Particle Formation.

Analysis of purified gp160 particles by electron microscopy revealed that they were protein-like, spherical 30-100 nM particles.

Purified gp160 is further analyzed by gel filtration to confirm the presence of particles.

Approximately 100 micrograms of gp160 was passed through an HR 10/30 Superose column of FPLC gel filtration (Pharmacia, Ine.). Prior to this, the column was calibrated using molecular weight protein standards. In this column, the protein elution profile reproduces very well; the elution volume is inversely proportional to the molecular weight of the protein standards. The column separates the monomeric gp160 from the polymeric forms and removes globular proteins with a molecular weight> 2x10 ⁶ . When working on this column, the entire gp160 protein is discharged in an empty volume so that the molecular weight is> 2x10 ⁶ (2,000,000).

EXAMPLE

A. gp! 60 adsorption on alum.

The efficacy of insoluble aluminum compounds as immunological adjuvants depends on the adsorption of antigens on the solid phase. One aspect of the present invention is the discovery that aluminum compounds can be made that effectively adsorb gp160. In this case, the pH should be such that the immunogenic activity of the gp160 -alum complex is not reduced. The following factors are controlled during the formation of this aluminum (aluminum phosphate gel) compound:

1. The optimum pH for adsorption of antigens on alum is approximately 5.0. However, gp160 was found at pH 6.5. loses immunogenicity more than at pH 7.5. Therefore, the alum was made at pH 7.l ± 0.1. At this pH, it was discovered that all 100% gp160 still adsorbed to alum.

2. The ionic force due to NaCl is relatively small and less than 0.15 M.

3. Use a molar excess of aluminum chloride relative to sodium phosphate to ensure that the supernatant is free of phosphate ions.

4. The gp160 protein is added to freshly made alum to stop crystal growth and reduce particle size.

200 ml of alum is prepared and the purified gp160 is adsorbed to the alum so that the final concentration of antigen is 40 µg / ml as below.

B. Reagent Preparation (200 ml series).

Prepare the following solutions in sterile depyrogenated bottles or glass bottles of 100 ml. Mix the salts for preparation of solution 1 and solution 2 with sodium hydroxide and filter through 0.2 micron diameter cellulose acetate filters into 100 ml sterile depyrogenated bottles.

Solution 1 A1C1 ₃ .6H /)

NaHAc.3HiO

Dissolve in 40 ml water for injections (VI), filter through a 0.2 micron filter

Solution 2 Na ₃ PO ₄ 12H /)

Dissolve in 40 ml VI, filter through a 0.2 micron filter

Solution 3 NaOH

0.895 g 0.136 g

1.234 g

2.0g

Dissolve in 100 ml VI, filter through a 0.2 micron filter

Solution 4 Tris 1.25 g

Dissolve in 100 ml VI, add 1 ml to 90 ml VI, adjust pH to 7.5 with 0.5N HCl and make up to 100 ml

Autoclave the solutions for 30 min; slowly release steam. Cool to room temperature.

C. Preparation of alum

1. Transfer 1 solution (Aluminum chloride-Sodium acetate) into the preparation vessel using 25 ml sterile disposable pipettes. Note the volume of solution 1 and start mixing the solution.

2. Add solution 2 (sodium phosphate) to the vessel using 25 ml sterile disposable pipettes and continue stirring in the fall. Note the volume of solution 2.

3. Add 3 ml of solution 3 (sodium hydroxide) and stir for a further 5 min. Take a sample of 0.5 ml and measure the pH. If the pH is less than 7.0, add 0.5 ml of sodium hydroxide, stir for a further 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 preparation vessel (solution 1 + solution 2 + solution 3), then add sterile VI to 100 ml.

5. Urgently Place 8.000 micrograms of purified gp160 in 100 ml of 1 mM Tris pH 7.5 directly into the preparation vessel.

6. Shake for at least another 20 minutes, then pour the reconstituted vaccine into sterile tubes.

EXAMPLE

Immunogenicity of alum adsorbed on gp! 60 (specific Ab response)

A suitable method for determining the immunogenicity of an antigenic preparation (vaccine) is to measure the specific immune response in a single dose of antigen in mice. At the end of 4 weeks, the mice are bled and the serum levels of antibodies against the indicated antigens (typically the antigens with which the animals were immunized) are measured by a standard assay, i.e. IFA (Immunoassay).

The immunogenicity of purified gp160 without adjuvant at pH 6.0 and 7.5 adsorbed to alum (as described in Example 9) or mixed with Freund's complete adjuvant is summarized below (Table 4).

TABLE

Group gp 160 IFA average Seroconversion a gp160 Adjuvant Series OT ² % (P / N) ³ 1 mcg None, pH 7.5 8702 0.140 57 4/6 None, pH 6.0 8702 0.110 26th 2/7 Alums 8702 1,000 90 9/10 Alums 8705 2.285 100 6/6 Freund 8604 1.108 83 5/6 Freund 8702 1.396 100 7/7 Freund 8604 0.434 67 4/6 0.1 mcg Freund 8604 0.434 67 4/6 Alums 8705 1.003 67 4/6 2 28 days after immunization, mice were bled and sera diluted 1:10; are tested by IFA before gp160 purified by gel filtration. Similar results were obtained using a commercial IFA (Genetic System Ine; EIA ™ ELISA) against native HIV1 proteins at a serum dilution of 1: 400.

Ratio of the number of seroconverted mice (P) to the total number of mice tested (N).

Mice immunized with a single dose of 1.0 microgram of gpl60 antigen without any adjuvant elicited an immune response to gp160 (see table below). Significantly stronger immune responses were observed in mice immunized with a single dose of 1.0 microgram of gp160 adsorbed on alum adjuvant. A single dose of less than 0.1 micrograms of gp160 mixed with Freund's complete adjuvant or adsorbed alum will cause seroconversion in> 50% of immunized mice. Although mild, the gp160 antigen without adjuvant was immunogenic in mice at pH 7.5 and pH 6.0. At lower pH, immunogenicity is lost.

EXAMPLE

Immunogenicity of gp! 60 adsorbed on alum (serum assay by IFA)

The ability of the vaccine to elicit an immune response is a very important biological property. The following experiments were performed to confirm that the gp160 vaccine prepared with alum is immunogenic in animals and that the alum adjuvant enhances this immunogenicity:

On day zero, mice (group of 10 individuals) received a single dose (0.5 micrograms, 1.0 micrograms and 5.0 micrograms) of a single gp160, gp160 adsorbed on alum or gp160 with Freund's complete adjuvant (PFA). On day 28, mice were bled and IFA (1:10 dilution) tested for serum antibodies to gp160.

The results in the table obtained after the given groups for more than days are summarized (Table 5)

The average is observed in all

28 below 50% seroconversion. Serum conversions (OT ₄₅₀ nm at 1:10, IFA) for seroconversions were greater at any dose of gp160 adsorbed on alum than when immunized with the corresponding single dose of gp160.

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

TABLE - 28 days after injection

0.5 µg dose 1.0 µg dose 5.0 µg dose Average Average Average P / N ⁴ OT ⁵ P / N OT P / N OT gpl60 9/10 0.407 7/10 0.699 7/10 0.430 gpl60 (alum) 9/10 0.547 8/10 0.797 10/10 0.347 gpl60 (CFA) 10/10 1.130 10/10 1.967 10/10 1.317

Seroconversion mice (P) versus 5 total mice tested (N) days after immunization with 0.5 micrograms, 1 micrograms and 5 micrograms of HIV-1 VaxSyn ™.

⁵ Average serokonvertavusių mice sera absorbance (OD _450), as measured by ELISA against gpl60 serum at 1:10 dilution.

EXAMPLE

Neutralization data

HIV neutralization assay is a recognized method for determining whether an antibody preparation inhibits HIV1 virus and protects against susceptible human lymphocyte culture cells. Antisera from animals immunized with gp160 were assayed for HIV-1 neutralization assay; the results are summarized in the table below (Table 6).

TABLE

Animal Identifi- cation Immunogen / A Micrograms ⁶ djuvant Neutralize duck titre ⁷ Rezus G55 gpl20 / Alums 16/8/8 1: 80-1: 160 Rezus H55 gpl20 / Alums 16/8/8 1: 80-1: 16 Rezus L55 gpl60 / Alums 16/8/8 £ 1: 80 Mice 1: 40-1: 80 Mixture 3 gpl20 / Freund's .25 / .25 / .25 Mice Mixture 8 gpl60 / Freund's .1 / .1 / .1 1: 40-1: 80 J.cow- Pure sex nint IgG gpl60 / Freund 10/10/10 1: 320

⁶ micrograms of gp160 or gp120 in the first / second / third immunization.

⁷ Maximum dilution of antiserum that inhibits infection by 50% compared to HIV-1 infected cells maintained in serum from non-immunized animals.

Guinea pigs, rabbits and rhesus monkeys were also immunized with gpl 60 (with alum or Freund's adjuvant). Overall, a good immune response to HIV-1 envelope proteins was observed after immunization of these animals.

EXAMPLE

Immunogenicity in chimpanzees

Genetically, chimpanzees are the closest relatives of humans. It is currently the only animal model suitable for testing for HIV-1 infection. Toxicity / immunogenicity tests were performed on three monkeys; two were immunized with 40 micrograms or micrograms of gp160 with alum-prepared vaccine. After 4 weeks, they were each immunized with 40 micrograms or 80 micrograms of gp160, respectively. The control animal was simultaneously injected with 1 ml saline. Blood samples from all three chimpanzees were taken weekly and tested for antibodies to gp160 and HIV-1 viral antigens. For this, three immunoassays were performed: IFA using purified gp160 developed by MicroGeneSys, Ine., Western Blot analysis and commercially available ZIV-1 IFA analysis. The data for these analyzes are presented below.

A. IFA (MGSearch HIV 160)

IFA Assay MGSearch HIV 160 (MGSearch is a trademark of MicroGeneSys, Inc. of Meriden, Connecticut, U.S.A. for immunosorbent assay against gp160) is described in co-pending and signed U.S. patent application Ser. No. 920,197 (now Nos. 585, 266).

Serum samples were taken prior to immunization and diluted 1:10 to 1: 100,000 for 11 weeks after primary immunization and incubated with nitrocellulose bands containing 100 µg of purified gp160 at each point. The final titer is the highest dilution of anti-gp160 antibody in which the assay yielded positive results using goat anti-human IgG conjugated to alkaline phosphatase.

Serum samples from control and experimental animals were negative prior to immunization. The chimpanzee at a dose of 80 micrograms was positive at 1: 100 dilution in the second week and the chimpanzee at a dose of 40 micrograms was positive at 1:10 in the fourth week. Antibody against gp160 titer increased to week 5, with final titers of 1: 100,000 and 1: 2,000,000, respectively. Antibody titers in both animals fell very slightly within 6-11 weeks.

This type of response is both qualitatively and quantitatively similar to the immune response observed in chimpanzees vaccinated against human hepatitis B.

B. Commercial IFA Test θ

Analysis of the sera of chimpanzees immunized with VaxSyn by MGSearch HIV 160 ELISA and Western blot revealed that they were seroconverted and contained antibodies to recombinant gp160. Sera before immunization and sera taken at week 1-11 were tested in a licensed commercial IFA assay kit to test for the presence of anti-HIV antibodies that recognize native viral envelope proteins. (LAV EIA ™ Continued Kit of Genetic System Corparation, Seattle, Washigton). The animal immunized with 80 micrograms of gp160 had a positive serum dilution of 1: 100 at week 2; antibody levels continued to increase throughout the 6 weeks. The serum of the animal immunized with 40 micrograms of gp160 diluted 1: 100 was positive at week 6.

EXAMPLE

Distribution of antibodies against gp! 20 and gp41

It is important to determine whether the immune response to gp160 in the vaccinated animal is directed against gp41, gp120 or both. Antibodies against various HIV-1

VaxSyn is MicroGeneSys, Ins. trademark of AIDS vaccine.

various immunoassays were used to determine and measure the levels of envelope protein regions, including radioimmunoprecipitation (RIP), immunofluorescence (IF), Western blot analysis (WB), and quantitative IFA against three different recombinant envelope antigens.

FIG. aggregated immune reactivity of three different recombinant antigens: [ART] [TAB] (1) gp120 - delta (truncated HIV-1 gp120 lacking approximately 40 amino acids at the C-terminus); [ART] [TAB] (2) gpl20 (full-length recombinant HIV-1 gpl20) and [ART] [TAB] (3) gp160.

sera from humans with HIV-1 antibodies and 3 mixed human sera responded well to gp160, moderately to gp120 and poorly or no response to truncated gp120. Truncated gp120, which accounts for more than 90% of the outer HIV1 glycoprotein, appears to have protective determinants. The fact that AIDS-positive human sera have low levels of antibodies to this region of the envelope is consistent with the fact that the immune response does not completely protect against viral infections, whereas positive human sera generally show low levels of neutralizing activity in vitro.

In contrast, sera from monkeys immunized with either the gp160 immunogen or gp120 truncated contain antibodies reactive only with the truncated gp120 portion of the HIV-1 envelope. Such differences in the distribution of antibody recognition sites in the viral envelope and higher titers observed in monkeys may be the cause of high neutralizing titers in monkey sera.

Quantitative serum immunoreactivity of these three recombinant envelope antigens in human and immunized rhesus monkeys is shown in Fig. 7. All monkey sera tested, including animals immunized with gp160, had high levels of antibody to truncated gp120 (gp120-delta).

These results indicate that recombinant gp160 induces an immune response in rhesus monkeys. This response is different from what usually occurs during natural infections. In the gpl60 region of the protein, gpl20-delta contains epitopes that are efficiently recognized in immunized monkeys and that have not been observed in the human immune system at the time of infection. These novel epitopes may be required for HIV-1 protection and should be an important property of recombinant gp160 for the prophylaxis and treatment of HIV-1 infection.

EXAMPLE

Application of the vaccine for treatment

The effect of vaccination with cloned HIV gp160 (synthesized in the baculovirus system according to the scheme described above) in HIV-infected subjects has been studied in clinical trials in 30 patients with HIV-positive sera.

Vaccination with recombinant gp160 induced enhancement of gp160's HIV-specific humoral and cellular immune response in 19 out of 30 (63%) HIV-1 volunteers with serum positive for HIV. Fourteen of the 15 (93%) volunteers who received 6 doses of the vaccine had elevated total anti-gp160 antibodies. Therefore, recombinant HIV proteins (i.e., rgp41, rgp120, rgp160 and their additives) can be successfully used in the treatment of HIV-infected patients.

The effective amounts of the HIV-1 protein used in the present scheme can be determined by the conventional methods described below. In general, these effective amounts may range from about 1 microgram to about 100 micrograms per kilogram of patient weight. Frequency of administration can also be determined by conventional means. The most suitable route of administration is parenteral, i.e. intravenous, peritoneal, intramuscular, subcutaneous, etc. known to those of skill in the art.

A. Volunteer Selection

Thirty volunteers infected with HIV were selected. Only subjects with serum positive and diagnosed early HIV infection known as Walter Reed Stage 1 or 2 (CD4 count of at least 400 with or without lymphadenopathy) were enrolled. (Redfield, et al., New Eng. J. Med. 314: 131-132 (1986). Additional criteria were selected from adult volunteers aged 18-50 with normal blood formula, with no apparent underlying organ disease, no alcohol abuse, and drug in the last 12 months and without antiretroviral and immunomodulatory medication.

Of the 30 volunteers, twenty-six were male and 4 were female. Among them were fourteen Caucasians, 13 Black and Hispanic races. Mean age 29 years (range 18-24). Volunteers were diagnosed with Walter Reed Stage 1 at baseline and 22 at Walter Reed Stage 2 at baseline, with an initial mean CD4 score of 668 (range 388-1639). The median time between initial diagnosis and study entry was 24 months (range 3 to 49 months).

B. Vaccine and immunization scheme

The test vaccine described herein consists of a non-infectious subunit glycoprotein derived from gp160, a recombinant protein expressed in baculovirus. The immunogenic protein was synthesized in Lepidoptera insect cells, purified by biochemistry and adsorbed to aluminum phosphate. This gave the final form of the vaccine.

Three doses of gp160 were used: 40 micrograms per milliliter, 160 micrograms per milliliter, and 320 micrograms per milliliter. The injection volume was 1 mL for both 40 µg and 160 µg doses; (320 µg / ml) A 2 ml volume was used for injections of 640 µg.

Thirty volunteers were divided into six groups of five volunteers each. Immunizations were carried out according to the following schedules: Scheme A, with vaccinations at days 0, 30 and 120; and Scheme B for vaccination at days 0, 30, 60, 120, 150, and 180.

The three immunization schedules (A or B) resulted in three groups of patients receiving different doses of the vaccine (Table 7). The vaccine was given into triangular muscles. Investigations lasted 10 months:

months at baseline and 8 months post-primary follow-up.

TABLE - Immunization Scheme

Injected gp160 quantity (hg) Day 0 30th 60 120 150 180 Scheme A Group 1 40 40 40 Group 3 160 160 160 Group 5 640 640 640 Scheme B Group 2 40 40 40 160 160 160 Group 4 160 160 160 640 640 640 Group 6 640 640 640 640 640 640

C. Safety and Toxicity Assessment

All volunteers were interviewed and examined at 0, 1, 2, 3, and 30 days after each injection. The volunteers were asked about fever, colds, nausea, nausea, arthralgia (joint pain), myalgia (muscle pain), ailments, hives, shortness of breath, dizziness or headache. Local reactions at the injection site were evaluated for erythema, swelling, itching, pain and hypersensitivity, skin discoloration, skin rupture, changes in regional lymphadenopathy, injection site extremities, injection site subcutaneous thickening.

In addition, complete blood formula, serum chemical parameters, blood coagulation rate, and urine analysis were performed monthly.

Cellular immune function was evaluated in vitro by phenotyping T-cells (common lymphocytes, CD4 and CD8 cell phenotypes) according to Rickman, et al., Clinical Iitununo. 52: 85-95, 1989; Birx, et al., J. Acguir. Immune Defic. Syndr. 4: 188-196 (1991). T cell proliferation induced by mitogens (American phytolac and Con A) and control antigens (Candida albicans and tetanus) was also evaluated. Birx et al., Supra. Cellular immune function was assessed in vivo by slow hypersensitivity skin tests against control antigens (i.e., mumps virus, tetanus toxoid, Candida albicans and trichophyton).

Peripheral blood mononuclear cells (PKVL) and plasma viral cultures were quantified according to Burke, et al., J. Acguir. Immune Defic. Syndr. 3: 1159-1167, 1991. DNA polymerase chain reaction (Wages, et al., J. Med. Virol. 33: 58-63, 1991) and serum p24 antigen were determined to assess HIV viral load in vitro. in vivo.

No significant systemic toxicity was observed, but local reactions were observed in 87% of patients (13 in each vaccine group). Local reactions included hardening, hypersensitivity, and the appearance of temporary subcutaneous nodules at the injection site; exacerbation of regional adenopathy was rare. No person withdrew from repeated injections. The incidence of local reactions was independent of primary immunization, booster injections and dosing.

No clear adverse effects on the immune system were observed, either by measurement of specific proliferative responses of mitogen and antigen in vitro, or by in vivo delayed hypersensitivity in skin reactions or accelerated CD4-like cell removal. The primary mean CD4 cell count was 716 and 605, respectively. The mean CD4 cell count in susceptible and non-susceptible vaccinees at days 180240 was 714 and 561, respectively. In the 240-day experiment, the net change in mean CD4 cell count was minus 0.2 percent in vaccine-sensitive patients, whereas in non-susceptible CD4 cells, the mean decrease was 7.3 percent (Figure 11). The vaccine-induced immunogenicity against HIV was not dependent on accelerated CD4-type cell clearance in any single case throughout the test cycle. Post-vaccination viral activity was assessed by quantification of plasma and PKLV viral cultures, PKLV DNA amplification by polymerase chain reaction, and serum p24 antigen for evaluation of HIV replication and virus accumulation. Quantitative analysis of the polymerase chain reaction of cultures and DNA amplification showed no changes throughout the assay. The p24 antigen was not found in the sera of these subjects.

D. Evaluation of immunogenicity

Antibodies against common HIV proteins were also measured using recombinantly produced viral gene products gp160, p66, p24 and the total prototype

HIV strain MN viral lysate. Dot blot and Western blot were performed according to Toubin, et al., Proc. Natl. Acad. Sci. USA 76: 4350-4354 (1979). In addition, immune responses to specific envelope epitopes were measured (see Figure 7).

FIG. Epitopes 88 (amino acids 8889 in gp120) and 448C (448514 in gp120) have been selected in 7 because antibodies to these regions of gp120 have been shown to interact with the 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 735752) were selected for their putative functional importance. Epitopes 106 and 422 were involved in CD4 binding; epitopes 241, 254 and 735 participated in group-specific neutralization, while epitopes 300 and 308 participated in type-specific neutralization.

Epitope 582 (amino acids 582-602) was selected as a control because it represents the immunodominant envelope domain in natural HIV infection. In addition, 49 (amino acids 49-128) and 342 (amino acids 342-405) epitopes were analyzed.

FIG. The dotted squares in 7a show a documented change in the immune response to the HIV envelope. Squares with (-) represent the primary humoral response; a dotted square with (+) indicates a secondary humoral response; (-) denotes antibodies that do not interact with specific epitopes before or after immunization; (+) denotes antibodies interacting with specific epitopes before and after immunization but without quantitative alteration. In dots, the column with (.) Represents the new T-cell proliferative response to gp160 immunization. One (.) Indicates the absence of an immune response to gp160, whereas hb indicates a high background (not interpretable); nd indicates not done.

Neutralization activity was measured against three prototype isolates (HIV-IIIB, RF, and MN) by assaying for syncytial inhibition according to Nara, Nature, 333: 469-470 (1988). HIV-specific cellular response was measured by a conventional lymphocyte proliferation assay using gp! 60, p24 and baculovirus expression system control proteins (Birx, supra).

E. Vaccine-sensitive and insensitive individuals

Individuals were considered vaccine sensitive only if the recurrent selective enhancement of the cellular and humoral immune response to HIV envelope-specific epitopes depended on the vaccination series (Fig. 7). Vaccine-induced humoral immunity was defined as seroconversion against specific epitopes and / or a secondary immune response against specific envelope epitopes. Vaccine induced cellular immunity was defined as re-echoes, vaccines induced a proliferative response to gpl60 emergence ^{* 9} insensitive individuals were considered to be those that did not neither humoral nor a cellular proliferative response or developed only a humoral or only a cellular proliferative response to gpl60 epitopes or HIV envelope.

HIV sheath booster

Vaccine individuals,

F. Vaccine-induced humoral response

Based on FIG. Vaccine-induced enhancement of the specific humoral and cellular immune response to gp160 in HIV was observed in 7, 19 out of 30 subjects (63%). These 19 individuals were considered vaccine sensitive. Four of the 11 vaccine insensitive individuals developed a humoral or cellular immune response alone. All 7 subjects who failed to detect any vaccine-induced responses received only 3 doses ⁹ Such a definition of a vaccine-sensitive individual is very limited in the light of the scientific objectives of this trial: e.g., to evaluate the feasibility of post-challenge immunization.

(Scheme A). No changes were found in antibody binding to HIV polymerase (p66), structural (p24) gene products, or non-HIV control tetanus antigen. No individual has antibodies to baculovirus-Lepidoptera proteins.

no system formed

An increase in envelope protein (gp160) was detected by Western blotting of 13 individuals,

HIV-MN. Changes

Three out of 15 individuals used Scheme A, and 10 of them used a common immunization scheme using the total virus lysate.

(20 percent) were vaccinated on an individual basis (67 percent) on Scheme B. Grafting on Scheme B resulted in increased antibody to envelope proteins (P = 0.025 by Fisher's exact test, two-sigma rule). All 13 individuals also seroconverted against specific envelope epitopes.

In contrast, none of the 10 subjects who failed to seroconvert to any specific envelope epitope showed increased envelope protein levels when analyzed by Western blot. The other 7 individuals who seroconverted to specific envelope epitopes showed no change in the overall antibody envelope antibody when analyzed by Western blot. Antibodies directed against non-enveloped HIV proteins were not altered in any individual.

Fourteen of the 15 individuals (93 percent) who received the schedule B (6 doses) had elevated total anti-gp160 antibodies. Meanwhile, in the group treated with Scheme A (3 doses), only 7 out of 15 subjects (47 percent) (P = 0.01 by Fisher's test, two-sigma rule) (Figure 7).

From FIG. the distribution of each gp160-specific epitope between pre-immunization and post-vaccination was as follows: epitope 49 (27 to 70 percent), epitope 88 (28 52 percent), epitope 106 (50 to 87 percent), epitope 214 (0 - 14 percent), epitope 254 (0 to 13 percent), epitope 300 (47 to 77 percent), epitope 308 (42 to 69 percent), epitope 342 (0 to 27 percent), epitope 422 (3 to 10 percent), epitope 448C ( 73 - 87 percent) and epitope 735 (17 30 percent). Vaccine-induced seroconversion was directed against all specific epitopes except epitope 582 (Fig. 7). Antibodies (seroconversion) directed against epitopes 241, 254, or 342 were detected only after the next vaccination.

The secondary immune response was directed against the following epitopes: 88, 106, 300, 448C, and 582. The prevalence of antibodies against epitope 582 was 100 percent before vaccination and only one individual (3 percent) had a secondary immune response.

The scale of vaccine-induced antibody to HIV envelope epitopes varied (Fig. 7). A primary immune response (seroconversion) was elicited to at least one epitope in 20 individuals; 14 of 15 treated with Scheme B and 6 of 15 randomized to Scheme A (P = 0.005 Fisher test, 2 sigma rule). Individuals treated with Scheme A seroconverted against only 15 of the 110 (14 percent) potential epitopes against which they did not have anti-immunization antibodies. Subjects treated with Scheme B seroconverted against 60 of the 129 (47 percent) (p <0.0001 Fisher test, 2 sigma rule) epitopes. Seroconversion against three or more envelope epitopes occurred in 9 individuals (60 percent) treated with Scheme B and only 2 subjects (13 percent) treated with Scheme A (p = 0.02 Fisher's 2-sigma rule).

Serum neutralizing activity against three different strains (HIV-IIIB, MN, and RF) was observed in 7 subjects at days 0, 90, and 195. Four of the vaccine-sensitive individuals showed increased neutralizing activity against one or more isolates. In addition, vaccine-responsive individuals inhibited syncytia formation better than vaccine-insensitive individuals.

G. Vaccine-induced cellular response

Abnormalities in the cellular immune response were determined by comparing the mean lymphocyte stimulation index (LSI) before grafting (baseline) and post-grafting using the Wilcoxon sequential summation method.

Twenty-one in 30 individuals (70 percent) developed a new T cell proliferative response to gp160 postimmunization (Fig. 7).

FIG. 9 illustrates proliferative responses to gp160, p24, and baculovirus control protein in four typical vaccine-sensitive individuals over time. For all individuals, gpl60-induced proliferation increased from a baseline mean LSI of 3 to a LSI of 10 (calculated from the mean of 4 values after the last immunization). In contrast, no proliferative responses targeting the HIV p24 protein or the baculovirus control protein were observed.

FIG. 10. shows the vaccine-induced changes in mean LSI values for all individuals and for individuals grouped by vaccine susceptibility and immunization schedule.

There was a clear difference in the proliferative response to gp160 in vaccine-sensitive and non-vaccinees (<0.001, Wilcoxon, single-sigma rule). The proliferative response of gpl60 to individuals treated with regimen B (6 doses) was stronger than that of individuals treated with regimen A (3 doses) (P <0.10, Wilcoxon, single sigma rule).

Nineteen of the 21 individuals who developed the proliferative response to gp160 also showed a humoral response (vaccine-responsive individuals). The maximum mean lymphocyte stimulation index (LST) i gp160 for all vaccine-sensitive individuals was 50.1. However, all responses in vaccine-sensitive individuals were variable (LSI values ranged from 3 to 171) (Figure 7). Also variable is the temporal relationship between the size and duration of the cellular response to gp160 (Figure 9) and vaccination.

H. Discussion of results

Despite the limited scope of this assay, several factors have been shown to be associated with vaccine immunogenicity. Six of the 15 (40 percent) subjects treated with Scheme A, compared with 13 of 15 treated with Scheme B, were vaccine sensitive (P = 0.02 Fisher's test, 2-sigma rule) (Figure 7). 13 of 16 individuals (percent) with mean baseline CD4 counts greater than 600 per milliliter were vaccine sensitive, whereas 6 of 14 (43 percent) individuals with mean baseline CD4 counts less than 600 per milliliter (p = 0.07) Fisher 's two sigmas) were insensitive to vaccines. Table 8 summarizes that multiple immunizations improved immunogenicity even in patients with mean baseline CD4 levels of less than 600 / ml. For example, 5 of the 6 individuals immunized with Scheme B (6 injections) were vaccine-sensitive compared with 1 of 8 treated with the three-injection regimen (Scheme A) (p = 0.03 Fisher's, two-sigma rule) (Table 8). .

TABLE

Immune response to qp! 60 vaccine based on CD4 baseline and immunizations scheme CD4 content N Sensitive individuals Of insensitive individuals number, % number, % SCHEME A > 600 7th 5 (71%) 2 (29%) 500-600 5 1 (20%) 4 (80%) <500 3 0 (0%) 3 (100%) Total 15th 6 (40%) 9 (60%) SCHEME B 600 9th 8 (89%) 1 (11%) 500-600 2 2 (100%) 0 (0%) <500 4 3 (75%) 1 (25%) Total 15th 13 (87%) 2 (13%) ALTOGETHER 30th 19 (63%) 11 (37%) For the treatment of vaccines In the 19th century started using Pasteur. He applied the vaccine acute rabies

for the treatment of infection. However, the suitability of vaccines for treating other infections has not been extensively studied. Although there are examples of post-infectious modification of virus-specific immunity (after Hepatitis A and B), unfortunately, there are no well-documented studies demonstrating the advisability of vaccines for the treatment of acute or chronic viral infections in humans.

Immunization modulated by active immunization after challenge according to the present invention. The gp160 vaccine enhanced humoral and cellular viral-specific HIV envelope gene-derived human viral-specific responses in 19 out of 30 individuals diagnosed with early HIV.

The present study compared qualitatively different immune responses to specific HIV epitopes in natural infection and post-infection immunization. Thus, accurate detection of the vaccine-induced humoral immunogenicity in already infected individuals has been documented to occur in 70 percent of individuals. For example, twenty individuals (19 vaccine-sensitive and 1 insensitive) seroconverted against specific envelope epitopes. Seroconversion dependent on grafting alone (epitopes 241, 254 and 342) was observed in 10 individuals.

In addition, the variations in the humoral response to this vaccine, as identified by the identification of perspectives for further analysis of the immune response, are prerequisites for non-causalization.

the potential of immunoregulatory natural infection to open up epitopes, effectively open up specific and unique mechanisms, the importance of serum neutralizing activity in vivo is not yet known; increased neutralizing activity against different HIV strains (IIIB, RF, MN) observed in 4 out of 5 vaccine-sensitive individuals post-infectious immunization assuming functional antibody abnormalities. The test vaccine caused an increase in serum neutralizing capacity against different strains of HIV and will likely help to identify group-specific neutralizing epitopes.

The proliferative response to HIV envelope proteins in natural HIV infection is rare. However, after immunization with gp160, a specific T-cell proliferative response was recorded in 21 (70 percent) individuals. The reason for this difference is not clear. One possible reason is that the novel proliferative response may be directed against the envelope epitope (s) that are unique to this vaccine (the result of vaccine production methodology or alternative antigen processing in vivo). In contrast, the protein used in the proliferation assay cannot stimulate the primary T-cell proliferative response to homologous wild-type envelopes of the native virus. However, additional evidence was found that the vaccine enhances the cellular immune response: selected vaccine-sensitive individuals developed type-specific cytoxic T-cell responses to HIV-IIIB following booster immunization.

Factors determining immune susceptibility to vaccine in HIV infected individuals should continue to be investigated. Even in the early stages of HIV infection, individuals respond less well to various vaccines than controls. Such low sensitivity is associated with early B-cell degeneration and T-cell dysfunction. Here, vaccine susceptibility was associated with baseline CD4 cell counts, which is consistent with the hypothesis that immunological host status is an important determinant of vaccine susceptibility. However, the immunization pattern (within a specific range of T cell counts) also influenced vaccine sensitivity:

(6 injections) was superior. In fact, vaccine sensitivity observed with lower CD4 cell counts was enhanced by increased vaccination rates. From this it can be assumed that a further decrease in dosage, regimen, adjuvant or dosage form may decrease the potency of the individual in Scheme B, and an increase in immune sensitivity can be expected. Although concerns have been raised about the safety of active immunization of HIV-infected vaccine subjects with HIV, immune-specific toxicity has not been demonstrated. Quantitative analysis of culture, chain polymerase DNA reaction and serum antigen showed increased levels of HIV in vivo. An excellent marker of HIV replication in vivo, the CD4 cell decline rate, has been adequately altered in patients, particularly those considered to be vaccine sensitive. The mean change in CD4 cell count was -0.2 percent in vaccine-sensitive individuals and -7.3 percent in non-vaccine susceptible individuals. The data showed that immune susceptibility after infection was not associated with increased CD4 cell destruction. This has been suggested to be associated with increased HIV replication in vivo.

Vaccination results at these stages were also compared with baseline data from ten infected and untreated individuals grouped by age, ethnic group, and baseline CD4 cell count. Mean CD4 cell counts decreased by 8.7 percent in this study group, decreased by 7.2 percent in subjects treated with Scheme A, and increased by 0.6 percent in individuals treated with Scheme B. These results indicate that vaccination with a recombinant HIV envelope after infection is possible. The results obtained are hopeful that such vaccines can be used for prophylaxis.

in the system.

purified, aluminum

Vaccine against Acquired Immunodeficiency Syndrome (AIDS), which contains human immunodeficiency virus type 1 (HIV-1) envelope proteins, is derived from the HIV-1 envelope gene, cloned in a baculovirus-insect cell vector Recombinant HIV proteins are assembled into particles and adsorbed on phosphate adjuvant. The resulting form of the adsorbed recombinant HIV-1 envelope protein (AIDS vaccine) is highly immunogenic in animals and stimulates the production of antibodies that bind to the HIV-1 envelope as well as neutralizing viral challenge in in vitro assays. The above AIDS vaccine elicits novel humoral and cellular immune responses in HIV-infected patients and is useful as a therapeutic vaccine to prevent or slow down the breakdown of the immune system.

DEFINITION OF INVENTION

Claims (15)

DEFINITION OF INVENTION A pharmaceutical composition suitable for use in the treatment of individuals infected with human immunodeficiency virus (HIV), comprising: a recombinant HIV envelope protein in an amount sufficient to enhance the HIV-specific cellular or immune humoral response. 2. The composition of claim 1, wherein the recombinant protein is produced in an expression system of a baculovirus-insect cell. 3. The combination of claim 1, wherein the recombinant protein has a molecular weight of about 145,000. 4. The composition of claim 1, wherein the HIV envelope protein is at least one of gp160, gp120 and gp41. 5. The composition of claim 1, wherein the recombinant protein is expressed by the baculovirus insect cell vector Ac3046. 6. The composition of claim 1, wherein the recombinant protein is agglomerated into particles having a molecular weight of at least about 2,000,000. 7. The composition of claim 1, wherein the recombinant protein is bound to an adjuvant. 8. A composition suitable for treating human immunodeficiency virus (HIV) -infected virus comprising a recombinant HIV envelope protein and an alum adjuvant, wherein the recombinant protein comprises particles having a molecular weight of at least about 2,000,000. 9. The composition of claim 8, wherein the recombinant protein is produced in a baculovirus-insect cell expression system. 10. The composition of claim 8, wherein the recombinant protein is selected from the group consisting of recombinant gp160, recombinant gp120, recombinant gp41, recombinant HIV envelope protein of about 145,000 molecular weight and recombinant protein expressed in Ac3046. 11. The composition of claim 8, wherein the recombinant protein is comprised of approximately 757 gp160 consecutive amino acids and has approximately 40 consecutive terminal amino acids completely removed. 12. A therapeutic composition of an anti-HIV vaccine comprising a recombinant HIV envelope protein and an alum adjuvant, wherein the recombinant protein is agglomerated into particles having a molecular weight of at least about 2,000,000. 13. The composition of claim 12, wherein the amount of recombinant HIV envelope protein is from about 10 µg to about 4000 µg per dose. 14. The composition of claim 13, wherein the recombinant protein is produced in a baculovirus-insect cell expression system. 15. The composition of claim 13, wherein the recombinant protein is comprised of approximately 757 gp160 consecutive amino acids, the gp160 protein is terminated at approximately 757 position, and is completely free of at least 40 gp160 terminal amino acids.