SI9200420A - Vaccine and treatment method for human immunodeficiency virus - Google Patents

Abstract

Produce !: We have acquired the syndrome vaccine immunodeficiency (AIDS) containing envelope proteins of human immunodeficiency virus type-1 (HIV-1) from of cloned HIV-1 envelope genes in a vector system baculovirus-insect cell. Recombinant proteins HIV-1 was purified, combined into particles and then adsorbed on aluminum phosphate adjuvant. Thus obtained form of adsorbed recombinant envelope protein of HIV-1 (an AIDS vaccine) is found in animals very immunogenic and extracts antibodies that bind to HIV-1 envelope and neutralize infectivity virus in vitro tests. This vaccine threatens A! DS induces new humoral and cellular responses in patients infected with HlV and is useful as a form vaccine treatments to delay or prevent destruction of the immune system.

Description

Human immunodeficiency virus type-1 (HIV-1) is a retrovirus that causes systemic infection with major pathology in the immune system and is the etiologic agent responsible for acquired immune deficiency syndrome (AIDS). Barre-Sinoussi, et al., Science, 220: 868-871 (1983); Popovich et al., Science, 224: 497-500 (1984). HIV-1 clinical isolates have also been published as a virus associated with lymphadenopathy (Feorino et al., Science, 225: 69-72 (1984) and an AlDS-related virus (Levy et al., Science 225: 840-842 (1984) ).

AIDS has become pandemic and the development of the vaccine is therefore a top priority for human health in the world. A high percentage of people infected with HIV-1 show a progressive loss of immune function due to the emptying of T4 lymphocytes. These T4 cells, as well as some nerve cells, have on their surface a molecule called CD4. HIV-1 differentiates CD4 molecules with receptors located on the envelope of the viral particles, penetrates these cells and finally replicates and kills the cell. It can be expected that the AIDS vaccine will extract antibodies that can bind to the HIV-1 envelope and prevent it from infecting T4 lymphocytes or other sensitive cells.

Vaccines are usually given to healthy individuals before being exposed to a diseased organism as an immune prophylactic. But it is also wise to consider using an effective AIDS vaccine in post-exposure immunization as immunotherapy for diseases. Salk, J., Nature 327: 47 3476 (1987).

The belief that the HIV-1 env is the most promising candidate for the development of the AIDS vaccine is widespread. Francis and Petricciani, Nev Eng. J. Med. 1586-1559 (1985); Vogt and Hirsh, Revievs of Infectious Disease, 8: 991-1000 (1986); Fauci, Away. Natl. Acad. Sci USA, 83: 9278-9283. The HIV-1 envelope protein is first synthesized as a glycoprotein with a molecular weight of 160,000 (gpl60). The precursor of gp 160 is then cleaved into an external glycoprotein of molecular weight 120,000 (gpl20) and a transmembrane glycoprotein of molecular weight 41,000 (gp41). These two envelope proteins are major antigens, targeting antibodies in rodents, goats, rhesus monkeys and chimpanzees. Robey et al., Off. Natl. Acad. Sci. USA 83: 7023-7027 (1986).

Due to the very low levels of native HIV-l envelope protein in infected cells and the dangers associated with the preparation of AIDS vaccine from HIV-1 infected cells, recombinant methods are used to produce HIV-l envelope antigen for use as AIDS vaccines DNA. Recombinant DNA technology appears to be the best choice for vaccine production from AIDS subunits because of its ability to produce large quantities of safe and economical immunogens. The HIV-1 gene was expressed in genetically modified recombinant vaccinia 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). Envelope proteins have also been expressed in bacterial cells (Putney et al., Science, 234: 1392-1395 (1986)), in mammalian cells (Lasky et al., Science, 23: 209212 (1986)), and in insect cells. Synthetic peptides derived from amino acid sequences in gp41 from HIV-1 have also been seen as candidates for the AIDS vaccine. Kennedy et al. (19 86). But, using these substances and methods, they did not produce successful AIDS vaccines.

Use of the baculovirus-insect vector system for the production of recombinant HIV-1 envelope proteins is one aspect of the invention disclosed in a protected and granted U.S. patent. patent application Serial No. 1 920,197 filed October 16, 1986 (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. As examples, the baculovirus of Autograph californica polyhedrosis virus nucleus (AcNPV) was used as a vector to express the full length of gp 160 and different parts of the HIV-1 envelope gene in infected Spodoptera frugiperada cells (worm species). Preliminary patent applications have also disclosed a cleaved gene for gpl60 (recombinant Ac3046), a protein made from recombinant Ac3046 and a purification technique for Ac3046 product, which includes affinity chromatography with lens lectin and gel filtration chromatography. The gpl60 protein has been found to be purified in this manner, which aggregates to form particles, highly immunogenic in rodents and primate species.

The ideal AIDS vaccine should allow for long-term protection against HIV-1 infection after one or more injections, in addition to the requirements for essential biological purity and non-pyrogenicity. This is usually the case for live diluted vaccines. When killed bacteria or viruses, or substances isolated from them, such as toxoids or proteins, are used to prepare the vaccine, the response with antibodies is often poor and the immunity is only short-lived. To overcome or reduce these vaccine deficiencies, an additional component, called an adjuvant, can be added to it. Adjuvants are substances that help stimulate the immune response. Adjuvants commonly used in human vaccines are gels or aluminum salts (aluminum phosphate or aluminum hydroxide), commonly referred to as galun adjuvants. Bomford et al., Adjuvants, Animal Whole Biotech. Vol 2: 235-250, Academic

Press Inc. (London: 1985).

The present invention provides a vaccine and methods of treatment for human immunodeficiency virus (HIV) comprising administering an iocombinant protein envelope to an HIV infected or susceptible person. In a preferred embodiment, the envelope protein can be purified, aggregated and combined with an adjuvant (e.g., galun) for use as a vaccine.

BRIEF DESCRIPTION OF THE DRAWINGS

The particulars of the present invention are explained below with reference to the accompanying drawings.

Figure 1 illustrates the cloning strategy used to isolate the env gene (env) of the HIV-1 envelope from the E. coli pNA2 plasmid. The dashed sections are DNA sequences from HIV-1 and the open sections are from cloning vectors. The black portion in plasma du pl774 was constructed from synthetic oligonucleotides and inserted as a Smal-KpnI fragment in Smal-KpnI into the sites of plasmid pl614. We have shown the sequence of this synthetic oligonucleotide.

Figure 2 illustrates the strategy used to construct the recombinant plasmid vector (p3046), which was then used to construct the baculovirus vector for Ac3046 expression. Plasmid pMGS3 contains sequences (transversely dashed surfaces) from AcNPV baculovirus on either side of the inclination site at position 4.00. This site has unique sites for Smal, KpnI and Bglll restriction endonucleases. The AcNPV polyhedrin promoter is located 5 'from position 4.00. The sequence 5TAATTAATTAA- 3 'is located in the 3' direction and has a translational codon for termination in all three reading frames. Plasmid pl774 and the sequence of the synthetic oligonucleotide region were described in Figure 1. Plasmid p3046 contains all of pMGS3 except the sequences between Smal and Bgll1 sites, where the HIV-1 envelope gene from pl774 is inserted.

Figure 3 shows the nucleotide sequence of DNA located at the ends of sequences encoding gpl60 Ac3046. The sequence of 3046 env DNA between +1 and +2264 is shown in Figure 4.

Figures 4a-4t show the actual DNA sequence of the HIV-1 env gene segment together with the synthetic oligonucleotide sequences at the 5 'end of the env gene in Ac3046 (between +1 and +2264). The locations of restriction endonucleases sites were entered above the DNA sequence and the predicted amino acid sequence was listed below the DNA sequence. The bases were marked with numbers on the right and left.

Figures 5a-5h compare the DNA sequences of the env gene from Ac3046 with the published sequence of the env gene from LAV-1. The LAV-1 sequence is at the top and Ac3046 at the bottom. Line (1) below sequence LAV-1 indicates that the sequence in Ac3046 is the same in this position. The DNA sequence numbering we used is that described by Wain-Hobson et al., Whole, 40: 9-17 (1985) for LAV-1.

Figures 6 and 6a show the final dilution titer in the ELISA test of human serum positive for HIV-1 antibodies (upper graph) and serum rhesus monkeys (lower graph) from animals immunized with gpl60 (IJ55, KL55) or gpl20 (AB55, CD55, GH55). The ELISA titers were measured against the highly purified gpl20 and gpl60 proteins. Specific bound antibody was measured with goat anti-human IgG HRP conjugate. The maximum serum dilution that gives a positive response in the test is the titer.

Figure 7 (a and b) is a table summarizing the immune responses induced by the gpl60-containing vaccine in vaccinated seropositive patients responding (7a) or non-responding (7b) to the vaccine.

Figure 8 (a, b, and c) shows antibody responses directed against specific epitopes on the HIV envelope. Figure 8b illustrates the reactions of patients from Program A, Figure 8c shows the reactions of patients from Program B, while Figure 8a provides values for both groups of patients.

Figure 9 (a - d) shows T-cell proliferation in vaccinated seropositive patients responding to a vaccine induced by a gpl60-containing vaccine. Figures 9a and 9c show the reactions of two typical patients from program A, while figures 9b and 9d illustrate the reactions of two typical patients from program B.

Figure 10 (a - c) shows lymphocyte proliferation in response to vaccination.

Figure ll is a graph showing the percentage change in CD4 cells in responders and non-responders over time.

SUMMARY OF THE INVENTION

Recombinant Hiv-i gpl60 (rgpieo) envelope protein, especially when adsorbed on an adjuvant such as alumina (e.g. aluminum phosphate), has been found to be particularly useful as an AIDS vaccine. One aspect of the present invention is an AcNPV expression vector having a sequence encoding a portion of the HIV-1 envelope gene surrounding amino acids 1-757 found in the recombinant clone no. 3046. Another aspect of the invention is the production of this recombinant HIV-1 envelope protein (and the protein itself) in insect cells - especially the rgpl60 protein encoded by amino acid sequences 1-757 (i.e. 03046).

Other aspects of the present invention include purification and particle formation of recombinant envelope protein from a product of the recombinant baculovirus gene that produces 3046 protein and adsorption of 3046 particles to aluminum phosphate aggregates.

The invention also includes prophylactic and / or therapeutic vaccines for AIDS or HIV infections and methods of preventing or treating AIDS or HIV infections.

DETAILED DESCRIPTION OF THE INVENTION

The following examples illustrate the invention without limiting its purpose.

The recombinant baculovirus of Autograph californica polyhedrosis virus core (AcNPV), which contains the cleaved gene for HIV-1 gpl60 encoding amino acids 1-757 of the HlV envelope protein (recombinant Ac3046), is described in U.S. patent application. application Serial No. 920,197 (now Serial No. 585,260) awaiting common recognition. The cloning steps used to form the recombinant baculovirus containing genes or gene portions of HIV-1 are also detected there and incorporated by reference.

The following is a detailed description of the genetic engineering phases used to form the Ac3046 expression vector. The substances used, including enzymes and immunological reagents, were obtained from commercial sources. We have also provided examples showing how the invention is made and used.

We have also considered other recombinant envelope proteins, collectively referred to as rgpl60, and these include recombinant gp! 2 0 and gp41. Ac3046 is just one example of an expression vector and a recombinant envelope protein of the invention.

EXAMPLE 1

Formation of Baculovirus Recombinant Ac3046 Carrying Sequences Coding for Amino Acids 1-757

Cloning and expression of a foreign protein in baculovirus as a vector requires that the coding sequence is aligned on one side with the polyhedrin promoter and sequences at the 5 'end and on the other with the coding sequences of baculovirus. The arrangement is such that recombination with the baculovirus genome results in the transfer of foreign coding sequences aligned with the polyhedrin promoter and the inactive polyhedrin gene.

Against this background, we have designed different insertion vectors to use to generate the HIV envelope gene. The MGS3 insertion vector described thereafter was designed to supplement the ATG initiation codon for translation. The insertion of foreign sequences into this vector must be performed in such a way that the translation framework established by the initiation codon is properly retained in the foreign sequences.

The MGS3 insertion vector was composed of a restriction fragment of an EcoRI-1 DNA clone isolated from a colony of purified AcMNPV isolate (WT-1). We designed the MGS3 to consist of the following structural characteristics; (a) 4000 bp sequence at the 5 'end of the ATG initiation codon of the polyhedrin gene; (b) a polylinker inserted by site-directed mutagenesis consisting of an ATG initiation codon at the position of the corresponding polyhedrin codon, Smal restriction sites, KpnI;

Bglll and the general stop codon segment; (c) a 1700 bp sequence extending from the restriction site of KpnI (located in the polyhedrin gene) through the terminal restriction site of the EcoRI clone of EcoRI-1. See, e.g.

Figure 2.

EXAMPLE 2

Formation of baculovirus recombinants carrying sequences encoding LAV env

The recombinant plasmid we designated as NA2 (Fig. 1) consists of a 21.8 kb segment of the entire HIV-1 provirus inserted into pUC18. They stated that this clone is infectious because it can produce the virus after transfection of some human cells. Adachi et al., J. virol. 59: 284-291 (1986). Whole gene sequences for the envelope proteins contained in NA2 were derived from the LAV strain of HlV. Barre-Sinoussi (1983).

The HIV-1 envelope protein gene was isolated and subjected to engineering as subsequently described and shown in Figure 1. The envelope protein gene was first isolated from NA2 as a 3846 bp long EcoRI / SacI restriction fragment and cloned into an EcoRI / SacI restriction fragment. pUC19 site. The plasmid thus obtained was designated p708.

The envelope protein gene was further re-isolated as a 2800 bp long KpnI restriction fragment and cloned into the KpnI restriction site pUC18. The clone we so debuted was designated pl6l4.

The kpnI restriction fragment in pl614 contains a slightly cleaved portion of the HIV envelope gene such that a 121 bp long sequence corresponding to the N-terminal portion is omitted. The omitted portion of the gene comprising the signal peptide sequences was replaced by 10 µl by the insertion of a two-stranded synthetic oligomer. We have determined that the inserted oligomer should be composed of the amino acid sequence of LAV, using a preferred polyhedrin gene codon. In order to facilitate the further process, we also introduced an ATG initiation codon into place. The ATG initiation codon will be supplemented with a baculovirus vector for insertion. The plasmid thus obtained was designated p! 774.

Referring to Figure 2, the restriction fragments of pl774 encoding the coding sequences of different HIV-1 envelope domains were encoded in the MGS insertion vectors (e.g., MGS3) such that the ATG initiation codon of the insertion vector was in the frame with the gene codons for proteins of the envelope. The p3406 formation consisted of a Smal / BamHI restriction fragment isolated from pl774 inserted into the Smal / Bglll site of the plasmid vector pMGS3. This clone contains sequences encoding amino acids 1 to 757 gpl60 and uses a termination codon supplemented with the MGS3 vector.

EXAMPLE 3

Preparation and selection of recombinant baculovirus

Plasmid p3046 recombination of the HIV env gene was precipitated with calcium phosphate together with AcMNPV DNA (WT-1) and added to uninfected Spodoptera f rugiperda cells. The chimeric gene was then inserted into the AcMNPV genome with homologous recombination. Recombinant viruses were identified by occlusal negative colony morphology. Such colonies exhibit a cytopathic effect that can be identified but not nucleus occlusion. Two additional colonies were purified to obtain pure recombinant virus. Recombinant viral DNA was analyzed for insertion at the specific site of the HvV env sequences by comparing their restriction and hybridization characteristics with those of the viral

Wild-type DNA.

EXAMPLE 4

Expression of HIV env from recombinant baculovirus in insect cells

The expression of HIV env sequences from recombinant viruses in insect cells should result in the synthesis of a primary translation product. This primary product will consist of amino acids that have been the result of translation from codons supplemented with a recombinant vector. The result is a protein that contains all the amino acids encoded by the ATG initiation codon of the expression vector at the 3 'end of the polyhedrin promoter to the translation termination signal on the expression vector (e.g., rgpl60). The primary translation product of Ac3046 should read Met-Pro-Gly-Arg-Val at the end of the chain where Arg (position 4) is Arg at position 2 in the original LAV clone. Met-Pro-Gly codons are updated as a result of the cloning strategy.

EXAMPLE 5

Nucleotide sequence of inserted gpl60 and DNA located at its ends

The nucleotide sequence of the inserted gpl60 and the DNA located at its ends were determined from restriction fragments isolated from the DNA of the viral vector for the expression of Ac3046. The sequencing strategy involved the following stages. The 3.9 kb EcoRV-BamHI fragment was purified by restriction digestion of Ac3046 viral DNA. The Ac3046 viral DNA was prepared from an extracellular virus present in the cell medium used for the vaccine production series.

As shown in Figure 2, the EcoRV-BamHl 3.9 kb fragment contains the whole gpl60 gene and 100 bp at the 5 'end and about 1000 bp at the 3' end of DNA located on either side of this gene. From this we determined the nucleotide sequence of the entire gpl60 gene, including 100 bp at the 5 'end and 100 bp at the 3' end of the DNA adjacent to the gene.

In short, the sequencing results revealed a chimeric formation that we predicted based on a cloning strategy. The gpl60 sequence was essentially the same as that reported by Wain-Hobson et al. (1985). The 2253 base sequence between putative translation initiation and termination codons predicts 751 amino acid codons and 28 potential N-linked glycosylation sites. The estimated molecular weight of this rgpl60, including sugars, is approximately 145,000.

Sequence analysis of 200 DNA bases located at both ends of the gene shows correct insertion, as shown in Figures 3,4 and 5.

EXAMPLE 6

Amino acid sequence of gpl6 0

With standard automated Edman degradation and HPLC procedures, we found that the N-terminal sequence of the first 15 gpl60 residues was identical to that predicted based on the DNA sequence. N-terminal methionine is not present on the gpl60 protein. This is consistent with the observation that AcNPV protein polyhedrin also forms without N-terminal methionine. The summary of the actual sequences of gpl60 DNA and Nterminal protein as determined by DNA analysis of AcNPV 3046 and purified gpl60 is as follows (Table l).

Table 1

env gen LAV's v AcNPV 3046 vector u for expression Remainder 2 3 4 5 6 7 8 9 10 11 12 13 14 Pro Gly Arg Val Lys Glu Lys Tyr Gin His Leu Trp Arg Trp Gly ATG CCC GGG CGT GTG AAG GAG AAG TAC CAA CAC CTG TGG CGT TGG

GGC

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

Table 2 env gene of LAV in the original clone of LAV-1

Remainder

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

Met Arg Val Lys Glu Lys Tyr Gin His Leu Trp Arg Trp Gly

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

EXAMPLE 7

Purification of recombinant gpl60

One aspect of the present invention is the process used to extract and purify the recombinant HIV-1 envelope protein encoded in the Ac3046 expression vector. Recombinant HIV-1 gpl60 envelope protein is made in S. frugiperda cells within 4-5 days after Ac3046 infection. Purification of this igpl60 protein involves the steps of:

1. Cell flushing

2. Cell lysis

3. Gel filtration chromatography

4. Affinity chromatography on the lectin of the lens

5. Dialysis

This example describes the purification of recombinant gpl60 from about 2 χ 10 ⁹ cells infected with Ac3046.

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

2. Cell lysis. The washed cells were lysed by sonication in 50 mM tris buffer (pH 8.0-8.5), 4% deoxycholate and 1% mercaptoethanol. Sonication was performed using standard procedures. After sonication, only the remnants of the core membrane are intact and were removed by centrifugation at 5000 rpm for 30 minutes. The supernatant containing the extracted gpl60 had no intact cells as determined by observation under a light microscope.

3. Gel filtration. Gel filtration was performed on 5.0 x 50 cm Pharmacia glass columns filled with Sephacryl resin (Pharmacia). The total useful volume of the column is approximately 1750 ml. In order to depyrogenate and sterilize the columns and tubing, at least 6 liters of 0.1 M NaOH were passed through the column for 24 hours. The eluent from the column was coupled to a UV flow cell, monitor and printer (Pharmacia) and then equilibrated with 4 liters of gel filtration buffer. Purified gpl60 was poured onto a column and developed with gel filtration buffer.

The column separates the crude mixture into three major UV light absorbing fractions. The first peak comes between about 500 and 700 ml, the second between 700 and 14000 ml and the third peak between 1400 and 1900 ml of buffer. This same profile was observed on small analytical columns, on the basis of which the molecular weight of the first peak was> 2,000,000.

This peak is transparent due to its high molecular weight lipid and lipid complexes. This peak also contains from 10% to 20% of gpl60 extracted from infected cells. Obviously, this gpl60 fraction is complexed with itself or with other cellular components to form large molecular weight aggregates.

The second broad peak contains most gpl60 and high molecular weight proteins between about 18,000 and 200,000.

The third peak contains little protein and most of the UV absorption is due to the / -meracaptoethanol present in the sample.

When the second peak was first observed on the UV absorbance record, the eluent from the column was directed straight to the lens lectin column. When the second peak came out of the column, we severed the bond between the eluent and the lectin column from the lens and directed it to the outflow,

4. Lectin from the lens. Affinity chromatography medium on lens lectin (Lentil Lectin-Sepharose 4B) was purchased in bulk from Pharmacia. Lectin from the lens was isolated by affinity chromatography on Sephadex to a purity greater than 98% and then immobilized by combining it with Sepharosa 4B using bromine for this purpose. This matrix contains about 2 mg of ligand ma ml of gel. The lens lectin column is a 5.0 x 30 cm glass column (Pharmacia) containing 125 ml of lens lectin-Sepharos 4B gel. This affinity matrix was reused after being extensively washed and regenerated by a process recommended by the caregiver. When not in use, the gel was stored in a column in a solution of 0.9% NaCl, 1 mM MnCl ₂ , 1 mM CaCl ₂ and 0.01% thimerosal. Before each use, the column is washed and equilibrated with 250 ml of lens lectin buffer described above.

Purified gpl60 was poured onto the column immediately after being eluted from the gel filtration column as previously described. After the crude gpl60 was bound to the column, it was washed with 800 ml of lens lectin buffer containing 0.1% deoxycholate. under these conditions all gpl60 binds to the column. The lens lectin buffer and 0.3 M ci-methyl mannoside were used to elute the bound glycoproteins and observed through a UV monitor at a wavelength of 280 nm.

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

Purification of gpl60 from l L infected cells can be summarized in the following table (Table 3).

Table 3 Summary of Purification

Phase purification a Total protein (mg) ¹ gpi6 0 protein (mg) % gpl60 from the whole Remove one impurities Precipitation 1-2000 20 1-2 Media for culture 1., 2. and 3. spiranj e 250 15 6 Serum albumin, most nucleic acids and soluble cellular proteins Gel filtration 120 12 12 Lipids, nucleic acids and aggregates large mol. m. Lectin from the lens 14 10 70 Non-glycosylated proteins Dialysis 13 9 70 Sugar, deok-

xicholate, excess Tris buffer.

^ Total protein was determined based on absorbance at 280 nm

In another embodiment of the experiment, ion exchange chromatography may be used instead of gel filtration. Similarly, the order of the phases is not critical: for example, gel filtration or ion exchange chromatography may follow the purification phase with lens lectin. Other reagents may be used according to the invention. For example, other detergents may be used to purify the recombinant protein instead of deoxycholate. This includes non-ionic detergents such as Tween 20 (polysorbate 20), Tween 80, Lubrol and Triton Χ-100.

EXAMPLE 8

A. Gpl60 particle pooling

As one aspect of the present invention, it has been discovered that the gpl6O antigen can be pooled into particles of molecular weight> 2,000,000 during purification. The gpl60 protein is extracted from the cell as a mixture of 80-90% monomeric (molecular weight 160,000) and 10-20% polymeric (particulate) form. The gel filtration phase removes the aggregated forms of gpl60. Attempts to purify the gpl60 protein from this fraction (the first peak from the gel filtration column) indicate that it is complexed with other cellular proteins, possibly even membrane fragments. However, the molecular weight of the gpl60 antigen located in the second peak emanating from the gel filtration column is approximately 160,000 300,000 and is therefore mainly in monomer or dimeric form.

The formation of aggregates or polymers of gpl60 occurs during the development of a lens lectin column. The antigen was found to form aggregates either when eluted from the lectin column in 0.5% deoxycholate, which is approximately 0.2% of the critical micelle concentration (CMC) for deoxycholate, or when gpl60 eluted from the column in 0.1% deoxycholate.

Aggregate size was measured on a FPLC Superose 12 high resolution column (Pharmacia). The sample sizes of representative quantities of purified gpl60 are substantially equal to or greater than 2,000,000 molecular weights in size of the blue dextran standard.

The research by Schwaller et al. (1989) by cross-linking showed that gpl60 produced by insect cells is a tetramer of identical subunits. This study also showed that gpl60 is tetrameric in cells infected with HlV and in viral particles. Thus, particles of recombinant gpl60 may have a tertiary and quaternary structure similar to that found in native HIV gpl60.

An accurate 3-dimensional structure could be important for epitope formation, which requires the proper spatial arrangement of gpl6 0. It is likely that, while binding to the lectin column from the lens and washing the column, the non-glycosylated proteins bound to gpl60 are removed. parts of gpl60 are beginning to form intermolecular associations. Deoxycholate is unlikely to bind to gpl6 0 when the concentration is maintained above CMC and the antigen will still form complexes. The fusion of this antigen into aggregates seems to be an intrinsic property of this protein once purified according to the invention. It may be that the highly hydrophobic N-terminal sequence located in the gpl60 protein contributes to the natural ability of this protein to form particles. After purification, the gpl60 complexes can be sterile filtered through a 0.2 m cellulose acetate filter without significant loss of protein.

B. Analysis of fragment formation

By analyzing the purified fragments formed by gpl60, electron microscopy revealed that these are spherical particles similar to proteins in the size of 30-100 nM.

An additional test for the presence of these fragments was the analysis of purified gpl60 by gel filtration. About 100 μg of gpl60 was poured onto a HR 10/30 Superose 12 (Pharmacia) column for FPLC gel filtration. This column was first calibrated with protein standards of known molecular weights. The protein profile from this column is highly reproducible; the elution volume is inversely proportional to the molecular weight of the preotinic standards. The column separates monomeric gpl60 from polymeric forms and eliminates globular proteins of molecular weight> 2 χ 10 ⁶ . When it is passed through this column, essentially all of the gpl60 elutes in the excretory volume and therefore has a molecular weight of> 2 x 10 ⁶ (2,000,000).

EXAMPLE 9

A. Adsorption of gpl60 onto the gallbladder

The effectiveness of insoluble aluminum compounds as immunological adjuvants depends on the complete adsorption of the antigen to the solid phase. As part of the present invention, we have discovered that it is possible to prepare galuncove compositions that will effectively adsorb gpl60, but at a pH that will not reduce the potency of the gpl60-galunov complex as an immunogen. The factors that are controlled during the formation of this galunov (aluminum phosphate gel) are:

1. The optimum pH for the adsorption of antigen per gallon is about 5.0. However, we found that gpl60 lost its immunogenicity at pH 6.5 compared to pH 7.5 by preparing the galunov to pH 7.1 + 0.1. We found that at this point, the pH of 100% gpl60 was still adsorbed on the galloon.

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

A molar excess of aluminum chloride relative to sodium phosphate is present to ensure that there are no free phosphate ions in the supernatant.

4. The gpl60 antigen is added to the freshly formed galleon to stop crystal growth and reduce particle size.

The procedure for the preparation of 200 ml of galunac and adsorption of purified gpl6 0 per gallon is such that the final antigen content is 40 pg / ml, as shown below in the main runs.

B. Preparation of reagents (200 ml whole formed quantity)

Prepare the following solutions in 100 ml sterile bottles or beakers of free pyrogen. The salts for solution l and solution 2 and sodium hydroxide are mixed and filtered through 0.2 µm cellulose acetate filters in 100 ml sterile pyrogen free bottles.

Solution 1

AlCl ₃ .6H ₂ O NaHAc.3H ₂ O

0.895 g 0.136 g

Dissolve in 40 ml of water for injection (WFI), 0.2 pm filter

2 Na ₃ PO solution ₄ .12H ₂ O

1,234 g

Dissolve in 40 ml WFI, 0.2 µm filter

Solution 3

NaOH

2,0 g

Dissolve in 100 ml WFI, 0.2 µm filter

Solution 4 Tris 1.25 g

Dissolve in 100 ml of WFI, add 1 ml in 90 ml of WFI, adjust the pH to 7.5 with 0.5 M HCl and make up to 100 ml with WFI.

The solutions were autoclaved for 30 minutes; let's slowly lower the steam. Cool to room temperature.

C. Formation of the Gallun

1. A solution of 1 (aluminum chloride-sodium acetate) is added to the molding vessel using sterile 25 ml volumetric pipettes. Record the volume of solution 1 and start mixing.

2. Add solution 2 (sodium phosphate) to the vessel using sterile 25 ml volumetric pipettes and continue stirring as a precipitate forms and record the volume of solution 2.

3. Add 3 ml of solution 3 (sodium hydroxide) and continue stirring for 5 minutes. Take 0.5 ml of the sample and measure the pH. If the pH is less than 7.0, another 0.5 ml of sodium hydroxide is added, stirred for a further 5 minutes and the pH is measured again. Continue until the pH is adjusted to between 7.0 and 7.2.

4. Determine the total volume added to the forming vessel (solution 1 + solution 2 + solution 3) and then add sterile WFI to adjust the volume to 100 ml.

5. Immediately add 8,000 µg purified gpl60 and 100 ml lmM Tris pH 7.5 straight to the molding vessel.

6. Continue stirring for at least 20 minutes and then dispense the formulated vaccine into sterile bottles.

EXAMPLE 10

Immunogenicity of gpl60 absorbed on galunovca (specific At response)

A recognized method for determining the immunogenicity of an anti-gene (vaccine) preparation is to measure the specific antibody response in a group of mice given a single dose of antigen. At the end of 4 weeks, the mice were lowered to blood and measured using standard antibody tests, e.g. ELISA (enzyme-linked immunosorbent assay) antibody level to a specific serum antigen (usually the antigen used to immunize animals).

The immunogenicity of purified gpl60 without adjuvant at pH 6.0 and pH 7.5 adsorbed with galun (as described in Example 9) or mixed with Freund's complete adjuvant determined in mice is summarized thereafter (Table 4).

The group

Serum conversion

Table 4 gpl60 sr. ELISA value

gpl6 0 Adj uvant Party OD ² % (P / N) ³ and ug None, pH 7.5 8702 0,140 57% 4/6 None, pH 6.0 8702 0,110 26% 2/7 Galunovec 8702 1,000 90% 9/10 Galunovec 8705 2,285 th most common 100% 9/6 Freundov 8604 1,108 th most common 83% 5/6 Freundov 8702 1,396 th most common 100% 7/7 0.1 pg Freundov 8604 0,434 th most common 67% 4/6 Galunovec 8705 1.003 67% 4/6 ² Mice we bled 28 days after immunization and serum tested at dilution 1 : 10 v test ELISA against gpl6 0 purified on gel. Similar results we got with use of the commercial ELISA (Genetic Systems Inc .; EIA ^thorn ELISA) test against native gout from HIV- 1 at serum dilutions 1: 400.

³ Number of mice with serum conversion (P) with respect to the total number of subjects tested (N).

Mice immunized with a single 1.0 pg dose of gp! 6 0 antigen without any added adjuvant will elicit an antibody response to gpl60 (see table above). However, a much stronger response with proiteles was observed in a group of mice immunized with 1.0 pg gpl6 0 adsorbed on a galleon adjuvant. A single dose of less than 0.1 pg gpl6 0 mixed with complete Freund's or formulated with a galun will seroconvert> 50% of the immunized mice. although less than this, gpl60 was immunogenic in mice as a non-formulated antigen at pH 7.5 and pH 6.0, but there was a loss of immunogenicity at lower pH.

EXAMPLE 11

Immunogenicity of gpl60 absorbed on the galun (serum test by ELISA test)

The ability of a vaccine candidate to elicit an immune response is a very important biological trait. The following experiments were performed to confirm that the gp! 60 vaccine formulated with galuns was immunogenic in animals, and to confirm that the adjuvant galuns increased this immunogenicity.

On day 0, a single dose (0.5 µg, 1.0 pg, or 5.0 pg) of gpl60 alone, gpl60 adsorbed on a galleon, or gpl60 in complete Freund's adjuvant (CFA) was injected into mice (groups of 10). On day 28, blood was collected from the animals and ELISA (1: 10 dilution) tested for antibodies to gpl60 in serum.

The results for the serum taken on day 28 were summarized in the table below (Table 5). In all groups, more than 50% of mice show serum conversion. At all doses, the number of serum conversions and the average serum absorbance (OD ₄ ie ₀ nm at a dilution of l: 10 in the ELISA test) were higher when gpl60 was adsorbed on the gallow than in mice immunized with gpl60 alone.

These results indicate that the galunc adjuvant significantly increases the immunogenicity of the gp! 60 antigen.

Table 5 - 28 days after injection Dose 0.5 pg Dose 0.5 pg 0.5 pg dose Wednesday, .value Mediumvalue Medium value

P / N ⁴ FROM ⁵ P / N OD P / N OD gpl6 0 9/10 .407 7/10 .699 7/10 .430 gpl6 0 (galun. ) 9/10 .547 8/10 .797 10/10 1,347 gpl6 0 (CFA) 10/10 1.130 10/10 1.967 10/10 1,317

⁴ Number of seroconverted (P) mice compared to the total number of mice tested 28 days after being immunized with 0.5 pg, 1 pg, or 5 pg VaxSyn ^tm HIV-1.

⁵ Mean absorbance (OD _45Q ) of a seroconverted mouse as measured by the sponsor's ELISA assay against gpl60 at a serum dilution of 1:10.

EXAMPLE 12

Neutralization data

The HIV-1 neutralization test is a recognized method for determining whether the preparation of an antibody will inhibit HIV1 virus from a human lymphocyte cell in an susceptible culture. Antiserum from animals immunized with gpl60 was tested in the HIV-1 neutralization assay and summarized in the table below (Table 6).

Table 6

animal Identifi- cation Imugen / Adj uvant g ⁶ Titer nev- trealizac. Rhesus G55 gpl20 / galun. 16/8/8 1: 80-1: 160 Rhesus H55 gpl20 / galun. 16/8/8 1: 80-1: 160 Rhesus L55 gpl60 / galun. 16/8/8 > 1:80 Mouse Pool 3 gpl20 / Freund's .25 / .25 / .25 1: 40-1: 80 Mouse Pool 8 gpl60 / Freund's .l / .l / .l 1: 40-1: 80 Marine pig Purified IgG gp! 60 / Freunds 10/10/10 1: 320

^b gpl60 or gpl20 administered during the first, second and third immunizations.

Maximum dilution of antiserum that will inhibit infection by 50% relative to HIV-1 infected cells exposed to serum of non-immunized animals.

EXAMPLE 13

Immunogenicity in chimpanzees

Genetically, the chimpanzee is the human's closest relative and is now the only animal model for HIV-1 infection. in the safety / immunogenicity experiment on three chimpanzees, two chimps were immunized with 40 pg and 80 ug gpl60, respectively. The control animal was simultaneously vaccinated with 1 ml of sodium chloride solution. Weekly, serum samples from each of the three chimpanzees were analyzed for the presence of antibodies to gpl60 and HIV-1 viral antigens using three immunological assays, an ELISA for purified gpl60 developed by MicrogeneSys, Inc., a Western blot analysis, and a commercial ELISA for HIV-l. The results of these analyzes are described in the text that follows.

A. ELISA (MGSearch HIV 160)

ELISA Test, MGSearch HIV 160, MGSearch is a trademark of MicxoGeneSys, Inc. from Meriden, Connecticur, U.S.A., is an immunosorbent assay against gpl60 and is described in U.S. Pat. Patent Application Serial No. 2 920,197 (now No. 585,166), which collectively awaits recognition,

Serum samples taken before immunization and diluted 1:10 to 11 weeks after primary immunization; 100,000 and then incubated with nitrocellulose strips containing 100 ug of purified gpl60 in spots. The final dilution titer is the highest dilution at which the assay was positive for anti-gpl60 antibodies, as observed with goat anti-human conjugate IgG-alkaline phosphatase.

Serum samples from the control animal and pre-immune serum of the immunized animal were negative. The chimpanzee receiving a dose of 80 ug was positive at a dilution of l: 100 per week and the chimpanzee receiving a dose of 40 ug was positive at a dilution of 1: 10 at week 4. The antibody titer against gpl60 continued to grow until week 5 when the final dilution titers were approximately 1: 100,000 and 1: 2,000,000, respectively. The antibody titer dropped slightly in both animals in weeks 6-11.

This type of response is both quantitatively and qualitatively similar to the antibody responses commonly seen in chimpanzees vaccinated with the human hepatitis B virus vaccine.

B. Commercial ELISA test

Based on the MGSearch HIV 160 ELISA and Western blot analysis of serum from VaxSyn ⁸ immunized chimpanzees, it became clear that they were serum-converted and had antibodies against recombinant gpl60. In order to identify or synthesize anti-HIV antibodies that differentiate native viral envelope proteins, pre-immune serum taken in weeks 1 to 11 was tested with a licensed, commercial ELISA test kit, the LAV ΕΙΑ ™ test kit, which it is manufactured by Genetic System Corporation, Seattle, Washington. An animal immunized with 80 pg gpl60 was positive at a 1: 100 dilution after 2 weeks and continued to increase antibody levels through week 6. An animal immunized with 40 pg was positive at a 1: 100 dilution at week 6.

EXAMPLE 14

Distribution of antibodies between gpl20 and gp41

It is important to determine whether the antibody response to gpl60 in the vaccinated animal is directed toward gp41, gpl20, or both. Different immunological methods including radioimmunoprecipitation (RIP), immunofluorescence (IF), Western blot analysis (WB) and quantitative ELISA against three different recombinant envelope antigens were used to detect and measure the distribution of antibodies against different parts of the HIV-1 envelope proteins.

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

Figure 6 summarizes the immunoreactivity of three different recombinant antigens: ART TAB (1) gpl20-delta (truncated recombinant gpl20 from HIV-1 with about 40 missing amino acids from the C-terminal portion of the molecule); [ARTJ [TABj (2) gpl20 (full length recombinant gpl20 from HIV-1); and [ARTj [TAB] (3) gpl6 0.

human serum from individuals positive for antibody 50 HIV-1 and 3 pooled human sera were highly reactive with gpl60, moderately reactive with gpl20, and little or no antibody reactive with truncated gpl20. Probably truncated gpl20, which accounts for more than 90% of the HIV-1 external glycoprotein, contains protective determinants. The observation that human AIDS-positive serum has few antibodies to this portion of the envelope proteins is consistent with the fact that the immune response to viral infection is not a complete protection and that human positive serum usually exhibits low levels of neutralizing activity in vitro.

In contrast, rhesus monkeys are immunized with either the immunogen gpl60 or truncated gpl20 antibodies, which react strongly with the truncated gpl20 portion of the HIV-1 envelope. This difference in the distribution of antibody-differentiated sites along the envelope of the virus and the higher titers observed in monkeys may be explained by the fact that monkey serum has higher neutralization titers.

A quantitative evaluation of the immunoreactivity of these three recombinant human and immune rhesus serum envelope antigens was shown in Figure 7. All monkey sera tested had high antibody titers against truncated gp! 20 antigen (gpl20-delta), including those from animals. which we immunized with gpl60.

These results indicate that recombinant gpl60 elicits an antibody response in rhesus monkeys that is different from that often occurring during natural infection. The gpl2031 delta part of the gpl60 contains epitopes that are effectively distinguished by immunized monkeys but are not visible to the human immune system during infection. These novel epitopes may be important for protection against HIV-1 and could be an important property of recombinant gpl60 for the prevention and treatment of HlV infection.

EXAMPLE 15

Therapeutic administration of the vaccine

We conducted a clinical trial of 30 HIV-seropositive human patients to determine the effects of vaccination with cloned gpl60 HlV (made in the baculovirus system as described above) on individuals infected with HlV.

Vaccination with recombinant gpl60 led to an increase in the specific humoral and cellular immune response to gpl60 of HlV in 19 of 30 (63%) HIV-positive volunteers. Fourteen of the 15 (93%) volunteers who received 6 doses of the vaccine showed an increase in their total antibodies to gpl60. Therefore, recombinant HlV proteins (i.e., rgp41, rgpl20, rgpl60, and admixtures thereof) can be useful in the treatment of a human patient infected with HlV.

The effective amounts of the HlV protein used in this embodiment of the invention can be determined by techniques well known in the art, such as those which are presented thereafter. Typically, such effective amounts are between about 1 pg and about 100 pg per kg of patient body weight. The frequency of administration can also be determined in known ways. In the preferred embodiment, administration is via the parenteral route, ie. intravenous, intraperitoneal, intramuscular, intradermal, etc. as is well known to those of ordinary skill in the art.

A. Selection of volunteers

Thirty volunteers have been identified who have been infected with HIV. Only seropositive volunteers with early-stage HIV infection, defined as Walter Reed stage 1 or 2, were eligible for the experiment (CD4 whole counts not less than 400 for more than 3 months, with or without lymphatic adenopathy ). (Redfield et al., Nev Engl. J. Med. 314: 131-132 (1986). An additional criterion limited volunteers to adults between the ages of 18 and 50, with a normal complete blood count, with no record of end-organ disease, without alcohol or drug abuse for the previous 12 months and for non-anti-retroviral or immunomodulatory drugs All patients underwent an assessment of their baseline status for 2 months before being randomly assigned to treatment groups. the volunteer received any antiretroviral or immunomodulatory medication.

Twenty-six volunteers out of 30 were men; 4 were women. fourteen were Caucasian, 13 were black, and 3 were of Spanish descent. The average age was 29 (range 18-49). At registration, 8 volunteers were in Walter Reed Stage 1 and 22 volunteers were in Walter Reed Stage 2. The baseline mean of CD4 counting was 668 (range 388-1639). The average time between initial diagnosis and admission to study was 24 months (range 3 months to 49 months).

B. Vaccine product and immunization schedule

As described herein, the test vaccine contains a non-infectious glycoprotein subunit derived from gpl60 as a recombinant protein expressed in baculovirus. Immunogenic pro33 tein was produced in Lepidoptera insect cells, biochemically purified, and adsorbed onto aluminum phosphate for final vaccine design.

Three formulations with different doses of gpl60 were used: 40 pg / ml, 160 pg / ml and 320 pg / ml. The injection volume was 40 pg / ml and 160 pg / ml 1 ml, respectively, and 2 ml 320 pg / ml was used to administer 640 pg injectable doses.

Thirty volunteers were divided into six groups of five volunteers. Two immunization regimens were studied: Scheme A, with vaccination at day 0, day 30, and day 120; Scheme B, with vaccination on Day 0, Day 30, Day 60, Day 120, Day 150, and Day 180. For each immunization regimen (Scheme A or B), there were three groups receiving different vaccine doses (Table 7 below). All vaccinations were performed by injection into the deltoid muscle. The trial lasted 10 months: 2 months of baseline evaluation and 8 months of benefit evaluation after initial vaccination.

Table 7 Immunization Schemes

Dana quantity gpl60 (pg) Day 0 30 00 120 100 100 The group I 40 40 40 The group - * » 160 160 160 The group 5 64 0 640 640 Scheme B The group 2 40 40 40 160 160 160 The group 4 160 160 160 640 640 640 The group 6 640 640 640 640 640 64 0

C. Assessment of fidelity and toxicity

Each of the volunteers was interviewed and screened on the day of the first injection and on days 1, 2, 3, 15, and 30 after each injection. We asked the volunteers about the possible occurrence of fever, chills, nausea, vomiting, arthralgia (painful joints), muscle rheumatism (muscle pain), nausea, hives (rashes), shortness of breath, dizziness or headache. Inspections were conducted to evaluate local reactions at the injection sites, red skin patches, swelling, itching, pain and tenderness, skin discoloration, skin lesions, changes in regional lymphadenopathy, functional changes at the injection site, and subcutaneous nodule formation. at the injection site. Monthly complete blood count, serum chemistry, coagulation profile and urine tests were performed and evaluated.

In vitro, cellular immune function was evaluated by T-cell phenotyping (whole lymphocyte, CD4, and CD8 cell phenotypes) as described in Rickman et al., Clinical Immuno. 52: 85-95, 1989; and in Birx et al., J. Acquir. Immune Defic. Sindr. 4: 188-196, 1991. Proliferative responses to mitogens (pokeveed and Con A) and control antigens (Candida albicans and tetanus) were also evaluated. Birx et al. supra. Cellular immune function was evaluated in vivo by hypersensitive delayed skin tests for antigen control (e.g., mumps, tetanus toxoid, Candida albicans and trichophyton).

Quantitative viral cultures of peripheral blood mononuclear cells (PBMCO) and plasma were evaluated as described in Burke et al., J. Acquir. Inanune Defic. Sindr. 3: 1159 -1167, 1991. DNA polymerase chain reaction (Wages et al., J. Med. Virol. 33: 58-63, 1991) and serum p24 antigen level were assessed by in vivo observation of HIV viral load.

There was no evidence of systematic toxicity, but local reactogenesis was observed in 87% of subjects (13 in each group). Local reactions included openings, tenderness, and transient subcutaneous nodules at injection sites; an increase in regional adenopathy has been rarely observed. No specimen declined additional injection. No difference was observed in the occurrence of local reactions, during primary immunization, additional injection or dosing.

There was no evidence of adverse effects on the immune system when measuring a specific proliferative response in vitro with mitogen and antigen, in vivo with a hypersensitive delayed skin test response, or by accelerating quantitative CD4 cell depletion. The baseline average of CD4 cell counts was 714 and 605 for vaccine susceptible and non-susceptible, respectively. The average CD4 cell counts during the days of the experiment (180-240) were 714 and 561 for the vaccine susceptible and non-susceptible, respectively. During the 240-day trial, the net difference in change in CD4 cell count averaged -0.2% for vaccine-susceptible, and for non-vaccine vaccine, the average varied by 7.3% (Figure 11). Vaccine-induced immunogenicity was not associated with evidence of aberrant CD4 cell depletion in any individual for the duration of the entire experiment.

To evaluate the possibility of accelerated replication of HIV and viral load as a consequence of vaccination, activity in quantitative avalanche and PBMC vital cultures, PBMS DNA polymerase chain reaction, and serum p24 antigen were measured in specimens in vivo. Quantitative cultures and DNA polymerase chain reaction assay showed that no changes were made during this experiment. Serum p24 antigen could not be detected in specimens.

D. Assessment of immunogenicity

Antibodies directed against whole HIV proteins were measured using both recombinantly obtained viral gene products gpl60, p66, p24 as well as the entire viral lysate of the HIV MN prototype strain. We used dot blot and Western blot techniques as described in Toubin et al. ,

Away. Natl. Acad. Sci. USA 76: 4350-4354 (1979). We also measured the antibody response to the specific epitope of the envelopes (see Figure 7).

In Figure 7, epitopes 88 (amino acids 88-98 in gpl20) and 448C (amino acids 448-514 in gpl20) were selected because we found that the antibody directed against these regions correlated with an early stage of HIV infection.

Epitopes 106 (amino acids 106-121 in gpl20), 241 (amino acids 241-272), 254 (amino acids 254-272), 300 (amino acids 300-340), 308 (amino acids 308-322), 422 (amino acids 422454) and 735 (amino acids 735-752) were selected because of the assumption of their functional importance. Epitopes 106 and 422 were included in the CD4 binding sites; epitopes 241, 254, and 735 were included in group-specific neutralization; epitopes 300 and 308 were included in type-specific neutralization.

Epitope 582 (amino acids 49-128) was selected for control because it represents an immunodominant envelope region for natural HIV infection, and other investigated epitopes include 49 (amino acids 49-128) and 342 (amino acids 342-405).

In Figure 7, shaded boxes represent a documented change in the envelope of the HIV-directed immune response. Shaded boxes with (=) represent the primary humorous response; shaded boxes with (+) represent a secondary humorous response; (-) represents antibodies negative to the specific epitope before and after immunization; (+) represents antibodies positive for the specific epitope before and after immunization, but without quantitative change. The shaded boxes with the sign (.) Represent a new T-cell proliferative response to gpl60 following immunization. The character (.) Itself represents a state without a cellular response to gpl60; while hb represents a high background (high

Ύ7 background) (cannot be interpreted); nd means not done.

Neutralization activity was measured against three prototype isolates (HIV-IIIB, RF and MN) in a syncytial inhibition assay, as described in Nara, Nature, 333: 469-470 (1988). HIV-specific cellular responses were measured using known lymphocyte proliferation assay test techniques, using gpl60, p24, and a control protein for the baculovirus expression system (Birx, supra).

E. Vaccine-susceptible and non-susceptible specimens

Specimens were considered vaccine susceptible only if the reproductive selective increase in both cellular and humoral immune responses against specific HIV envelope epitopes was related to vaccination series (Figure 7). Vaccine-induced humoral immunity is defined as seroconversion to specific HIV envelope epitopes and / or a secondary, additional immune response to specific envelope epitopes. Vaccine-induced cellular immunity is defined as the development of novel, reproducible, vaccine-induced proliferative responses to gp! 60 ⁹ . Subjects who develop neither a humoral nor a cellular proliferative response to the gpl60 or HIV envelope, or those who develop only a humoral or only cellular proliferative response to the gpl60 or HIV envelope are designated as vaccine-free.

F. Humoral response induced by vaccine

According to Figure 7, 19 out of 30 specimens (63%) indicated that with ⁹ This definition of susceptible vaccine is very restrictive in light of the scientific objectives of this experiment, ie. to achieve the effectiveness of post-infection immunization.

vaccine-induced, enhancement of both specific immune responses (humoral and cellular) to HIV gpl60. These 19 specimens were characterized as vaccine susceptible. Four, out of 11 vaccine-free, developed only a humoral or cellular response. All 7 subjects who did not show either of the two vaccine-induced responses received only three doses of vaccine (Scheme A). No changes were observed in the binding of the antibody to HIV polymerase (p66), the products of the structural gene (p24), and the non-HIV control antibody for tetanus. None of the specimens developed the antibody for the anti-baculovirus Lepidopteran cell control protein.

An increase in envelope antibodies (gpl60) was observed in 13 Western blot subjects using whole HIV-MN lysate. The changes were related to the immunization schedule. Three of the 15 specimens in Scheme A (20%) and 10 of the 15 specimens (67%). Scheme B shows an increase in antibodies for proteins from the envelope (P = 0.025 by Fisher's two-way accuracy test). All 13 specimens also seroconverted to specific envelope epitopes.

In contrast, 10 specimens that did not seroconvert to any specific envelope epitope showed no increase in Western blot antibody for the envelope. The remaining 7 specimens that seroconverted to only some specific envelope epitopes showed no antibody change for the entire viral envelope. No changes were observed in antibodies for HIV proteins without envelope.

Fourteen of the 15 specimens (93%) of Scheme B showed an increase in antibodies for total gpl60, as opposed to only 7 of 15 (47%) of Scheme A specimens (3 doses) (P = 0.01 Fisher, two-way). (Figure 7).

As shown in Figure 8, pre-immunization is dominated by post-vaccination for each specific gpl60 epitope, respectively, as follows: epitope 49 (27-70%), epitope 88 (28-52%), epitope 106 (50-87%) , epitope 214 (0-14%), epitope 254 (0-13%), epitope 300 (47-77%), epitope 308 (42-69%), epitope 342 (0-27%), epitope 422 (3 -10%), epitope 448 (73 -87%), and epitope 735 (17 -33%). Vaccine-induced seroconversion was observed against all specific epitopes except for epitope 582 (Figure 7). Antibodies (seroconversion a) directed against epitopes 241, 254, or 342 were detected only when accompanied by vaccination.

A secondary immune response was detected for the following epitopes: 88, 106, 300, 448C, and 582. The prevalence of antibodies directed against epitope 582 was 100% before vaccination, and only one subject (3%) showed a secondary immune response.

The pattern, with vaccination induced, of antibodies for the envelope epitopes was variable (Figure 7). Primary antibody response (seroconversion) to at least one epitope occurred in 20 specimens; 14 of 15 under Scheme B and 6 of 15 randomly under Scheme A (P = 0.005, Fisher, two-way). Schedule A specimens seroconverted to only 14% (15 of 110) of potential epitopes for which they had no pre-immunization antibodies. Schedule B specimens seroconverted to 60 of 190 epitopes (47%) (P <0.0001, Fisher, two-way). Seroconversion to three or more epitopes of the envelope occurred in 9 specimens (& 0%), randomly under Scheme B, but only in 2 specimens (13%) randomly under Scheme A (P = 0.02, Fisher, two-way).

Serum neutralization activities against three different strains (HIV-IIIB, MN, and RF) were determined on days 0, 9 0, and 195 in 7 specimens. Four out of five vaccine susceptible patients showed increased neutralizing activity on one or more isolates. We have also shown an increased ability to inhibit syncytium formation compared with non-syncytials.

G. Vaccine-induced cellular response

Changes in the cellular immune response were based on a comparison of the mean, pre-vaccination (baseline) and post-vaccination, lymphocyte stimulation indices (LSI), using the Wilcoxon test.

Twenty-one of 30 specimens (70%) developed a novel cellular proliferative response to gpl60 after vaccination (Figure 7).

Figure 9 shows the proliferative response to gpl60, p24, and baculovirus control protein in 4 typical susceptible specimens. In all specimens, gpl60 induced proliferation above baseline average from LSI 3 to LSI 10 (calculated using 4 values following last immunization). In contrast, no change was observed in the proliferative responses against the HIV p24 protein or for the control baculovirus protein.

Vaccine-induced changes in the average LSI values were illustrated in Figure 10 for all specimens, for specimens grouped by vaccine dependence, and for specimens classified by immunization regimens.

The change in proliferation response to gpl60 was clearly different between susceptible and non-susceptible subjects {<0.001, Wilcoxon, one-way). The proliferative responses to gpl60 induced by Scheme B (6 doses) were greater than those induced by Scheme A (3 doses) (P <0.10, wilcoxon, one-way).

Nineteen of the 21 specimens that developed proliferative responses to gpl60 also developed a humoral response (vaccine susceptible). The highest average LSI for gpl60 was 50.1 for all susceptible specimens. However, each response of susceptible individuals was different (the interval of values obtained was between 3 and 171) (Figure 7), as were the time and size ratios of vaccinations (Figure 9).

H. Discussing Results

Notwithstanding the limited number of samples in this experiment, many factors have been shown to be associated with vaccine immunogenicity of jo. six out of 15 (40%) specimens under Scheme A versus 13 of 15 (87%) under Scheme B were vaccine susceptible (P = 0.02, Fisher, bidirectional) (Figure 7), Out of 16 specimens with baseline average CD4 values greater than 600 / ml, 13 were susceptible (81%), in contrast, 6 individuals out of 14 had a baseline average CD4 value less than 600 cells per milliliter (P = 0.07, Fisher, two-way) ).

Multiple immunizations increase immunogenicity, even among patients with a baseline CD4 average of less than 600 ml, as shown in Table 8. For example, 5 of 6 specimens under Scheme B (6 injections) were susceptible compared to only l of 8 specimens. who received the vaccine under Scheme A (3 injections) (P = 0.03, Fisher, two-way), Table 8.

Table 8

Immune responsiveness to the gpl60 vaccine Baseline; CD4 and immunization regimens

CD4 value N ft Susceptible (%) tt Susceptible ..... (11

SCHEME A > 600 7 5 (71%) 2 (29%) 500-600 5 1 (20%) 4 (80%) <500 3 0 3 (100%) In total 15 6 (40%) 9 (60%) SCHEME B > 600 9 8 (89%) 1 (11%) 500-600 2 2 (100%) 0 <500 4 3 (75%) 1 (25%) In total 15 13 (87%) 2 (13%) TOTAL 30 19 (63%) 11 (37%)

The therapeutic use of vaccines was introduced by Pasteur in the 19th century for the treatment of rabies. The use of this method of treating other infections has quickly become widespread, although there are other examples of post-infection modification of viral-specific immunity (such as after hepatitis A or B), there are no well-known human studies to show that this method can be used to treatment of known or chronic viral infections.

Herein, the invention provides a viral-specific immune modification by active immunization after infection. A specific HIV envelope gene derived from the HIV envelope vaccine increased the human virus-specific humoral and cellular response of a human host in 19 of 30 specimens in the early stages of infection.

In the present study, we quantitatively and quantitatively measured different antibody responses to specific HIV epitopes in natural infection against postinfection immunization. In this way, we determined precisely, with vaccine-induced, humoral immunogenicity in 70% of already infected persons. For example, 20 subjects (19 susceptible and 1 non-susceptible) seroconverted to specific epitopes. Seroconversion, which occurred only due to vaccination, occurred in 10 people.

In addition, variations in the humoral responses of this vaccine, as characterized in the epitope map, will allow the development and efficient analysis of specific antibody responses, and will lead to unique opportunities to identify potential immunoregulatory mechanisms not exacerbated by natural infection.

Although the importance of serum neutralizing activity is unknown to date, observations of increased neutralization of serum activity against different strains of HIV (HIB, RF, MN) at 4 or 5 for vaccine-susceptible individuals suggest that postinfection immunization induces changes in functional antibodies. The test vaccine induces an increase in serum neutralizing capacity against certain HIV strains and will be a potential aid in defining a group of specific neutralizing epitopes.

The proliferative response to HIV envelope proteins rarely occurs with natural HIV infection. However, after immunization with gpl60, specific T-cell proliferation responses in 21 (70%) subjects were documented. The reason for this difference is unknown. A possible explanation is that the new proliferative response is directed toward the epitope of the vaccine envelope (as a result of the method for producing the vaccine or in vivo processing of the antigen). Alternatively, the protein used in the proliferation assay may not stimulate the proliferative response of primary T cells against the homologous envelopes of the wild-type natural virus. Evidence that vaccination stimulates the host cell's immune response exists: vaccine-susceptible specimens gave the HIV-IIIB type-specific cytotoxic T-cell response after additional immunization.

The factors responsible for the immunoresponse of the vaccine in HIV-infected individuals have not yet been elucidated. Even in early HIV infection, individuals respond suboptimally to different vaccines compared to appropriate controls. This hypersensitivity is correlated with early disturbances in B and T-cell function. Here, immunoresponsiveness was associated with baseline CD4 cell count, which is consistent with the hypothesis that the host immune status is an important indicator of vaccine responsiveness. The immunization schedule, with specific T-cell intervals, also affected the response to the vaccine: Scheme B (6 injections) was the best. The reduced vaccine response in subjects with a lower CD4 cell count can be corrected by an increased number of vaccinations; this indicates that further modifications to the dosage, regimen, adjuvants or formulations are needed to increase host immunoresponsiveness.

Although there have been many concerns about the safety of the HIV-infected host with active immunization with the HIV-specific vaccine, there is no evidence of immuno-specific toxicity. Quantitative cultures, DNA polymerase chain reaction assays, and serum antibody tests show increased in vivo HIV burden. An excellent indicator of HIV replication in vivo, CD4 cell depletion, was very useful in particularly susceptible subjects. The change in mean CD4 values was -0.2% for susceptible and -7.3% for non-susceptible. The data indicate that the postinfection immune response is not associated with an increase in CD4 cell destruction and suggests an association with decreased HIV replication in vivo.

The results of the vaccination were compared with data from ten infected and untreated persons of similar age, ethnic groups and similar baseline average of CD4 values. The mean CD4 values decreased by 8.6% in this group, 7.3% in scheme A group and increased by 0.6% in scheme B. These results indicate that postinfection vaccination with recombinant protein is possible HIV envelopes, moreover, the results are encouraging with regard to the prophylactic use of such vaccines.

FOR

Claims (12)

PATENT APPLICATIONS 1. A vaccine against infection caused by human immunodeficiency virus (HIV), characterized in that it contains the recombinant HIV envelope protein. Vaccine according to claim 1, characterized in that the recombinant protein is produced by insect cells by a baculovirus expression system. A vaccine according to claim 2, characterized in that the recombinant protein is expressed using the vector Ac3046, which is a baculovirus vector of insect cells. Vaccine according to any one of the preceding claims, characterized in that the recombinant protein is a recombinant envelope glycoprotein with a molecular weight of 160,000, a recombinant outer envelope glycoprotein with a molecular weight of 120,000, a recombinant transmembrane glycoprotein with a molecular weight, or a molecular weight. A vaccine according to any one of the preceding claims, characterized in that the recombinant protein contains about 757 consecutive amino acids of a glycoprotein with a molecular weight of 160,000, with 40 consecutive terminal amino acids of this glycoprotein being almost completely excluded. A vaccine according to any one of the preceding claims, characterized in that the molecular weight of the recombinant protein is about 145,000. 7. A vaccine according to any one of the preceding claims, characterized in that the recombinant protein agglomerates to form particles with a molecular weight of at least about 2,000,000. Vaccine according to any one of the preceding claims, characterized in that the recombinant protein is combined with an adjuvant. A vaccine according to claim 8, characterized in that a galun-based adjuvant is used. A vaccine according to any one of the preceding claims, characterized in that it is produced in single doses containing from about 10 to about 4000 ug of recombinant protein. A vaccine according to claim 10, characterized in that the individual dose contains from about 40 to about 1280 µg of recombinant protein. A vaccine according to any one of the preceding claims, characterized in that it is used to prevent and / or treat an infection caused by human immunodeficiency virus (HIV).