HUT68355A - Method for production of vaccine serving for treatment of hiv infection - Google Patents

Description

Human Immunodeficiency Virus Type 1 (HIV-1) is a retrovirus that causes an entire systemic infection with severe damage to the immune system and is responsible for Acquired Immune Deficiency Syndrome (AIDS) as a pathogen (etiologic agent). . [Barrera

-Sinoussi et al., Science, 220, 868-871 (1983); Popovic et al., Science 224: 497-500 (1984)]. HIV-1 isolated from clinical cases has been designated as Lymphadenopathy-Associated Virus = LAV; Feorino et al., Science, 225, 69-72 (1984)] and the AIDS-related virus (AIDS-related virus; Levy et al., Science, 225, 840-842 (1984)].

AIDS has become a worldwide epidemic, making the development of a vaccine a major priority for public health worldwide. A large percentage of people infected with HIV-1 have a gradual loss of immune function as a result of T4 lymphocyte depletion. T4 cells, like certain neurons, carry a molecule called CD4 on their surface. HIV-1 recognizes the CD4 molecule through one of the receptors in the envelope of the virus particles, enters these cells where it replicates and kills the cells. An effective AIDS vaccine is expected to produce antibodies that bind to the HIV-1 envelope, preventing T4 lymphocytes and other sensitive cells from being infected.

Vaccines are usually given to healthy individuals as an immunoprophylaxis before being exposed to a pathogenic organism. However, it would be wise to consider immunization with an effective AIDS vaccine after infection as an immunotherapy against the disease (J.Salk, Natur. 327: 473-476 (1987)).

The prevailing perception is that the HIV-1 envelope (env) is the most promising candidate for the development of an AIDS vaccine. Francis and Petricciani, New Engl.J.Med., 1586-1559 (1985); Vogt and

Hirsch, Reviews of Infectious Disease, .8, 991-1000 (1986); Fauci, Proc. Natl.Acad.Sci. , 83, 9278-9283 (1986)]. The HIV-1 envelope protein is first synthesized as a 160,000 molecular weight glycoprotein (gp160). The gp160 precursor is then cleaved to form an external glycoprotein (gp120) with a molecular weight of 120,000 and a transmembrane glycoprotein (gp41) with a molecular weight of 41,000. These envelope proteins are the major target antigens for antibodies produced in AIDS patients (Barin et al., Science 228: 1094-1096 (1985)). Native HIV-1 gp120 was immunogenic and was able to induce neutralizing antibodies in rodents, goats, Rhesus monkeys and chimpanzees. [Robey et al. , Proc. Natl. Acad. Sci. , 83, 7023-7027 (1986)].

Given that infected cells contain very few native HIV-1 envelope proteins and that there are risks associated with producing HIV vaccine from HIV-1 infected cells, recombinant DNA methods have been used to produce HIV-1 envelope antigens for use as AIDS vaccines. Recombinant DNA technology appears to be the best choice for the production of the AIDS subunit vaccine, as it allows the economical production of large quantities of harmless immunogens. HIV-1 envelope protein was expressed in genetically modified recombinant vaccine viruses. [Chakrabarti et al. Naturre 220: 535-537 (1986); Hu et al., Natur. 320, 537-540 (1986); Kieny et al., Biotechnology, 4, 790-795 (1986). The envelope protein in bacterial cells (Putney et al., Science, 234, 1392-1395 (1986)), mammalian cells (Lasky et al., Science, 23, 1986). 209-212 (1986)] and expressed in insect cells. HIV-1 gp41 •; . ··. ......

* ··· ·· ··· synthetic peptides of amino acid sequence origin have also been considered as AIDS vaccine candidates. (Kennedy et al., 1986). However, no effective AIDS vaccine has yet been produced using these materials and methods.

The use of a baculovirus-insect cell system for the production of recombinant HIV-1 envelope proteins is one embodiment of U.S. Patent Application No. 920,197, filed October 16, 1986; current Serial No. 585,266. (See also number 151,976).

The baculovirus system has been found to be universally useful in the production of HIV-1 proteins and other proteins. For example, the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has been used as a vector to express the full length gp160 and various portions of the HIV-1 envelope gene in infected Spodoptera frugiperda (Wall armyworm) cells (Sf9). Techniques for purifying the truncated gp160 gene (recombinant vector number Ac3046), the protein produced by the recombinant Ac3046 vector, and the gene product Ac3046, which include lens-lectin affinity chromatography and gel filtration chromatography, are also disclosed in the aforementioned patents. The gp160 protein thus purified and aggregated into particles has been found to be highly immunogenic in rodents and non-human primates.

The ideal AIDS vaccine, in addition to the requirements of being essentially biologically pure and pyrogen-free, should provide lifelong protection with a single or

after a few injections against HIV-1 infection. This is usually the case with live attenuated vaccines. When killed bacteria or viruses, or substances isolated from them, such as toxins or proteins, are used in the preparation of vaccines, they often have a weak antibody response and only a short duration of immunity. In order to overcome or minimize these deficiencies in a vaccine, an additional component, a so-called adjuvant, may be added. Adjuvants are substances that help stimulate the immune response. Adjuvants commonly used in human vaccines are gels made of aluminum salts (aluminum phosphate or aluminum hydroxide), commonly referred to as aluminum salt adjuvants. [Bomford et al., Adjuvants, Anim Cell Biotech. Volume 2, pp. 235-250. Academic Press Inc. (London, 1985). ]

The present invention relates to a vaccine against human immunodeficiency virus and methods of treatment. Treatment includes the use of recombinant HIV envelope protein in an infected or susceptible individual. In a preferred embodiment, the envelope protein is purified, aggregated and combined with an adjuvant (e.g., aluminum phosphate) for use in a vaccine.

The following is a detailed description of the invention and an explanation of the accompanying drawings.

Figure 1 depicts the cloning strategy used to isolate the HIV-1 envelope gene (env) from E. coli pNA2. The bold sections represent the HIV-1 DNA sequences and the blank sections represent the clones from the cloning vectors. The black region in plasmid p774 was constructed from synthetic oligonucleotides introduced as the Smal-KpnI fragment into the Smal-KpnI site of plasmid p1614. The sequence of the synthetic oligonucleotide is shown.

Figure 2 depicts the strategy used to construct the recombinant plasmid vector (p3046). The p3046 vector is then used to construct the Ac3046 baculovirus expression vector. Plasmid pMGS3 contains sequences from the AcNPV baculovirus (cross-hatched region) on both sides of the cloning site at position 4.00. This is the only site for the restriction endonucleases Smal, KpnI and BglII. The AcNPV polyhedrin promoter is located 5 'to position 4.00. The 5'-ΤΑΆΤΤΑΑΤΤΆΑ-3 'sequence is in the 3' direction and contains a translation terminator codon in each of the three reading frames. Plasmid p774 and the sequence of the synthetic oligonucleotide region are shown in Figure 1. Plasmid p3046 contains the entire plasmid pMGS3 except for the sequences between the SmaI and BglII sites, where the p774 HIV-1 envelope gene was inserted.

Figure 3 shows the nucleotide sequences of DNA flanking the Ac3046 gp160 coding sequences. The 3046 env DNA sequence between +1 and +2267 is shown in Figure 4.

Figures 4a-4t show the actual DNA sequence of the HIV-1 env gene segment along the synthetic oligonucleotide sequences located at the 5 'end of the env gene in Ac3046 (between +1 and +2267). The restriction endonuclease sites are inscribed above the DNA sequence and the predicted amino acid sequences are listed below the DNA sequence.

Figures 5a-5h compare the DNA sequence of the env gene in the Ac3046 expression vector with the reported sequence of an env gene from LAV-1. Above is the LAV-1 sequence, below is the Ac3046 sequence. Lines below the LAV-1 sequence (|) indicate that the Ac3046 sequence is identical at these positions. The DNA sequence numbering is the same as that described for LAV-1 by Wain-Hobson et al., Cell, 40, 9-17 (1985).

Figure 6 shows the dilution titers of ELISA endpoints of human HIV-1 antibody-positive sera (top drawing) and Rhesus monkey immunized with gp160 (IJ55, KL55) or gp120 (AB55, CD55, GH55) (bottom drawing). . ELISA titers were measured against highly purified gp120 and gp160 proteins. The specifically bound antibody was measured with a goat anti-human IgG horseradish peroxidase (HRP) conjugate. The highest dilution of serum giving a positive response in the assay is the titre.

Figure 7 summarizes the gp160 vaccine-induced immune responses of vaccinated seropositive patients.

Figure 8 (A and B) shows vaccine-induced antibody responses against specific HIV envelope epitopes.

Figure 9 depicts the vaccine-induced T cell proliferation response in vaccinated seropositive individuals.

Figure 10 (A-C) depicts vaccination-dependent lymphocyte proliferation response.

Figure 11 shows the percentage change in CD4 cells in responding and non-responding individuals as a function of time.

• ·

Recombinant HIV-1 gp160 envelope protein (rgp160), particularly when adsorbed on an aluminum compound to an adjuvant (e.g., aluminum phosphate), has been found to be particularly useful as an AIDS vaccine. The present invention relates to an AcNPV expression vector comprising a coding sequence for a portion of the HIV envelope gene. Said gene defines amino acids equivalent to sequences 1 to 757 of the sequence of Wain-Hobson cited above, which are amino acids 1 to 752 of recombinant clone 3046. The present invention further relates to the production of the recombinant HIV envelope protein of interest in insect cells (and the protein itself), in particular the rgpl60 protein of 752 amino acid residues (encoded by recombinant clone 3046) as defined by members 1 to 757 of the Wain-Hobson amino acid sequence. .

The invention further relates to the purification and preparation of recombinant envelope protein particles from the gene product of the recombinant baculovirus producing protein 3046 and the adsorption of the particles 3046 onto aluminum phosphate aggregates.

The present invention encompasses vaccines for the prevention or treatment of AIDS or HIV infection and methods for preventing or treating AIDS or HIV infection.

The following examples illustrate the invention, but are not intended to limit the scope thereof.

U.S. Pat. The cloning steps used to construct a recombinant baculovirus containing genes or portions of HIV-1 origin are also incorporated herein by reference.

The genetic engineering steps used to construct the Ac3046 expression vector are described in detail below. Materials used, including enzymes and immunological reagents, were commercially available. Examples illustrating the practicability and applicability of the invention are also provided.

Other recombinant proteins, collectively referred to as rgp160 and containing recombinant gp120 and gp41, are also contemplated. Ac3046 is merely an example of an expression vector and recombinant envelope protein of the invention.

Example 1:

1 -757. construction of Ac3046 recombinant baculovirus encoding HIV-1 sequences.

For cloning and expression of foreign protein coding sequences into baculovirus, it is necessary to attach the polyhedrin promoter and 5 'sequences on one side and the baculovirus coding sequences on the other. The alignment is such that homologous recombination with the baculovirus genome results in the transfer of the foreign coding sequence linked to the polyhedrin promoter and an inactive polyhedrin gene.

Thus, various insertion vectors have been designed for use in HIV envelope constructs. The MGS3 insertion vector described below was designed to replace the translational ATG codon. The insertion of foreign sequences into this vector is performed such that the translation frame generated by the start codon is correctly maintained along the foreign sequences.

The MGS3 insertion vector was constructed from an EcoRI restriction fragment of an isolated and purified AcMNPV plaque (WT-1). The MGS3 vector comprises the following elements: a) a 4000 bp long sequence 5 'to the ATG initiation codon of the polyhedrin gene; b) a site-directed mutagenesis-containing polylinker comprising an ATG initiation codon at the position of the corresponding polyhedrin codon, as well as SmaI, KpnI and BglII restriction sites and a universal stop codon segment; c) a 1700 bp sequence extending from the KpnI restriction site (within the polyhedrin gene) to the last EcoRI restriction site of the EcoRI-Ι clone (see Figure 2).

Example 2:

Construction of recombinant baculoviruses carrying LAV env gene coding sequences.

The recombinant plasmid NA2 (Figure 1) was used, which contains a 21.8 kb segment of a complete HIV-1 provirus inserted into pUC18. The clone was claimed to be contagious because it was able to produce virus when certain human cells were transfected with it. (Adachi et al., J. Virol., 1986, 59, 284-291). The complete envelope gene sequences in NA2 were derived from the HAV LAV strain (Barre-Sinoussi (1983)).

* · «· 4«

- 11 - ...... ... *

The HIV envelope gene was isolated and manipulated as described below (see also Figure 1). The envelope gene was first isolated from NA2 in the form of a 3846 bp EcoRI / SacI restriction fragment and then cloned into the EcoRI / SacI restriction site of pUC19. The resulting plasmid was designated p708.

Subsequently, the envelope gene was isolated as a 2800 bp KpnI fragment and cloned into the KpnI restriction site of pUC18. The resulting clone was designated p1614.

In the p1614 clone, the KpnI restriction fragment contained a slightly truncated fragment of the HIV envelope gene: 121 bp long corresponding to the N-terminal sequence. The missing part of the gene containing the signal peptide sequences was replaced by the insertion of a double-stranded synthetic oligomer.

The incorporated oligomer was constructed based on the amino acid sequence of LAV using the preferred codon structure of the polyhedrin gene. To facilitate further manipulation, a new Smal restriction sequence was introduced in place of the ATG initiation codon. The ATG initiation codon was provided by the baculovirus insertion vector. The resulting plasmid was designated p1774.

Pl774 restriction fragments containing the coding sequences of the various envelope domains of HIV-1 (see Figure 2) were cloned into the MGS insertion vectors (e.g., MGS3) such that the AT initiation codon of the insertion vector was aligned with the codon of the envelope gene. The p3046 construct was obtained by inserting the Smal / BamHI restriction fragment isolated from p774 into the Smal / BglII site of plasmid vector pMGS3. This clone contains sequences coding for amino acids 1 to 757 of gp160 in which the termination codon is provided by the MGS3 vector.

Example 3:

Production and selection of recombinant baculovirus.

Recombinant plasmid p3046 containing the HVV env gene and AcMNPV DNA were co-precipitated with calcium phosphate and added to uninfected Spodoptera frugiperda cells. The chimeric gene was then introduced into the AcMNPV genome by homologous recombination. Recombinant viruses are identified by occlusion-negative plaque morphology. Such plaques have a measurable cytopathic effect, but do not contain inclusion bodies in the nucleus. Two further consecutive plaques were purified to obtain the pure recombinant virus. Analysis of recombinant viral DNA for site-specific insertion of HIV env sequences was performed by comparing their restriction and hybridization properties with wild-type viral DNA.

Example 4:

Expression of the HVV env gene in infected insect cells from recombinant baculoviruses.

Expression of HVV env sequences from recombinant viruses in insect cells results in a primary translation product. This primary product consists of • ·· * · «♦ resulting from the translation of the codons available in the recombinant vector. The result is a protein encoded by all the amino acids in the sequence from the ATG initiation codon of the expression vector 5 'to the polyhedrin promoter to the translation termination signal of the expression vector (e.g., rgp160). The first translation products of Ac3046 end with Met-Pro-Gly-Arg-Val residues, where Arg (position 4) corresponds to Arg at position 2 in the original LAV clone. Met-Pro-Gly codons were provided by the cloning strategy.

Example 5:

Nucleotide sequence of gp160 insert and adjacent DNA.

The nucleotide sequence of the gp160 insert and the adjacent DNA were determined by restriction fragments isolated from the DNA of the Ac3046 viral expression vector. The sequencing strategy included the following steps. The 3.9 kb EcoRV-BamHI fragment was purified by restriction digestion of Ac3046 viral DNA. Ac3046 viral DNA was isolated from an extracellular virus present in a cell culture used for a vaccine batch.

Figure 2 shows that the 3.9 kb ExoRV-BamHI fragment consists of the entire gp160 gene and 100 'bp in the 3' direction and approximately 1000 bp in the 5 'direction. From this, the nucleotide sequence of the entire gp160 gene was determined, including the 3 'upstream 100 bp long and the 5' up 100 bp long flanking DNA sequences.

Briefly, sequencing revealed a chimeric structure that was predictable from the cloning strategy. The sequence of gp160 was substantially identical to that of Wain-Hobson (1985). A sequence of 2256 bases between the putative translation start and stop codons is expected to have 752 amino acids and 28 potential N-glycosylation sites. This rgp160 glycopeptide has an estimated molecular weight, including sugar moieties, of about 145,000.

Sequence analysis of the 200 bases of the flanking DNA showed that the insertion was correct, as shown in Figures 3, 4, 4. and Figure 5.

Example 6:

The 0 amino acid sequence of gp160.

The first 15 residues of the gp160 N-terminal sequence were determined by standard automated Edman degradation and HPLC and these were predicted residues from the DNA sequence. There is no N-terminal methionine in the gp160 protein. This is consistent with the observation that the AcNPV polyhedrin protein is also produced in the absence of N-terminal methionine. Table 1 summarizes the actual DNA and N-terminal protein sequences based on analysis of the AcNPV 3046 DNA sequence and purified gp160.

Table 1

LAV env gene in the AcNPV 3046 expression vector.

Group

2 3 4 5 6 7 8 9 10 11 12 13 14 Pro Gly Arg With Lys Glu Lys Tyr Gin His Leu Trp Arg Trp Gly ATG CCC GGG CTG GTG AAG GAG AAG TAC CAA CAC CTG TGG CGT TGG GGC

J · © · ·· »f

··· ···· «« tfg ·. * ·· '<

• · ··

These results are similar to the original LAV-1 clone (see Table 2).

Table 2

LAV env gene in the original LAV-1 clone.

Group

1 2 3 4 5 6 7 8 9 10 11 12 13 14 Met Arg With Lys Glu Lys Tyr Gin His Leu Trp Arg £ tr Gly ATG AGA GTG AAG GAG AAG TAT CAG CAC TTG TGG AGA TGG GGG

Example 7:

Purification of recombinant gp160

The present invention relates to a process of the invention

Used to extract and purify recombinant HIV-1 envelope protein encoded by the Ac3046 expression vector. The gp160 recombinant HIV-1 envelope protein is produced in S. frugiperda cells infected with the Ac3046 vector. Cells were cultured for 4-5 days after infection. Purification of the rgpl60 protein involves the following steps:

1. Washing of cells

2. Cell lysis

3. Gel filtration chromatography

4. Lens-lectin affinity chromatography

5. Dialysis.

In this example, purification of recombinant gp160 from approximately 2 x 10 4 Ac3046-infected cells is described.

1. Cell washing:

Infected cells were treated with 50 mM Tris pH 7.5, ···: · -.

···. *. . · ·· ·· ···· *

Wash in buffer containing mM EDTA and 1% Triton Χ-100.

Cells were resuspended in this buffer, homogenized by standard methods, and centrifuged at 5000 rpm for 20 minutes. The procedure is repeated three times.

2. Cell lysis:

The washed cells are lysed by sonication in 50 mM Tris pH 8.0-8.5, 4% deoxycholate and 1% beta-mercaptoethanol. Ultrasonic treatment is performed by standard methods. After treatment, only the nuclear membrane residues are intact and are removed by centrifugation at 5000 rpm for 30 minutes. The supernatant containing the extracted gp160 lacks intact cells by light microscopy.

3. Gel filtration:

Gel filtration was performed on a 5.0 x 50 cm glass column packed with Sephacryl resin (Pharmacia) from Pharmacia. The total bed volume is approximately 1750 ml. At least 6 liters of 0.1 M sodium hydroxide solution was flushed through the column for 24 hours to purify the column and tube connections. The column flow fluid is connected to a UV cell, monitor, and chart recorder (Pharmacia). The column was then equilibrated with 4 L of gel filtration buffer. The crude gp160 was loaded onto the column and eluted with the buffer used for gel filtration.

The column separates the crude mixture into three main UV absorbing fractions. The first peak falls between about 500 and 700 mL of buffer, the second peak between 700 and 1400 mL and the third

Between 1400 and 1900 ml. The same image was obtained on small analytical columns, whereby the molecular weight of the first peak material was determined, which proved to be £ 2,000,000.

This peak is translucent due to the concentration of high molecular weight lipids and lipid complexes and also 10-20% of the gp160 extracted from the infected cells. This fraction of gp160 is likely to complex with itself and other cellular components to form high molecular weight aggregates.

The second, broad peak contains most of the gp160 and proteins of molecular weight between 18,000 and 200,000.

The third peak contains little protein, most of the UV absorption is from the beta-mercaptoethanol content of the sample.

When the appearance of the second peak is first observed from the UV absorption pattern, the effluent from the column is directly applied to the lens-lectin column. As soon as the second peak comes off the column, the eluate is disconnected from the lens-lectin column and allowed to drain.

4. Lens-Lectin Affinity Chromatography:

Lentil Lectin Affinity Gel (Lentil Lectin - Sepharose 4B) was purchased in bulk from Pharmacia. The lens lectin was purified by affinity chromatography on Sephadex to obtain a purity greater than 98% and then immobilized by binding to Sepharose 4B using bromine. The matrix contained about 2 mg of ligand per ml of gel. The lens-lectin column was a 5.0 x 30 cm glass column (Pharmacia) containing 125 ml of lens-lectin-Sepharose 4B gel. The affinity matrix can be reused after thorough washing and the manufacturer's recommended regeneration procedure. When not in use, the gel should be stored in the column in the following composition: 0.9% sodium chloride, 1 mM manganese chloride, 1 mM calcium chloride, and 0.01% thiomersal. Prior to use, the column was washed and equilibrated with 250 ml of the lens-lectin buffer described above.

Crude gp160 eluted from the gel filtration column was directly applied to the column as described above. As soon as the crude gp160 bound to the column, the column was washed with 800 ml of 0.1% deoxycholate in lens-lectin buffer. Under these conditions, the total amount of gp160 is bound to the column. Bound glycoproteins are eluted with lens-lectin buffer and 0.3 M alpha-methyl-mannoside. The elution is monitored at 280 nm on a UV monitor.

5. Dialysis:

The sugars and deoxycholates are removed by conventional dialysis.

The purification steps of gp160 from 1 liter of infected cells are shown in Table 3.

Table 3

Cleaning (summary).

Cleaning step Total Protein (mg) ¹ gp160 protein (mg) Sum- -gp16 0 (%) Removed impurities Cell sediment 1-2000 20 1-2 broth 1. 2nd, 3rd wash 250 15 6 Serum albumin, most nucleic acids and soluble cellular proteins Gel filtration 120 12 12 Lipids, nucleic acids, high molecular weight aggregates Lentil lectin 14 10 70 Non-glycolysed proteins Dialysis 13 9 70 Excess sugar, deoxycholate, tris buffer

Total protein was determined from absorbance at 280 nm.

In another embodiment of the invention, conventional ion exchange chromatography (anion or cation exchange) may be used instead of gel filtration. The order of the steps is also not critical: for example, gel filtration or ion-exchange chromatography can follow the lens-lectin purification step. Other reagents may be used according to the invention. For example, other detergents may be used instead of deoxycholate to purify the recombinant protein.

• ·

Examples include nonionic detergents such as Tween 20 (polysorbate 20), Tween 80, Lubrol and Triton X-100.

Example 8:

A. Assembly of the gp160 particles.

It has been found that the gp160 antigen can be assembled into particles of molecular weight> 2,000,000 during purification. This is considered to be an element of the invention. 80-90% of the protein extracted from the cells is monomeric (molecular weight 160,000) and 10-20% is polymeric (particles). Gel filtration removes aggregated forms of gp160. Purification of gp160 from this fraction (first peak from the gel filtration column) was attempted and it was concluded that gp160 complexed with other cellular proteins, possibly membrane fragments. However, in the second peak coming from the gel filtration column, the gp160 antigen has a molecular weight of 160,000 to 300,000, i.e., the majority is monomeric or dimeric.

The gp160 aggregates or polymers are formed during the development of the lens-lectin column. It was found that the antigen forms aggregates with up to 0.5% deoxycholate - which at deoxycholate corresponds to about 0.2% critical micelle concentration (CMC) - from the lectin column or up to 0%. , Dissolve gp160 in the column with 1% deoxycholate.

The size of the aggregates was determined on a high-resolution FPLC Superose 12 column (Pharmacia). Aggregate sizes in representative batch samples of purified gp160

They have a molecular weight of 2,000,000 or more, compared to a blue dextran standard.

A study on cross-linked structure (Schwaller et al., 1989) showed that gp160 produced in insect cells is a tetramer consisting of identical subunits. The study also showed that gpl60 is a tetramer in HIV-infected cells and virus particles. Thus, the recombinant gp160 particles may have a tertiary and quaternary structure similar to that found in native HIV gp160.

An appropriate three-dimensional structure may be important for epitope formation and requires proper folding of gp160. Since the non-glycosylated proteins are removed from the gp160 antigen by binding to the lens-lectin column and washing the column, the hydrophobic moieties of gp160 are expected to initiate intermolecular linkages. Deoxycholate is unlikely to bind to gp160 as its concentration can be maintained above CMC and the antigen will still form complexes. It appears that the assembly of this antigen into aggregates, once purified according to the invention, becomes an intrinsic property of this protein. It is possible that the highly hydrophobic N-terminal sequence present in the gp160 protein contributes to the natural property of this protein to form particles. After purification, the gp160 complexes can be sterile filtered on a 0.2 micron cellulose acetate filter without significant loss of protein.

B. Analysis of Particle Formation:

Electron microscopic analysis22 of the purified gp160 particles did not show that they were proteinaceous spherical particles of 30-100 nm.

Purified gp160 was subjected to gel filtration as an additional test for the presence of particles. Approximately 100 μg of gp160 was applied to a Superose 12 FPLC HR 10/30 gel filtration column (Pharmacia, Inc.). The column was previously calibrated with protein molecular weight standards. The protein profile of this column is highly reproducible; the elution volume is inversely proportional to the molecular weight of the protein standards. The column separates the monomeric gp160 from the polymer form and excludes globular proteins of molecular weight> 2 >10θ. When the column is developed, substantially all of the purified gp160 elutes in the effluent volume, i.e., has a molecular weight greater than 2 million (> 2x10 ⁶ ).

Example 9:

A. Adsorption of gp160 on aluminum phosphate gel.

The efficacy of insoluble aluminum compounds as immunological adjuvants depends on the extent to which the antigen is completely adsorbed on the solid phase. Part of the invention is the discovery that aluminum compositions can be prepared that effectively adsorb gp160a at a pH η that does not reduce the immunogenic activity of the gp160-aluminum compound complex. During the preparation of this aluminum composition (aluminum phosphate gel), the following factors are controlled:

1.) For adsorption of antigens to aluminum phosphate ··· * 4 44 4 • ♦ «4 44 • · 4 4 4 * 4 · 4

- 23 - '··' · '* ··· * ♦ ·' ··· requires an optimum pH of about 5.0. However, it has been observed that gp160 loses its immunogenicity at pH 6.5 compared to pH 7.5, i.e. the pH of aluminum phosphate.

Adjusted to 7.1 ± 0.1. It was found that gp160 is essentially 100% adsorbed to aluminum phosphate at this pH.

2.) The ionic strength from the sodium chloride content is relatively low, less than 0.15 M.

3.) There is a molar excess of aluminum chloride in the sodium

- relative to phosphate to avoid free phosphate ions remaining in the supernatant.

4.) gp160 is added to freshly formed aluminum phosphate to prevent crystal growth and to minimize particle size.

A method of making 200 ml of aluminum phosphate by adsorbing gp160 to a final concentration of 40 µg / ml of antigen is described below.

B. Preparation of Reagents (for 200 ml of formulated preparation):

The following solutions are prepared in 100 ml sterile, pyrogen-free bottles or beakers. The salts are weighed into solutions 1 and 2 and sodium hydroxide into solution 3, and the solutions are filtered through a 0.2 g porous cellulose acetate membrane into 100 ml sterile, pyrogen-free bottles.

• ·

Solution 1 Aluminum chloride-water (1/6)

0.895 g.

Sodium acetate-water (1/3)

0.136 g.

Reconstitution with 40 ml water for injections.

0.2 μιη filtration.

Solution 2 Trisodium phosphate-water (1/12)

1.234 g.

Reconstitution with 40 ml water for injections.

0.2 μιη filtration.

Solution 3 Sodium hydroxide

2.0 g.

Reconstitution with 100 ml water for injections.

0.2 μιη filtration.

Solution 4 Tris

1.25 g.

Dissolution in 100 ml water for injections, of which 1 ml is added to 90 ml water for injections and adjusted to pH 7,5

0.5 M hydrochloric acid and make up to 100 ml with water for injection.

The solutions were autoclaved for 30 minutes, slowly draining, cooling to room temperature.

C. Preparation of Aluminum Phosphate Gel

1.) Solution 1 (Aluminum Chloride + Sodium Acetate) is weighed into the forming vessel using 25 ml sterile disposable pipettes. Record the volume of solution 1 and begin stirring.

2.) Weigh the solution 2 (sodium phosphate) into the vessel ♦♦ ·· ···· 4 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Using sterile, disposable pipettes, continue mixing until a precipitate is formed and record the volume of solution 2 · · · · · · · · · · · · · · · · · · · · · · · · · · ·

3.) 3 ml of solution 3 (sodium hydroxide) is added to the vessel and stirring is continued for 5 minutes. A 0.5 ml sample is taken for pH measurement. If the pH is less than 7.0, 0.5 ml of sodium hydroxide is added again, stirring is continued for another 5 minutes, and the pH is again measured. The alkaline addition is continued until the pH is between 7.0 and 7.2.

4.) Determine the total volume (solution 1 + solution 2 + solution 3) and make up to 100 ml with sterile water for injection.

5.) Immediately afterwards, 8.000 Mg of purified gp160 in 100 ml of 1 mM Tris pH 7.5 is added directly to the forming vessel.

6.) Stirring is continued for at least 20 minutes and the vaccine is dispensed into sterile vials.

Example 10:

Immunogenicity of gp160 adsorbed on aluminum phosphate (specific antibody response).

An accepted method for determining the immunogenicity of an antigenic preparation (vaccine) is by measuring the specific antibody response in groups of mice receiving a single dose of antigen. At the end of the fourth week, the mice are bled and serum antibody levels to a specific antigen (usually the antigen used to immunize the animal) are measured by a standard antibody assay. For example, by ELISA (enzyme linked immunosorbent assay).

Table 4 summarizes the results obtained for the immunogenicity of purified gp160 in mice. Purified gp160 vaccine was prepared without adjuvant adsorbed at pH 6.0 and pH 7.5 on aluminum phosphate (formulation described in Example 9) or mixed with complete Freund's adjuvant.

Table 4

Group gp160 number ELISA mean OD ¹ Serum conversion gp! 60 Give me uvant % (P / N) ² 1 pg without pH 7.5 8702 0,140 57 4/6 without pH 6.0 8702 0,110 26 2/7 alum.foszf. 8702 1,000 90 9/10 alum.foszf. 8705 2.285 100 6/6 Freund 8604 1,108 83 5/6 Freund 8702 1,396 100 7/7 0.1 pg Freund 8604 0,434 67 4/6 alum.foszf. 8705 1,003 67 4/6 ^ The mice are vaccinated after 28th on the sun bled

and sera were tested at 1:10 dilution against gel-purified gp160 by ELISA. Similar results we got in a trade! ELISA (Genetic Systems Inc., EIA ^tm ELISA) against native HIV-1 proteins at 1: 400 serum dilution.

»·« · ··· * ·················································· ·

- 27 - ....... '· ··· ² Number of mice showing serum conversion from the number of mice tested (N) (P).

Mice immunized with a single 1.0 μg dose of gp160 without adjuvant produced an antibody response to gp160 (see table above). However, a much stronger antibody response is seen in the group of mice immunized with 1.0 µg of gp160 adsorbed on aluminum phosphate. More than 50% of mice immunized with 0.1 M9 of gp160 mixed with complete Freund's adjuvant or aluminum phosphate formulated antibody. The gp160 antigen was found to be immunogenic in mice, although in unformulated form, although at lower pHs of pH 7.5 and pH 6.0, but lower pH η showed reduced immunogenicity.

Example 11:

Immunogenicity of gp160 adsorbed on aluminum phosphate (serum assay by ELISA).

A very important biological property of a vaccine formulation is the ability to elicit an immune response. This is evidence that the aluminum phosphate-formulated gp160 vaccine is immunogenic in animals. To confirm that aluminum phosphate as an adjuvant enhances immunogenicity, the following experiment was performed.

On day zero, mice (10 mice per group) were inoculated with gp160 mixed with gp160 antigen or gp160 adsorbed on aluminum phosphate or complete Freund's adjuvant. Immunization was carried out with a single vaccine (0.5 μg, 1.0 μg, 5.0 μg). On day 28, the mice were bled and the sera were bled (1:10 ·······

- 28 ~ ** ** ···· ······ was tested by ELISA for the presence of antibodies to gp160.

The results of the sera taken on day 28 are summarized in Table 5. In each group, more than 50% of the mice showed serum conversion. At each dose, the number of serum conversions and the mean of serum absorption values (OD45Q _{nm at} 1:10 dilution by ELISA) were higher in mice vaccinated with gp160 adsorbed to aluminum phosphate than in mice immunized with gp160 only.

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

Table 5 (Day 28 after vaccination)

Dose

0.5 Mg 1.0 μς 5.0 M9 Mean Mean Mean

P / N ¹ OD ² P / N OD P / N OD gpl60 9/10 .407 7/10 0.699 7/10 0.430 gpl60 (Alum) * 9/10 0.547 8/10 0,797 10/10 1.347 gpl60 (FA) ** 10/10 1,130 10/10 1,967 10/10 1,317

* Aluminum Arch * Complete Freund's Adjuvant ···

- 29 µg seroconversion mice, number (P) versus total number of mice tested (N), 0.5 µg, 1 µς and 5 µg VaxSyn ^tm HIV-1 vaccine on day 28.

² The mean absorbance (OD ₄₅₀ ) of the serum conversion mice serum ELISA (1:10 dilution serum against gp160).

Example 12:

Neutralization studies.

The HIV-1 neutralization assay is an accepted method for determining the ability of an antibody formulation to inhibit HIV-1 virus to infect cultured sensitive human lymphocyte cells. Antisera from animals immunized with gp160 were tested in an HIV-1 neutralization assay and the results are summarized in Table 6.

Table 6

neutralization

Animal Identification Immunogen / adjuvant Mikrogramm ^ · titer ² rhesus G55 gplőO / alum.foszf. 16/8/8 1:80 - 1: 160 rhesus H55 gpl60 / alum.foszf. 16/8/8 1:80 - 1: 160 rhesus L55 gpl60 / alum.foszf. 16/8/8 1:80 Mouse half 3 gpl20 / Freund 0.25 / 0.25 / 0.25 1:40 - 1:80 Mouse 8pm gpl20 / Freund 0.1 / 0.1 / 0.1 1:40 - 1:80 tgm tiszt.IgG gpl60 / Freund 10/10/10 1: 320

The amount of gp160 or gp120 administered in the first / second / third immunization vaccine, in μg.

² The highest dilution of antiserum capable of inhibiting HIV-1 infection by 50% compared to serum-treated cells from non-immunized animals.

Guinea pigs, rabbits and Rhesus monkeys were also immunized with gp160 (using aluminum phosphate or Freund's adjuvant as adjuvant). Immunization of these animals generally elicited a good antibody response against HIV-1 envelope proteins.

Example 13:

Immunogenicity in chimpanzees.

Chimpanzees are genetically the closest relatives of humans and are currently the only animal model for HIV-1 infection. A safety and immunogenicity study was performed on three chimpanzees. Two chimpanzees were immunized with 40 μg or 80 μg of gp160 in the form of a vaccine formulated with aluminum phosphate. At the fourth week, each animal received 40 µg and 80 µg of gp160 as a booster dose, respectively. A control animal was, however, vaccinated with 1 ml of saline. Weekly serum samples from the three chimpanzees were tested for antibodies against gp160 and HIV-1 viral antigens. Three types of immunoassays were performed: ELISA against purified gp160 (developed by MicroGeneSys, Inc.), Western bioassay and a commercially available HIV-1 ELISA. The results of these assays are described below.

···· · * 4 ^ • · ·· · * · «• · 4» • · «* ·« 4 »* · _ 31 - * · · * ··· ♦ ·· · · ·

A. ELISA (MGSearch NEWS 160):

MGSearch NEWS 160 (MGSearch is a trademark of MicroGeneSys, Inc. (Meriden, Connecticut, USA)) ELISA assay for an immunosorbent assay for gp160, which is described in U.S. Patent Application No. 920,197 (currently No. 585,266).

Serum samples taken prior to immunization and 11 weeks after the first immunization were diluted 1: 10 to 1: 100,000 and then incubated with nitrocellulose strips containing 100 purified gp160 per spot. The limit dilution titre is the highest dilution where the assay is positive for anti-gp160 by detection with a goat anti-human IgG alkaline phosphatase conjugate.

Serum samples from control animals and before immunization were negative. The chimpanzee who received 80 µg at a 1: 100 dilution was positive after 2 weeks and the chimpanzee who received 40 µg at a 1:10 dilution became positive at 4 weeks. Antibodies to gp160 increased by week 5, at which time dilution titers of about 1: 100,000 and 1: 2,000,000 were obtained. The antibody titer in both animals was slightly reduced to levels 6-11. during the week.

This type of response is similar in quantitative and qualitative terms to the antibody responses generally observed in chimpanzees vaccinated with the human hepatitis B virus vaccine.

B. Commercial EKISA test:

With the VaxSyn Vaccine (VaxSyn is MicroGeneSys, Inc., described here. • · ··· «

- 32 - * '.....

MGSearch's HIV 160 ELISA and Western biot analysis revealed that the sera had been converted and contained antibodies to recombinant gp160. viral envelope proteins are also recognized, pre-immunization sera, and serum samples from 1 to 11 weeks are assayed with the registered commercial ELISA kit and the LAV EIA ™ test kit (Genetic System Co. Seattle, Washington, USA). The serum of the animal immunized with 80 Mg gp160 was positive at a 1: 100 dilution in the second week and increased the antibody titer by 6 weeks. The serum of the animal immunized with 40 μς gp160 at 1: 100 dilution became positive at week 6.

Example 14:

Distribution of antibodies between gp120 and gp41.

It is important to determine whether the antibody responses to gp160 in a vaccinated animal are directed against gp41 or gp120, or both. Various immunoassays - radio-immunoprecipitation (RIP), immunofluorescence (IF), Western biotest (WB), and quantitative ELISA - were used to detect and measure against three different recombinant envelope antigens against different HIV-1 envelope proteins. distribution of antibodies.

Figure 6 depicts the immunoreactivity of three different recombinant antigens. These are: (1) the gpl20 delta (truncated recombinant HIV-1 gp120; around the C-terminus of the molecule33) · · · · · · · · · · · · · · · · · · · 4 ···· • • 44 »··· · * ······ ····· within 40 amino acids); (2) gp120 (full length recombinant HIV-1 gp120); (3) gp160.

Fifty HIV-1 antibody positive human sera and 3 mixed human sera were highly reactive with gp160, slightly reactive with gp120, and few or no antibodies reacted with truncated gp120. Truncated gp120, which represents more than 90% of the HIV-1 outer glycoprotein, is likely to contain protective determinants. The observation that human AIDS-positive sera contain little antibodies to this envelope is consistent with the fact that the immune response to the viral infection is not completely protective and that positive human sera exhibit low neutralizing activity in vitro.

In contrast, serum from Rhesus monkeys immunized with both gp160 immunogen and truncated gp120 contained antibodies that reacted strongly with the truncated gp120 portion of the HIV-1 envelope. The differences in the distribution of antibody recognition sites along the viral envelope and the higher titers observed in monkeys make it clear that monkey serum neutralization titers are high.

Quantification of the immunoreactivity of these three recombinant envelope antigens in human and immunized Rhesus sera is shown in Figure 7. All monkey sera tested had high antibody titers against truncated gp120 antigen (gp120 delta), as did sera derived from an animal immunized with gp160.

These results indicate that the recombinant gp160 ♦ ·· ♦ ··· <* · ·· 9 ··· · · · · · · · · · · · · · · · · · · · «·« «· Elicits an antibody response in Rhesus monkeys that is different from that most commonly encountered during natural infection. In the gp160 delta region of gp160, there are epitopes that are effectively recognized by the serum of immunized monkeys, but not recognized by the human immune system during infection. These novel epitopes may be important for protection against HIV-1 and may be an important property of recombinant gp160 in preventing and treating HIV infection.

Example 15:

Therapeutic use of the vaccine.

A clinical trial was conducted in 30 HIV seropositive human patients to determine the efficacy of vaccination with cloned HIV gpl60 (produced in the baculovirus system described above) in HIV infected individuals.

Vaccination with recombinant gp160 resulted in an increase in the gp160-specific humoral and cellular immune response in 19 (63%) of the 30 HVV seropositive volunteers. Fourteen of the fifteen volunteers (93%) who received 6 doses of the vaccine had elevated total gp160 antibody levels. Thus, recombinant HIV proteins (e.g., rgp41, rgp120, rgp160, and mixtures thereof) can be successfully used in methods of treating HIV infected patients.

The effective amount of the HIV protein used in the embodiment of the invention may be determined using techniques well known in the art, as described below.

· «# ··« ··· · * · «···· ·····························································································································• «··

Generally, an effective amount will be from 1 to 100 µg / kg body weight per patient. The frequency of administration is also determined in a known manner. In a preferred embodiment, the administration is by parenteral route, for example, intravenously, intraperitoneally, intramuscularly, intradermally, and the like. in a manner well known to those skilled in the art.

A. Selection of volunteers:

Thirty volunteering HIV infected patients were recruited.

Only seropositive volunteers who were at an early stage of HIV infection with Walter Reed Grade 1 or 2 (no CD4 cell count of less than 400 with or without lymphadenopathy) were included [Redfield et al. (New Engl. J. Med. 314: 131-132 (1986)). The inclusion criteria for volunteers were selected from adults 18-50 years of age with normal blood cell counts, no end-stage disease, no alcoholics or drug abusers in the previous 12 months, and no antiretroviral medication or immunomodulators. All patients were subjected to a 2-month baseline evaluation before being randomly assigned to the treatment groups.

Of the 30 volunteers, 26 were male and 4 female. Of these, 14 were Caucasian, 13 were Black and 3 were Hispanic. The mean age of men was 29 years (18-49). At the time of enrollment, 8 volunteers belonged to Walter Reed Grade 1 and 22 volunteers to Walter Reed Grade 2. The mean CD4 count was 668 (388-1639). The median time from initial diagnosis to enrollment was 24 months (3-49 months).

···· ···· ·· • · · • ·· "·· ♦ • * * * · · · · · · · ·· ·· ·· ····

Β. Vaccine formulation and immunization scheme:

The vaccine described herein contained a non-infectious glycoprotein subunit, i.e., the gp160 protein expressed in baculovirus. The immunogenic protein was produced in Lepidopteran insect cells, purified by biochemical methods, and adsorbed on aluminum phosphate at the time of formulation of the final vaccine.

Three doses of gp16 were formulated: 40 µg / ml, 160 µg / ml and 320 µg / ml. The injection volume was 1 ml for the 40 and 160 μς doses and 640 µg for the 320 gg dose in 2 ml.

The 30 volunteers were divided into 6 groups, each consisting of 5 volunteers. Two immunization schedules were examined. Scheme A: Vaccination on days 0, 30 and 120 and Scheme B: Vaccination on days 0, 30, 60, 120, 150 and 180. Both immunization schedules (A and B) had three groups, each receiving different doses of vaccine (see Table 7). Vaccination was administered by intramuscular injection into the deltoid muscle. The duration of the study was 10 months, with a baseline evaluation for 2 months and a continuous evaluation for 8 months after the first vaccination.

Table 7

Immunization scheme

Amount of gp160 injected (μς)

Nap 0 30 60 120 150 180

The scheme

First group 40 40 40 Third group 160 160 160 5th group 640 640 640 B schema Second group 40 40 40 160 160 160 4th group 160 160 160 640 640 640 6th group 640 640 640 640 640 640

C. Harmlessness and toxicity testing:

On day 0, 1, 2, 3, 15 and 30 post-injection, each volunteer was interviewed and examined. The volunteers were asked about fever, chills, nausea, vomiting, arthralgia, myalgia, malaise, rash (urticaria), asthma breathing, dizziness or headache. When testing for local reactions at the injection site, we examined the following: erythema (pink rash), swelling, itching, pain and tenderness, skin discoloration, cracked skin, regional lymphadenopathy change in vaccinated limb function and subcutaneous nodule formation.

- 38 - .............

site. We performed monthly, whole blood count, done. serum, chemistry, coagulation, and urine.

In vitro cellular immune functions were assayed by T cell phenotyping (oste lymphocyte, CD4 and CD8 cell phenotypes) as described by Rickman et al., Clinical Immunol., 52, 85-95 (1989) and Birx et al., J.Acquir.Immune. Defic-Syndr. 4: 188-196 (1991). The Τεεί t proliferative response to mitogens (pokeweed and ConA) and control antigens (Candida albicans and tetanus) was also evaluated (Birx et al., Supra). In vivo immune function was tested by a delayed hypersensitive wine test against control antigens (e.g., mumps, tetanus toxoid, Candida albicans and trychophyton).

Quantitative viral cultures of peripheral blood mononuclear cells (PBMC) and plasma were performed as described by Bürke et al., J. Acquir. Immune Defic.Syndr., J 1159-1167 (1991). DNA polymerase chain reaction was performed [Wages et al. , J.Med. Virol. , 33, 58-63 (1991)] and serum p24 antigen levels were determined to monitor viral load in vivo.

No evidence of general toxicity was observed, but local reactivity was observed in 87% of patients (13 in each vaccine group). The local reactions were: hardening, tenderness, transient subcutaneous ballooning at the injection site; increased lymph node abnormalities were rarely observed. No patient refused a booster dose. There was no difference in the frequency of local reactions with • · ·

- between the first 39 immunizations, booster doses, or doses.

No adverse effects on the immune system were detected. This was measured with mitogen and antigen-specific proliferative responses in vitro and in vivo delayed hypersensitivity skin test responses or quantitative CD4 cell depletion acceleration.

The mean baseline CD4 cell count was 716 for vaccine responders and 605 for non-responders. During the 180-240 days of the study, the mean CD4 cells were 714 and 561. During the 240-day study, the net change in CD4 cell count was minus 0.2%, while the mean CD4 cell count among non-vaccine non-responders was 7.3% (Figure 11). Vaccine-induced HIV immunogenicity was not accompanied by a sign of faster CD4 reduction in any patient throughout the study.

To examine the possibility of increased HIV replication and viral load in patients as a result of vaccination, viral activity was measured in vivo by quantitative plasma and PBMC viral culture, PBMC polymerase chain reaction, and serum levels were measured. Measurements by quantitative cultures and DNA polymerase chain reaction showed no change in this assay. No p24 antigen could be detected in the patients' serum.

D. Immunogenicity test:

Antibodies to total HIV proteins were measured using recombinant viral gene products gp60, p66, p24 and the complete viral lysate of the prototype HIV MN strain. Dot biotech and Western biotech techniques as described by Towbin, et al., Proc-Natl.Acad, Sci., 7, 6, 4350-4354 (1979). Antibody responses to specific envelope epitopes were also measured (see Figure 7).

Figure 7 shows the amino acids 88 (amino acids 88-98 in gp120) and

448C (amino acids 448-514 in gp120) epitopes were selected because these regions of gp120 were reported to be involved in the early stage of HIV infection.

The 106 (in gp120, 106-121).

amino acids)

At 241 (241-272.

amino acids), amino acids) 300 (300-340.

amino acids), a

308th {308-322.

amino acids) at 422 (422-454).

amino acids) and 735 (amino acids 735-752) were chosen because of their likely functional importance. Epitopes 106 and 422 play a role in CD4 binding; epitopes 241, 254 and 735 play a role in group-specific neutralization; and epitopes 300 and 308 in type-specific neutralization.

The epitope 582 (amino acids 582-602) was chosen as a control because it represents the immunologically dominant envelope domain in natural HVV infections. The epitope 49 (amino acids 49-128) and 342 (amino acids 342-405) were also examined.

In Figure 7, the shaded boxes represent a documented change in the immune response to the Call envelope. The shaded cubes (-) represent a primary humoral response; shaded cubes with a (+) sign represent a secondary humoral response; the (-) signals indicate a specific epitope negative antibody response before and after immunization; (+) marks the specific epitope-positive antibody response before and after immunization with no quantitative change. The shaded boxes (.) Represent a novel T-cell proliferative response to gp160 following immunization. The (.) Sign only indicates that there is no cellular response to gp160; hb (high background) meaning: unintelligible; nd (female done) meaning: not tested.

Neutralization activity was measured against three isolated prototypes (HIV-IIIB), RF and MN by a syncope inhibition assay as described by Nara (Natura, 333: 469-470 (1988)). Call specific cellular responses were measured using known lymphocyte proliferation assays using gp160, p24, and a baculoviral expression system control protein (Birx, supra).

E. Vaccine responders and non-responders:

Patients were classified as vaccine responders only if there was a reproducible, selective increase in both cellular and humoral immune responses against HIV envelope-specific epitopes (Figure 7). Vaccine-induced humoral immunity was defined as seroconversion to HIV-envelope-specific epitopes and / or as a secondary memory immune response to envelope-specific epitopes. Vaccine-induced cellular immunity is defined as a novel, reproducible, proliferative response against gp160 associated with vaccination. (This definition of vaccine responders is a very restrictive definition for the scientific purpose of this series of tests: for example, to determine the applicability of post-infection immunization.) Two non-responders who did not give either a humoral or cellular proliferative response, or only a humoral or cellular proliferative response to the gp160 epitopes or HVV envelope, were non-responders. group.

F. Vaccine-Induced Humoral Responses:

Referring to Figure 7, 19 of the 30 patients (63%) showed vaccine-induced increase in the gp160 HIV-specific humoral and cellular immune response. The 11 non-responders developed either a humoral or only a cellular immune response. The seven patients who did not show any detectable vaccine-induced response received only 3 doses (Scheme A). No change in antibody binding to HVV polymerase (p66) or structural (p24) gene product or to non-HVV control tetanus antigen was observed. No patient developed antibodies to the baculovirus Lepidopteran cell control protein.

Thirteen patients showed envelope antibody (gp160) growth by Western blot analysis using complete HIV-MN virus lysate. The changes were related to the immunization schedule: Three out of 15 patients (20%) in Scheme A and 10 out of 15 patients (67%) in Scheme B. Scheme B induced an increase in antibody levels to the envelope proteins (P = 0.025, Fisher's exact test, two-sided). 13 patients also had seroconversion to specific envelope epitopes.

In contrast, in the 10 patients without seroconversion, no envelope-specific epitope was elevated in envelope antibody levels by Western biot analysis. The remaining 7 patients who had seroconversion to the specific envelope epilope ···· ···· · · · · · · · · · · · · · · · · · · · · No changes were observed in antibody levels to non-envelope Hiv proteins in any patient.

In Scheme B, 14 (93%) of the 15 patients had elevated total gp160 antibody levels, whereas in Scheme A (3 doses), only 7 of the 15 patients had increased (P = 0.01 Fisher, bilateral) (7 . figure).

Figure 8 shows the post-vaccination variation in pre-immunization levels against specific gp160 specific epitopes: epitope 49 (from 27 to 70%), epitope 88 (from 28 to 52%), epitope 106 (From 50 to 87%), epitope 214 (from 0 to 14%), epitope 254 (from 0 to 13%), epitope 300 (from 47 to 77%) ), Epitope 308 (from 42 to 69%), epitope 342 (from 0 to 27%), epitope 422 (from 3 to 10%), epitope 448C (from 73 87%) and epitope 735 (from 17 to 33%). Seroconversion was detected against each specific epitope except epitope 582 (Figure 7). Antibodies (seroconversion) to epitopes 241, 254 or 342 were detected only after vaccination.

Secondary immune responses were observed at the following epitopes: 88, 106, 300, 448C and 582. Antibody levels of epitope 582 were 100% pre-vaccine and only one patient (3%) had a secondary immune response.

The HIV antibody against vaccine-induced envelope epitopes showed a diverse pattern (Figure 7). Primary antibody response (seroconversion) to at least one epitope occurred in 20 patients; in 14 of the 15 patients who were vaccinated according to Schedule B and 6 of 15 patients who were vaccinated according to Schedule A (P = 0.005 according to Fisher, two) 44 · · ······························································ · · ·· · * ···· · · ··· page). Patients in Scheme A responded to seroconversion to only 15 of the 110 potential epitopes without pre-immunization antibodies. Of the patients vaccinated according to Scheme B, 60 responded by seroconversion (47%; P <0.0001 by Fisher, bilateral). 9 (60%) of patients in Scheme B responded to three or more envelope epitopes by seroconversion, while only 2 patients randomized to Scheme A (13%; P = 0.02 Fisher, bilateral) ·

On days 0, 90 and 195, serum neutralization activity was detected in 7 patients against three different strains (HIV-IIIB, MN and RF). Of the 5 patients who responded to the vaccine, 4 showed increasing neutralization activity against one or more cultured material. Vaccine responders showed better inhibition of syncope formation compared to non-responders.

G. Vaccine-Induced Cellular Responses:

Changes in cellular immune response were based on a comparison of pre-vaccination foot) and post-vaccination lymphocyte stimulation index (LSI) using the Wilcoxon rank sum test.

In 21 of the 30 patients (70%), a new T-cell proliferative response was initiated after immunization with gp160 (Figure 7).

Figure 9 depicts the time course of proliferative responses to gpl6, p24 and a baculovirus control protein in four typical vaccine responders. In all patients, gpl60-induced proliferation from a baseline mean of 3 LSI to 10 LSI ·················································································································· ~ ** ·· ···· ····· (calculated as an average of 4 values since the last immunization). In contrast, there was no change in the proliferative responses to the HIV p24 protein or the control baculovirus protein.

Figure 10 depicts vaccine-induced changes in mean LSI values for all patients, patients grouped by vaccine response, and patients grouped by immunization schedules.

There was a significant difference in the change in proliferative response to gp160 between vaccine responders and non-responders (P <0.001, Wilcoxon, 1-sided). The gpl60 proliferative responses induced by Scheme B (6 doses) were greater than the responses induced by Scheme A (3 doses) (P <0.10, Wilcoxon, one-sided).

Of the 21 patients who gave a proliferative response to gp160, 19 had a humoral response (vaccine responders). The maximum mean lymphocyte stimulation index (LSI) for gp160 observed in all vaccine responders was 50.1. However, the response of each patient responding to the vaccine was different (peak values ranging from LSI 3 to 171) (Figure 7), and the time course of cellular responses to gp160 differed in time (Figure 9).

H. Evaluation of results:

Despite the limited sample size of this assay, a number of factors have been reported that are related to the immunogenicity of the vaccine. Six out of 15 patients (40%) who were vaccinated under Scheme A and thirteen (87%) of 15 patients who were vaccinated under Scheme B

- 46 - .............

cina responders (Ρ = 0.02 Fisher, two-sided) (Figure 7). Thirteen of the 16 patients with baseline CD4 counts greater than 600 / ml (81%) and six of the 14 patients with baseline mean CD4 counts of less than 600 / ml (P = 0, 07 Fisher, two-sided). Table 8 shows that multiple immunization vaccines improve immunogenicity, even in patients with baseline CD4 counts of less than 600 / ml. For example: 5 of the 6 patients who were vaccinated according to Schedule B (6 vaccinations) were responding to the vaccine, whereas of the 8 patients who received 3 vaccinations (Scheme A), only one (P = 0.03 Fisher, bilateral) (Fig. spreadsheet).

Table 8

Influence of baseline CD4 count and immunization schedule on immunological reactivity of gp160 vaccine.

CD4 number

N Number of respondents (%)

Number of non-respondents (%)

The scheme

600 7 5 (71) 2 (29) 500-600 5 1 (20) 4 (80) 500 3 0 (0) 3 (100) Altogether 15 6 (40) 9 (60)

·································································•

Table 8 (continued)

Number of CD4 N Respondents Number of non-respondents (%) (%)

Scheme B

600 9 8 (89) 1 (11) 500-600 2 2 (100) 0 (0) 500 4 3 (75) 1 (25) Altogether 15 13 (87) 2 (13) Altogether: 30 19 (63) 11 (37)

The therapeutic use of vaccines was introduced by Pasteur in the 19th century to treat acute rabies infection (rabies). The utility of this procedure in treating other infections has not been thoroughly researched so far. Although there are other examples of modifying virus-specific immunity following infection (such as after the occurrence of hepatitis A and B infection), there are no well-documented human studies demonstrating the feasibility of this approach for an emerging or chronic viral infection.

The present invention provides for virus-specific immune modification by active immunization after infection. More specifically, an HIV-envelope gene-derived gp160 vaccine enhanced viral-specific humoral and cellular responses in the human host in 19 of the 30 HIV-infected individuals.

In this study, individual antibody responses against specific HVV epitopes were measured qualitatively and quantitatively.

• ·. - · «····· ·

- 48 ~ * ♦ ·· ···· ·· ··· for multiple infections and post-infection immunizations. In this way, vaccine-induced humoral immunogenicity could be demonstrated in 70% of patients in already cooked subjects. For example, 20 patients (19 responding to the vaccine and 1 not responding to the vaccine) had seroconversion to specific envelope epitopes. Ten patients had seroconversion related solely to vaccination (epitopes 241, 254 and 342).

In addition, the variety of humoral responses to the vaccine, as examined by epitope mapping, allows future analysis of the cause and effect of specific antibody responses and provides unique opportunities for exploring potential immune regulatory mechanisms that have not yet been discovered in natural infections.

Although the in vivo significance of serum neutralizing activity is currently unknown, increased neutralization activity against various HIV strains (IIIB.RF, MN) in four of the five vaccine responders suggests that post-challenge immunization induces changes in functional antibodies. The test vaccine induces an increase in serum neutralizing capacity against various HIV strains and may be a potential aid in determining group-specific neutralization epitopes.

A proliferative response to HIV envelope proteins is rare in natural HIV infection. However, after immunization with gp60, specific T cell proliferation responses were observed in 21 patients (70%). The reason for the difference is unclear. One possible · «·· ··· <* · * ♦ ♦ * *

- 49 that the new proliferative response is directed against epitope (s) that are specific to the vaccine (as a result of vaccine production methodology or alternative in vivo antigen processing). Alternatively, the protein used in the proliferation assay may not stimulate primary T-cell proliferative responses against the wild-type envelope of the natural virus. However, further evidence was obtained that vaccination induces a host cellular immune response: HIV-IIIB type-specific cytotoxic T-cell responses were elicited in response to immunized vaccines in response to a selected vaccine.

The factors responsible for the immunological reactivity of the vaccine in HIV infected individuals still need to be clarified. Even with early HIV infection, individuals give suboptimal responses to many vaccines compared to appropriate controls. This low reactivity is associated with early B cell dysregulation and T cell dysfunction. In this case, the immunological reactivity of the vaccine was related to basal CD4 cell counts, which is consistent with the hypothesis that host immunological status is an important determinant of vaccine reactivity. However, the immunization pattern - within specific T cell count limits - also influences the reactivity of the vaccine: Scheme B (6 injections) was outstanding. Poorer response to vaccine in patients with low CD4 counts! indeed, skill could be improved by increasing the number of vaccinations, suggesting that further modifications of dosage, application, adjuvants, or formulation may result in a decrease in A further improvement in the immunological response of 4 «· · teg teg vár vár vár vár vár vár vár is expected.

Although concerns have been raised regarding the safety of active immunization of HIV-infected individuals with HIV-specific vaccine formulations, there was no evidence of immuno-specific toxicity. Quantitative cultures, DNA polymerase chain reaction assays, and serum antigen assays showed an increase in in vivo HIV load. An excellent in vivo marker of HIV replication, that is, a favorable effect on the extent of CD4 cell depletion, was observed especially in the patients classified as vaccine responders. Respondents had a change in mean CD4 of -0.2% and non-responders -7.3%. These data indicate that post-infection immunoreactivity is not associated with increased CD4 cell death and is thought to be accompanied by a decrease in in vivo HIV replication.

Vaccination results obtained in this study were compared with data from ten infected and untreated individuals with similar age, ethnicity, and baseline CD4 cell counts. In this reference group, the mean CD4 count decreased by 8.7%, with a 7.2% reduction in Schedule A vaccinated patients and a 0.6% reduction in Schedule B vaccinated patients. These data demonstrate that post-infection vaccination with the recombinant HIV envelope protein is reasonable, and the result even encourages the prophylactic use of such vaccines.

Claims (9)

A process for the preparation of a HIV vaccine composition, said composition comprising as an adjuvant aluminum phosphate and recombinant HIV envelope protein particles having a molecular weight of at least about 2,000,000, and comprising the steps of: a) infecting cultured insect cells with recombinant baculovirus, said recombinant baculovirus comprising a gene encoding a HIV envelope protein expressing a recombinant HIV envelope protein; b) isolating said recombinant HIV envelope protein from said insect cell culture; c) isolating and assembling the purified recombinant HIV envelope protein particles by eluting the resulting recombinant protein from an affinity matrix containing gel-immobilized lens selectin; and d) adsorbing said purified recombinant envelope protein particles to said aluminum phosphate adjuvant. 2. The method of claim 1, wherein the recombinant protein particles comprise the consecutive 757 amino acids of gp160, truncated at position 757, and substantially lacking about 40 consecutive terminal amino acids of said protein. The method of claim 1, wherein the protein particles are spherical and have a diameter of 30 to 100 nm. 4. The method of claim 1, wherein: ν · the recombinant baculovirus is the Ac3046 vector. 5. The method of claim 1, wherein the isolated purified recombinant protein is dialyzed prior to step d). The process according to claim 1, wherein the pH of step d) is from 7.0 to 7.2. The method of claim 1, wherein step b) comprises: i) washing and homogenizing the cultured infected insect cells; ii) disrupting the cells; and iii) isolating the recombinant protein from the disrupted cells. The process of claim 1, wherein step c) comprises: i) transferring the resulting protein to said affinity matrix; ii) washing the matrix with the first lens-lectin buffer; and iii) eluting the protein from the matrix with the second lens-lectin buffer. 9. The method of claim 8, wherein the first lens-lectin buffer comprises deoxycholate and the second lens-lectin buffer comprises alpha-methyl-mannoside.