JPH06501851A - Smallpox seedlings (vaccine) and treatment methods for human immunodeficiency syndrome viruses - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Variola seedlings (vaccine) and treatment method for human immunodeficiency syndrome virus The present invention , a continuation-in-part of U.S. Patent Application No. 151,976, filed February 3, 1988. (currently Patent Application No. 585.266). and U.S. Patent Application No. 151 ,976 is U.S. Patent Application No. 920°197, filed October 16, 1986. It is also a partial continuation application.

Background of the invention Type 1 human immunodeficiency syndrome virus (HIV-1) is a retrovirus. Acquired immunodeficiency syndrome (acquired immunodeficiency syndrome), which causes infections with severe pathology of the immune system. Barre-3 inoussi is the pathogen of AIDS). et al.), Science Magazine 220:868-871 (1983) ; Popovicetal, Science Magazine 224: 479-500 (1984)). Clinical HIV-1 isolation occurs in lymph nodes Flame-related viruses (Feorino etal, ), Scien 225:69-72 (1984)') and AIDS-related viruses (Res. Levy et al., Science Magazine 225: 840 -842 (1984).

AIDS has become an epidemic, and the development of smallpox seedlings (vaccine) is the best option from a global health perspective. This is a pressing issue. A large number of people infected with AIDS have a deficiency of T-lymphocytes. Immune function is gradually lost. Like certain nerve cells, T-lymphocytes It has a molecule called CD4 on its surface. HIV-1 is a virus particle table Recognizes CD4 through receptors located in the face membrane, enters the inside of cells and proliferates, As a result, cells die. An effective AIDS smallpox vaccine is HI’V-1 infection of lymphocytes and other sensitive cells by attracting antibodies that attach to their surface membranes. To prevent.

Smallpox vaccines are administered to healthy people before they become infected with pathogenic bacteria as a means of immunoprevention. Generally, it is given. However, post-infectious therapy is recommended as immunotherapy against disease. In part, consideration should be given to using an effective AIDS vaccine for Ku, J. (SalK, J.), Nature, 327: 473-476. (1987)).

In developing smallpox seedlings (vaccines), the envelope (“env”) of HIV-1 is the most important. Francis, Bettricia 12 (Fra. ncis, Petricciani), New England Journal Mail Disson, 1586-1559 (1985); Vogt and Hirsch (v. ogt, Hirsh), Infectious Disease Review, 8:991-1000 (19 86); Fauci, National Academy of Sciences Bulletin USA, 83:9278-9283), the envelope protein of HIV-1 has a molecular weight of 160.0 It was first synthesized as a 00 glycoprotein (gp160). This early glycoprotein White (gp160) is envelope glycoprotein (gp120) with a molecular weight of 120.000. and a transmembrane glycoprotein (gp41) with a molecular weight of 41,000. this The envelope protein of these proteins is the main target antigen for antibodies in AIDS patients (e.g., valine, etc.). Barin et al.), Science Magazine 228: 1094-109 6 (1985)). HIV-1gp120 itself exhibits immunogenicity and is found in rodent necks, sheep It has the ability to induce neutralizing antibodies in rhesus monkeys and chimpanzees. (Robey et al.) National Academy of Sciences Bulletin USA, 83: 7023-7027 (1986)).

The level of the envelope protein of HIV-1gp120 itself in infected cells is low, and -1 Due to the risks associated with extracting AIDS pox seedlings (vaccine) from infected cells, Recombinant DNA methods have been applied to produce AIDS I (IV) for use as a vaccine. -1 envelope antigen has been generated. The technology of recombinant DNA methods is This is the best method for producing a vaccine. This is safe and economical This is because they have the ability to produce immunogens. HIV-1 envelope protein is a gene This is done by recombining the V7 Kushina virus that has been replaced (Chakrabar). Chakrabarti et al., Nature, 320:5 35-537 (1986); Hu et al., Nature Journal, 320:537-540 (1986); Kfeny et a. 1), Biotechnology, 4: 790-795 (1986)). child The envelope protein is produced within bacterial cells (Putney et al. t al.

), Science Magazine 234:1392-1395 (1986)), Mammal Microscope Cells (Lasky et al., Science Magazine, 23: 209-12 (1986)) and in insect cells. HIV-1gp41 A synthetic peptide derived from the amino acid sequence within is a promising candidate for an AIDS vaccine. (Kennedy et at, ) (19 86)) o However, no effective AIDS vaccine can be produced from this method or substance. could not be built.

Production of recombinant HIV-1 envelope protein using the rod-shaped viral insect cell vector system It is an aspect of this invention, which is pending with this application, that the assignor Co-owned U.S. Patent Application No. 920.197, filed October 16, 1986 (now (now U.S. Patent Application No. 585,266). See also Patent Application No. 151,976).

The rod-shaped virus system is useful for producing HIV-1 proteins and other proteins. It turns out that there is something. For example, the autografa caliph, which is a rod-shaped virus. Nuclear polyhedral structure of Autograph Ca1ifornica AcNPV was used as a vector to obtain the full-length gp160 and infected Spodoptera frugipe cells Various proteins were produced in Sf9 cells (Sf9 cells). Please note that the application is pending The patent application inside contains the dicot-shaped gp160 gene (recombination number Ac5O-4). 6), protein produced from recombinant number Ac3046, or protein produced from Ac3046 gene. A purification technique for manufacturing is disclosed. This purification technique includes lentil-lectin (lentil extract type cell aggregation resident) (lentil 1ectin) affinity cucumber Includes chromatography and gel permeation chromatography.

Of the 1 gp160 protein purified in this way, the one that is aggregated and made into cyanoblasts. was found to be an effective immunogen against rodents and primates.

The ideal AIDS vaccine would be biologically pure and non-pyrogenic. In addition, permanent protection against infection should be possible after several doses.

This requirement also applies to vaccines with attenuated activity. dead bacteria and bacteria When viruses and other substances are separated, substances such as poisons and proteins can be used as vaccines. However, these drugs have a weak antibody response and only have a short-term immune effect. I often don't. To compensate for this shortcoming, additional ingredients called adjuvants are required. Ru. This adjuvant has the effect of stimulating an immune response. Used in human vaccines Common adjuvants include aluminum salt gel (aluminum phosphate, hydroxide aluminum chloride) and is commonly referred to as an aluminum adjuvant (Ponford et al. (Bomford et al.), “Adjuvant”, Animal Cell Biotic Volume 2: 235-250, Academic Press, London: 1985).

The present invention provides smallpox seedlings (vaccines) and vaccines against human immunodeficiency virus (HIV). The purpose is to provide treatment methods for infected people and those who are likely to be infected. The essential requirement is to administer a modified HIV envelope protein. Preferred embodiment and Then, the envelope protein is purified, collected, and assisted for use as a vaccine. treated with a chemical agent (for example, aluminum).

Brief description of the drawing The details of the invention are described below in conjunction with the accompanying drawings.

Figure 1 shows the HIV-1 envelope gene (en) from E. coli plasmid pNA2. The cloning method used to separate v) is shown. The shaded area is HIV-I DNA In the array, blank parts indicate parts from the cloning vector. Plasmid p177 The black part in 4 is the synthesized oligonucleotide. plasmid p1614 at the SmaT position of the plasmid p1614. It was introduced as the I/KpnI piece. This synthetic oligonucleotide The array is shown.

Figure 2 shows the vector used to construct the recombinant plasmid vector (p. 3046). This method is used to construct a rod-shaped virus generation vector (Ac3046). used for Plasmid pMG S3 has a clone represented by 4.00. Sequences generated from rod-shaped viruses AcNPV at either of the printing positions (shaded areas) minutes). This position is unique to SmaI, KpnI, and Bgll. Contains nuclease restriction enzymes. A c N P V polyhedral promoter is 4 ．． It is in the direction of 5' from the 00 position. This array 5″-TAATTAATTAA- 3= is in the 3' direction and all three reading systems have a transcription stop codon (specific It has three consecutive nucleotides that form the genetic information that creates amino acids. plastic The sequences of Sumid p1774 and the synthetic oligonucleotide portion are shown in Figure 1. be. Plasmid p3046 contains the inserted HIV-1 envelope gene of p1774. Contains all of pMGS3 except for the sequence between Smar and BgI11 positions. I'm here.

Figure 3 shows the DNA sequences on both sides of the Ac3046 gp160 coding sequence. Reotide sequence is shown. 3046 env DNA sequence between +1 and +2264 is shown in FIG.

Figures 43 to 4 show en of Ac3046 (between +1 and +2264). HIV-1e shown with a synthetic oligonucleotide at the 5' end of the V gene. This is the actual DNA sequence of nv. The position of the endonuclease restriction enzyme is The amino acid sequences listed above are shown above the columns and the aforementioned amino acid sequences are shown below the DNA sequences. to each base are numbered on the left and right.

Figures 5ae to 5d show the sequence of env gene from Ac3046 with known L It is a figure which compares with the sequence of env gene from AV-1. The LAV-1 sequence is and Ac3046 is shown below. The lower line of the LAV-1 array (1 ) indicates that the sequences of AC3046 are identical at this position. DNA Sequence numbers are from Wine-Poplin et al.) The same cells as those described for AV-1 are used (cells, 40: 9-17 (1985)') Figure 6 shows human HIV-1 antibody positive serum (upper group). rough) and gp160 (IJ55, KL55) or gp120 (AB Rhesus monkey serum from animals immunized with 55, CD55, GH55) ( ELISA endpoint dilution titration for (bottom graph) is shown. This ELI SA end point Dilution titrations are performed on highly purified gp120 and gp160 proteins. . Specifically bound antibodies were measured against a sheep-anti-human IgG HRP pair.

The maximum dilution of serum that will give a positive response by this test is a titration.

Figure 7 shows gp160 vaccine induction for vaccinated seropositive patients. This is a table summarizing the immune responses caused by

Figure 8 (A and B) shows the work done directly against specific HIV enveloped epitopes. Showing cutin-induced immune response.

Figure 9 shows vaccine induction against gp160 for vaccinated seropositive patients. Shows conductor cell proliferation reaction.

FIG. 10 (A-C) shows the lymphocyte proliferation response associated with vaccination.

FIG. 11 shows changes over time in CD4 cells in responders and non-responders.

Summary of the invention Recombinant HIV-1gp160 envelope protein ('rgp160''), especially phosphorus When aluminum acid etc. are adsorbed to the adjuvant, it is particularly difficult to use as an AIDS vaccine. has been found to be effective. In one aspect of this invention, recombinant clone 30 The protein of the HIV-1 envelope gene related to amino acids 1-757 found at position 46. An AcNPV expression vector containing the coding sequence for the AcNPV expression vector is used. In other aspects, This invention relates to the production of recombinant HIV-1 envelope protein (and the protein itself) in insect cells.

In particular, the amino acid sequence encoded by amino acid sequence 1-757 (i.e. 03046) rgl) 160 protein.

In another aspect of the invention, to collect the 3046 protein and aluminum phosphate. Recombinant envelope protein from gene copies of recombinant rod-shaped viruses adsorbed by 3046 particles. Includes white refining and purifying.

The present invention provides prophylactic and therapeutic vaccines for AIDS and HIV infection, and It also includes methods of prevention and treatment against cancer and HIV infection.

Detailed description of the invention The following examples are not intended to limit the invention.

Recombinant rod-shaped autographa californica nuclear polyhedral virus (AcNPV) ) is the dicot-shaped HIV-1gp16 of the HIV envelope protein (recombinant Ac3046). 0 code, which indicates that pending patent application bundle no. 920.197 ( Currently, the patent application number is 585,260). Construct recombinant rod-shaped virus-containing genes. The cloning procedures used to create the It is also included here as a document.

Below are the genetic techniques used to construct the Ac3046 expression vector. The order is described in detail. The substances used here are enzymes and immune reactants. It is commercially available. The manufacturing method and application examples of the present invention are also described. .

Other recombinant envelope proteins, collectively referred to as rgp16o, are considered to be Contains recombinant gp120 and gp41 proteins. AC3046 is An example of an expression vector according to the present invention is also a recombinant envelope protein.

Example 1 Rod virus recombination with HIV-1 code sequence for amino acids 1-757 Generation of A c 30 = 46 The heteroprotein expression coding sequence in rod-shaped virus vectors is, on the one hand, Polyhedrin promoter and upstream sequence On the other hand, they are aligned with the rod-shaped virus chord sequence.

In this sequence, the recombination corresponding to the rod-shaped virus gene is Trigger transcription of foreign coding sequences alongside phosphoprotein promoters and inactive injection genes. Rub.

Therefore, various insertion vectors have been created for the production of HIV envelope genes. ing. The insertion vector, NfGS3, is an ATG transcriptional opener, as described below. It is used to supply the start codon. When inserting a foreign object array into this vector, to ensure that the transcription system generated by the transcription start codon or the foreign sequence is maintained correctly overall. It is carried out in a state where Insertion vector NlG53 purifies plaque EcoRI-I restriction fragment clone of 7' DNA obtained from the AcMNPV isolate (WT-1,). 20\1Gs3 has the following structure It has the following characteristics.

(a,) 400C1bl upstream from the ATG start codon of the pleiotropic gene) There is an array of

(b) It has a high degree of linkage that induces the generation of variants in the positional index form, and corresponds to polyhedral codons. A T G start codon located at the corresponding position, restricted ISma L Kpn L It consists of Bgl1■ and a universal stop codon fragment.

(C) EcoRI-r clone from the Kpnl restriction position inside the polyhedral gene. (1700 bl) extending to the terminal EcoRI restriction site 1 (see Figure 2). .

Example 2 Rod virus with LAV env coding sequence for amino acids 1-757 Generation of string replacement The recombinant plasmid, shown as NA2 in Figure 1, contains the entire plasmid inserted into pUc18. 21. of HIV-1 tropic viruses. Consists of skb sections. to certain human cells This clone is considered infectious because it becomes a virus after being infected. (Adachi et al., Journal of Virology, 59: 284-291 (1986)). The complete envelope gene sequence contained in NA2 is , derived from the LAV species of HIV (Barre Shinosi). -3inoussi), 1983).

The HIV-1 envelope gene is isolated and processed as follows (see Figure 1). . This envelope gene is a 3846 bp EcoRI/5acI restriction fragment from NA2. It was initially isolated as an EcoRI/5acl restriction site pUc19. -Yun be logged. The resulting plasmid is identified as p708.

The envelope gene was then reisolated as a 2800 bp KpnI restriction fragment. , cloned into the KpnI restriction position of pUC18. The resulting crawl The code is identified as p1614.

This KpnI restriction fragment at p1614 is a small fragment of the HIV envelope gene. Since it contains a shape part, the sequence corresponding to 121bp at the N end is lost. . This missing portion of the gene contains a derivatized peptide sequence and is a double-helix monomer. (oligomer) has been inserted. The inserted monomer is The LAV amino acid sequence sequence is created using the appropriate polyhedral gene codons. This legacy To facilitate gene recombination operations, a new Sma in place of the ATG start codon I restriction sequences are introduced at the same time. ATG start codon is inserted into the rod virus insertion vector. Powered by Tor. The resulting plasmid was identified as p1774. It will be done.

In Figure 2, p1774 contains coding sequences in various regions of the HTV-1 envelope. These restriction fragments were cloned into the NiG5 insertion vector (e.g. MGS3). Therefore, the ATG start codon of the insertion vector is assembled with the codon of the envelope gene. It is included. The structure of p1774 is Sma of plasmid vector pMGS3. SmaI/Bam isolated from p1774 inserted at position I/BglII Consists of the HI restriction fragment. This clone contains amino acids 1-7 of gl) 160 57 and a termination code supplied by the 5fGS3 vector. I am using de.

Example 3 Creation and selection of recombinant rod-shaped viruses HIV-1 env gene silk recombinant virus Rasmid p3046 was precipitated with AckiNPV (WT-1). Subodoptera frugiberda (Spo. doptera frugiperda) cells. Then , the hypothetical gene is inserted into the AcMNPV gamete by homologous recombination. recombination virus has an obstructive negative morphology. It is identified by e plaque morpholOg. This kind of disease , exhibits a cell-destructive effect but does not cause nuclear occlusion. Two additional evil purifications followed. is carried out to obtain pure recombinant viruses. This recombinant virus DNA By comparing these limitations and hybrid characteristics with wild viral DNA, It is analyzed to identify the insertion position into the env sequence.

Example 4 Expression of HI V e n v from recombinant rod-shaped viruses in infected 1-worm cells Expression of HIV env sequences from recombinant viruses in insect cells results in The primary transcription substance is synthesized. This primary transcription material is derived from the recombinant vector. Consists of amino acids transcribed from supplied codons. As a result, polyhydrin protein Downstream from the white promoter to the transcription end guide on the expression vector (e.g. rgl) 160) A protein containing all the amino acids for the AGTI start codon of the expression vector located in is generated. AC3046, which is the primary transfer substance, is originally from Arg (position 4). In the LAV clone of , it is Arg at position 2, but at the terminal position it is Met. -Pro-G　y-A　r　g　-Va　1. Met-Pro- Gly:+t is provided as a result of cloning techniques.

Example 5 gp160 insert and DN A nucleotide sequence on both sides DNA and g The nucleotide sequence of the p160 insert was derived from the viral expression vector Ac3046. Determined by restriction fragments isolated from DNA. Such an array technique follows the steps below. by. The 3.9 kb EcoRV-BamHI fragment was isolated from the Ac3046 virus. Purified by limited incorporation of form DNA. This Ac3046 virus type D NA is an extracellular virus present in the vector cells used for vaccine production. It was created from.

As shown in Figure 2, the 3.9 kb EcoRV-BamHI fragment contains the entire gp 160 genes, from 100 bp upstream and about 1000 bp downstream of both sides of DNA Become. Of these, the entire gp160 nucleotide gene sequence is the upstream 100 bp and 1000 bp downstream of both sides of the DNA.

In short, the sequencing results show that the hypothetical structure is as predicted by cloning techniques. Appeared. The sequence of gp160 is based on the general information reported by Wein-Poplin et al. (1985). It was ri. The 2253 groups existing between position 1, which is thought to be the transcription initiation site, and the termination codon The sequence reveals 751 amino acid codons and 28 potential N-linked glycosylation positions. is predicted. This rgp160 calculated molecular weight is approximately 14 including the remaining sugar content. It is 5.000. Through continuous analysis of 200 groups on both sides of pNA, Figures 3 and 4 were obtained. As shown in FIG. 5, it can be seen that correct insertion is shown.

Example 6 Amino acid sequence of gp160 Automated Edman degradation and HPL Using the C method, we determined whether the N-terminal part of the first 15 residual parts of gp160 was a DNA sequence. It was determined that the result was the same as that predicted. The N-terminal methionine is gp16 It is not present in the o protein. This means that the AcNPV polyhedral protein has only one N end. This is theoretically consistent with the finding that it is produced without the terminal methionine. In the end, the fruit The sequence of gl)160 DNA and N-terminal protein is AcNPV 3046 As determined by analysis of DNA and purified gp160, the (Table 1).

Table 1 LAV env gene remnants in AcNPV 3046 expression vector 2 3 4 5 6 7 F3 9 10 11 12 13 1.4Pro Gly Arg Val Lys Glu Lys Tyr Gln His Leu　Trp　Arg　Trp1y ATG CCCGGG CGT GTG to AG GAG AAG TACCA To CACCTG TGG CGT TGGGG Table 2 is obtained by comparing these results with the original LAV-1 clone.

Table 2 LAV env gene remnants in the original LAV-1 clone 1, 2 3 4 5 6 7 8 9 10 11 12 13 14Met ArgVal Lys Glu Lys Tyr Gin His Leu T rp Arg Trp Gly, to TG AGA GTG ~ AG GAG AG TAT C to G CACTTG TGG AGA TGG GGG Example 7 Purification of recombinant gp160 In one aspect of the present invention, an A.C3046 expression vector encoded A manufacturing method is used in which the HIV-1 envelope protein is extracted and purified. Recombinant HrV -1 envelope protein gp160 is expressed in S, Frugiperda ( It is produced in the cells of S. frugiperda). Purification of rgl)160 is , is performed in the following steps.

1. Cell washing 2. Cell lysis 3. Gel permeation chromatography 4. lentil -Lectin (Lectin obtained from lentils) Affinity chromatography 5. dialysis This example shows recombinant gp16 obtained from 2×10′ infected cells of Ac3046. A method for purifying 0 is described.

1.Cell washing Infected cells were treated with 50mM Tris buffer (Tris buffer pH 7.5). , in a buffer containing 1 mM EDT A and 1% Triton X-100. and then washed. Cells were suspended in this buffer and homogenized by standard techniques for 5 Centrifuge for 20 minutes at 000 rpm. Repeat this procedure three times.

2. Cell lysis Infected cells were grown in 5QmM Tris buffer (pH 8,0-8.5), 4% deoxyco Deoxycholate and 1% beta-mercaptoethanol It is dissolved in the chamber by sonication. This impulse sonic method is carried out using conventional techniques. After performing the shock sonic method, only the remnants of the nuclear membrane remained intact and these were removed by centrifugation at 5000 rpm for 30 min. removed. Surface floats containing extracted gp160 are intact cells and optically Confirmed by microscopic observation.

3. Gel penetration The gel penetration operation was performed using a gel filled with Sephacryl resin (manufactured by Pharmacia). Pharmacia's 5. Performed in an OX50cm glass column. The total thickness volume is It is approximately 1750m1. This glass column and joints are made non-heat generating and can be cleaned. Add at least 6 liters of 0.1N sodium hydroxide to the glass to Leave it on the pillar for 24 hours. The effluent from this glass column is sent to a UV-flow cell, monitor and a chart recorder (Pharzacia), with a 4-liter gamer. equilibrate with permeation buffer. Untreated gl) 160 was packed in a glass column. and treated with gel permeation buffer. This glass column allows this untreated The mixture is separated into three large UV adsorbing components. The first peak is the buffer It appears between 500m1 and 700m1. Then, the second peak is about 700m 1 and 1400 m1, and the third peak is approximately between 1400 m1 and 1900 m1. appear. This same phenomenon shows that the first peak is essential and has a molecular weight of 2,000,000 It is also observed by a small analytical column that determines to show 0 or more.

This peak is translucent due to precipitation of high molecular weight lipids and lipid:mixtures. Ru. This peak contains 10% to 20% of gp160 extracted from infected cells. include. Apparently, this part of gp160 is complexed with self or nl. is combined with other components to form a high molecular weight precipitate.

The second broad peak contains most of gp160 and has a molecular weight of approximately 18,000. Contains about 200,000 open white matter.

The third peak contains beta-mercaptoethanol (β-mercap) in the data. toethanal), most of it is UV adsorbed, except for a small amount of protein. It is.

As a result of tracking UV adsorption, when the second peak was first detected, the glass column The effluent from the lentil-lectin column was column) directly. When the second peak disappears from the glass column The effluent was collected from a lentil lectin column. Remove it and throw it away.

4.Lentil Lectin (obtained from lentils) cell aggregating substance) lentil-lectin affinity gel medium (Lentil Lec tin-3epharose 4B) is purchased in bulk from a pharmacy. lentil - Lectin is Sephadex (spherical particles for chromatography) (Sephadex) dex) and is separated to a purity of over 98% by affinity chromatography. Then , to Sepharose 4B using boron cyanide. It is fixed by joining. This mother liquor is approximately 2 mg per 1 ml of gel. Contains lentil-lectin (as a ligand). lentil-lectin The column was a 5.0 cm x 30 cm glass column (Pharmacia). 125ml of lentil-lectin Sepharose 4B (Lentil L ectin-Sepharose 4B) gel (low-resolution of substances with high molecular weight) filtration media).

This mother liquor is reused after being thoroughly washed, using the method specified by the supplier. be reproduced. If not used, the gel will contain 0. 9% NaCl, 1mM nCl2.1mM CaC1□ and 0.01% thimerosal (thimmer osal) is stored in a glass column containing a solution of This glass column is Washed after each use as previously described and added with 250 ml of lentil-lectin buffer. is balanced.

Untreated gp160 is exposed to gas as it flows out of the gel permeation column as described above. Transferred to Raskaram. Untreated gp160 is connected to this glass column and 800ml containing 0.1% deoxycholate of lentil-lectin buffer. Under these circumstances, all g p160 is attached to a glass column. 0.3M α-methyl-manocide The attached glycoproteins are dissolved by using a lentil-lectin buffer containing Let go. This is observed via a UV monitor in the wavelength range of 280 nm.

5. Dialysis Sugars and deoxycholate (deoxycholate) can be separated by standard dialysis techniques. s) is removed. Purification of gp160 from 1 liter of infected cells was , are summarized as in Table 3 below.

In the pond embodiment of the present invention, instead of gel permeation, conventional ion exchange chromatography is used. Rafi (anionic and cationic) is used. Also, the order of the steps may vary. It's not important. For example, gel permeation methods or ion exchange chromatography are This may be followed by a lectin-lectin purification procedure. According to the invention, other reaction reagents may be used. For example, in recombinant protein purification, deoxycholate (deoxycholate), other cleaning agents may be utilized. child For these, Tween 20 (Polysorbate 20 ), Twe e n 80. Lubrol and Triton X Contains non-ionic cleaning agents such as -100 (Triton X-100) .

Table 3 Summary of purification procedure Example 8 Components of A0gp160 particles In one aspect of the invention, during the purification step the gp160 antigen has a molecular weight of 2. Oo It was found that the particles were composed of o, ooo or more particles. This gp160 protein is , 80-90% monomer (molecular weight 160,000) and 80-90% multimer (grain) It is a protein extracted from cells as a mixture of particles. Through the gel penetration process, g Remove p160 aggregates. Due to the purification process of gp160 (gel permeation glass filter) gp160 is complexed with other cells. , or the possibility that it is composited with membrane fragments. However, gel soaking For the gp160 antigen, the second peak in the clear glass column was approximately 160.00 Since it has a molecular weight of 0 to 300,000, it becomes a monomer or a dimer. Being is dominant.

gp160 aggregates and multimers are formed during the lentil-lectin treatment process. . At a critical micelle concentration (CMC) of 0.2% for deoxycholate. Lectin color in 0.596 deoxycholate or 0.1% deoxycholate. The antigen may take an aggregated form depending on how it is eluted from the glass column in can be determined.

The size of the aggregates was determined by high-resolution FPLC Superose 12 2) Measured using a column (Phannacia). purified gp16 A representative sample of 0 is approximately 2,000,000 mol. It has a size that is approximately equal or slightly larger. Schbara et al. According to cross-linking studies by Waller et al. (1989), insect cells gl) 160, which occurred within the same group, was found to be a trimer within the congener. This lab Studies have shown that gp160 in HIV-infected cells and virus particles is a trimer. I also know that. Thus, compared to what is seen in untreated HIV gp150, Similarly, recombinant gp160 particles should also exhibit tertiary and quaternary structures. It is.

Appropriate tertiary structures form the epitope necessary to properly wrap gp160. It is important for Non-glycated protein is used as gp160 antigen during binding and washing steps. gp16 is removed from its attachment to the lentil lectin column and transferred to the lentil-lectin column 0 non-hydrophilic proteins begin to form intramolecular bonds. The concentration is greater than or equal to the CMC. , deoxycholate (deoxycholate) because it forms a complex with antigens. late) will not bind to gp160. The product purified according to the present invention Therefore, it is an inherent property of this protein itself that the components of this antigen form an aggregate. Dew. Due to the extremely non-hydrophilic N-terminal sequence part of the gp160 protein, It is thought that particle formation may contribute to the neutralization of this protein. After purification, gp 160? J (coalescing <0.2 micron cellulose without substantial loss of protein) Sterilely filtered through an acetate filter.

Analysis of B0 particle formation According to the analysis results of purified gT) 160 particles by electron microscopy, 30 to 1100 It has been found that it is a spherical particle in the protein state of n.

Additional experiments confirming the presence of this particle that purified gp160 is This was analyzed using the transmission method. Approximately 100 micrograms of gp160 is 12 on a FPLC gel permeation HRIO/30 column (Pharmaeia). Added. The column is initially calibrated with a protein molecular weight standard solution. child The protein formers from the column are highly reproducible and the adsorption volume is inversely proportional to the molecular weight of the protein standard solution. This column allows monomeric gp160 to be Globular proteins with a molecular weight of 2×10 6 or more are separated from multimer formations and removed. child column, almost all of the purified gp160 was eluted. There will be no capacity. Therefore, the molecular weight is 2X106 (2,000,000) That's all.

Example 9 A. Adsorption of gp160 to aluminum The effectiveness of the insoluble conjugate as an immunological adjuvant depends on the completeness of the adsorption of the antigen to the solid phase. depends on. In some aspects of the invention, the aluminum composite comprises gp160 aluminum. at a pH that does not reduce the potential of the micomplex as an immunogen and which inhibits gp160. It has been found that it is actually adsorbed. This aluminum complex (concentrated acid The elements to be controlled in the process of forming aluminum are as follows.

1. The optimum pH for antigen adsorption to aluminum is approximately 5.0. however, It was found that gp160 loses its immunity at pH 6.5 compared to pH 7.5. Therefore, aluminum is formed in the pH range of 7.1±0.1. In this pH range It has also been found that almost 100% of gp160 is adsorbed by aluminum.

2. The ionic force generated from NaCl is relatively low, less than 0.15M.

3. A molar excess of aluminum chloride compared to sodium phosphate has a negative effect on surface floaters. This indicates that phosphate ions are not included.

4. gp160 is added to newly formed aluminum to inhibit crystal growth and reduce grain size. Minimize child size.

Prepare 200ml of aluminum using the above procedure and adsorb it onto purified gp160. Therefore, the final concentration of antigen is 40 μg/ml as shown below.

Preparation of B1 reaction reagent (200ml total standardization solution) The following solution is sterilized and non-pyrogenic. Prepare a 100ml bottle or beaker. Then, add the salt to solution 1 and solution 2. and sodium hydroxide, and 0.2 micron cellulose acetate fibres. After filtering with a filter, it is sterile and non-C, forming aluminum. 1. Add 25ml of sterilized solution 1 (aluminum-sodium-acetate). Add to the standard container using a disposable pipette. At this time, keep the volume of solution 1. Stir solution 1 carefully.

2. Add 25ml of solution 2 (sodium phosphate) using a sterile disposable pipette. and add it to the standard solution container. Precipitate while continuing to stir. In addition, at this time, the Note the volume of liquid 1.

Add 3.3 ml of solution 3 (sodium hydroxide) and continue stirring for 5 minutes. 0゜5 Take a ml sample and measure the pH. If the pH is below 7.0, add 0° Add 5 ml of sodium hydroxide and continue stirring for another 5 minutes to adjust the pH of the sample again. Measure. This operation is continued until the pH is between 7.0 and 7.2.

4. Determine the total volume to be added to the container for the reference solution (solution 1 + solution 2 + solution 3) Then add sterilized WFI to make 100ml.

Purified gp160 in 100ml of 5.1mM Tris pH 7.5 was added to 8.000ml of 5.1mM Tris pH 7.5. Add the icrogram directly to the standard container.

6. Continue stirring for 20 minutes and then dispense the reference vaccine into the sterile vial.

Example 10 Immune ability of aluminum adsorbed gp160 (special Ab reaction) created antigen (vaccine) A common method for determining immunity is to administer a single dose of antigen to a group of mice. The purpose of this study is to measure specific antibody responses to specific antibodies. Four weeks after administration, mice Blood is exsanguinated to determine serum antibody levels against specific antigens using, for example, ELISA. Measured using standard tests. Here is the specific antigen that normally immunizes the animal. An antigen used for In addition, ELISA uses Enzyme 1 inked i Abbreviation for mmunosorbent assay (enzyme-linked immunosorbent sample). Ru.

For murine immunity of purified gp160, pH 6.0 and pH 7.5, for aluminum adsorption as described in Example 9 and When mixed with Freund's complete adjuvant, the following (Figure 4) ) is summarized as follows.

Table 4 1 μg None, pH 7,587020, 14057% 476 None, pH 6,0 87020, 11026% 27'7 aluminum 8702 1.000 90% 9/10 Aluminum 8705 2.285 100% 6/'6 Freund 86 04 1.10883% 576 Freund 8702 1.396 100% 7/70.1μg Freund 8604 0.434 67% 47'6a Rumi 8705 1.003 67% 4/62, mice 28 days after immunization Blood was drawn, gel purified, and 1=10 in ELISA reagent for gp160. Serum is tested by dilution. Commercially available ELISA reagents (Dientic's EIA Even when using ELISA), the ratio of 1:400 to untreated HIV-1 protein was Similar results were obtained for diluted serum.

3. It is the ratio of the number of seroconverted mice (P) to the total number of tests (N).

Mice immunized with a single dose of 1.0 micrograms of gp160 received no supplementation. Even in the absence of adjuvants, antibody responses against gp160 are elicited as shown in Table 1. death However, a more potent antibody response appears to be due to gp160 adsorbed with aluminum adjuvant. 1. In the case of O microgram administration. Freund's complete adjuvant mixture or Single throw of less than 1.0 microgram of gp160 when adjusted by aluminum It can be seen that the seroconversion rate was over 50% among immunized rats. .

The antigen of gp160 is an unprepared antigen at pH 7.5 to pH 6.0. There is no immunity in this species, but immunity is lost in low pH ranges.

Example 11 Immune ability of aluminum adsorbed gp160 (ELISA serum study) Candidate vaccine or immunity Having the ability to elicit a response is a very important biological property. Al The gp160 vaccine prepared by the human body confers immunity to animals and this The following experiment was conducted to confirm that Lumil supplement enhances immunity.

First, on the day zero (day 0), a group of 10 mice was given 0.5 μg 1 Single administration of 1.0 μg or 5.0 μg of gp160, aluminum-adsorbed gp16 0 and a single dose of Freund's complete adjuvant (CF A) treated gp160. was given. After 28 days, the rats were bled and the serum was analyzed by ELISA (dilution: 1:10) was used to test for the presence of antibodies against gp160.

Results from sera tested on day 28 are summarized in Table 5 below. all A seroconversion rate of more than 50% was observed in mice. in all administrations , number of serum conversion rates and mean serum adsorption (O values at 1=10 dilutions in ELISA reagents). The results show that aluminum adjuvant significantly improves the immunocompetence of gp160 antigen. are doing.

Table 5 28 days after injection Dose 0.5μg Dose 1.0μg Dose 5.0μg Average value Average value Average value P/N' OD5 P/N OD P/N ODgp160 9/10. 40 7 7/10. 699 7/10. 430gp160 (alum) 9/1 0. 547 8/10. 797 10/10 1.347gp160 (CF A) 10/10 1.130 10/10 1.967 10/10 1.3 17': VaxSyn″m0HIV-1 0.5 μg, 1.0 μg, 5. The number of converted mice (N) relative to the total number (N) at 28 days after immunization with a dose of 0 μg. Sponsor's ELIS for gp160 in serum dilutions of ratio 5.1.10 of P) Average adsorption (OD45o) of seroconverted mice as measured by reagent A Example 12 Neutralization materials The HIV-1 neutralizing reagent allows the created antibody to inject Hr into cultured human lymphocytes. Common methods used to determine whether to prevent the V-1 virus from infecting This is a great method. Antiserum from animals immunized with gp160 was used as an HIV-1 neutralization assay. It was tested in medicine and the results are summarized in Table 6 below.

Indicates the amount of micrograms.

7: Only 50% infected compared to HIV-1 infected cells exposed to serum from non-immunized animals The maximum dilution of antiserum that inhibits is shown.

Guinea pigs, rabbits, and monkeys are also made of aluminum or Immunization with gl) 160 using Freund's adjuvant It was done. Generally, immunization of these animals is effective against HrV-1 envelope protein. A good antibody response was shown.

Example 13 Immunity to chimpanzees Genetically speaking, chimpanzees are the closest relatives to humans; is the only animal example of HIV-1 infection.

In a safety/immunity experiment in three chimpanzees, two chimpanzees immunized with 40 μg and 80 μg of aluminum-adjusted vaccine gp160. . These include 40 μg and 80 μg of gp160 for 4 weeks or boosting immunity. It was administered as follows. The remaining control chimpanzees received 1 ml of saline.

Serum samples were analyzed from each chimpanzee on a weekly basis and analyzed for gp160 and HIV-1 viruses. Antibodies against virus antigens were investigated. At this time, three immune samples, namely pure 1601. : On the other hand, Temiron Energy Co., Ltd. (MicroGeneSy) ELISA reagent developed by S., Inc.), Western plot analysis Reagent (Jestern Blot analysis) and commercially available HIV-I Each ELISA reagent was used. The results of these analyzes are shown below.

A, ELISA (7g search HI V 160 (MGSearch) I lff 160)) ELISA reagent is ELISA reagent and MagSearch H IV160 is an immunoadsorbent for gp160, which is described in this application and Both applications are pending and are listed in patent application No. 920.197 with the same assignee. (now patent application no. 585.266). In addition, mug search is is a trademark of Microgenesis, Inc., Meriden, Connecticut. .

Serum samples taken before immunization and 11 weeks after the first immunization vaccination were 1:10 to 1:100.000 and then 100 μg per spot cultured on nitrocellulose pieces containing purified gl) 160. The end of this case Endpoint dilution titration detected with goat anti-human TgG alkaline phosphatase complex The highest dilution that tested positive for anti-gp160 antibodies, as shown. The rate was

control chimpanzees and vaccinated pre-immune chimpanzees. Serum samples were negative. Chimpanzees receiving 80 μg of 1: A chimpanzee that tested positive at a dilution of 100 and received 40 μg A weekly dilution of 1:10 was positive. Infusion of antibody against gp160 Continued until overuse, at which point endpoint dilution titrations were approximately 1:100 and 0, respectively. 00 and 1:2.000,000. Antibodies in both chimpanzees Titration decreased slightly with overuse from week 6 to 11.

This type of reaction occurs in chimpanzees vaccinated against the human hepatitis B virus. The antibody response is similar both quantitatively and qualitatively to the antibody response commonly observed in G.

B. Commercial ELISA test VaxSyn-immunized chimpanzee serum Stern plot analysis and MagSearch HIV 160 ELISA ( As is clear from MGSearch HIV 160 ELISA), Lindsay has been seroconverted and now has antibodies against recombinant gp160. Ta. Additionally, vaxSyn refers to the AIDS vaccine described here. It continued to be added under the trademark name of Microsienesis, Inc. 40 μg of gp160 was administered. The chimpanzee fed tested positive at a 1:100 dilution for 6 weeks.

Example 14 Distribution of antibodies between gp120 and gp41 against gp160 in vaccinated animals Is the antibody response shown to gp120 or gp4 or both? It was important to check whether this was the case. This immunological method involves Epidemiological precipitation method (RIP), immunofluorescence method (IF), Western plot analysis ( WB) and quantitative ELISA methods.

Figure 6 summarizes the immune activity against three different recombinant antigens.

The three different recombinant antigens are: CART] [TAB] (1) gp120 ~Delta (approximately 40 amino acids are missing from the C-terminus of the molecule; recombinant HIV-1gp120); f:ART] [TAB] (2) (recombinant [ART] [TAB] C 3) Refers to gp160.

Sera from 50 HIV-1 antibody-positive humans and pooled sera from 3 humans. Serum is highly active against gp160 and moderately active against gl)120. There are few or no antibodies against gp120. Met. Dimoid gp120 accounts for more than 90% of the coat glycoprotein of HIV-1. , likely to contain determinants for protection. Human AIDS positive serum is found in this package. The finding that there are almost no antibodies against the proteinated region indicates a viral infection. The immune response may not be sufficiently protective or, in routine experiments, positive human sera. This is consistent with the fact that the compound shows only low neutralizing activity.

In contrast, lees immunized with gp160 or truncated gp120 Sas monkeys have antibodies that strongly react with the HIV-1 envelope protein of gp120. do. Differences in the distribution of antibody recognition sites along the viral envelope and in monkeys The relatively high titer explains that monkey serum has a high neutralizing titer. .

Immunology of these three recombinant envelope antigens in human serum and immunorhesus monkey serum. Quantitative evaluation of infectious disease activity is shown in Figure 7. Animals immunized with gp160 sera from all monkeys tested contained sera from the gp120 antigen (gp1). 20-delta).

These results demonstrate that recombinant gp160 occurs during natural infection in rhesus monkeys. It has been found that this drug induces an antibody response that is different from the antigen response that occurs most often. These are immune The epitope in the gp120-delta region of gp-160 is often recognized in these are not found in the human immune system during infection. this These novel epitopes are important for protection against HIV-1 and This may be an important feature of recombinant gp160 in the prevention and treatment of infections.

Example 15 Therapeutic vaccination A clinical trial was conducted on 30 (IV) seropositive patients, and the HIVgp160 cloned against (as mentioned above, rod-shaped virus The efficacy of the vaccine produced by the system was determined.

Vaccination with recombinant gp16cl resulted in the death of 30 HIV-seropositive volunteers. Of these, 19 (63%) reported gp160HIV-specific body fluid and cellular responses. There was an increasing trend. Of the 15 volunteers who received six doses of the vaccine. 14 (93%) showed an increase in total gp160 antibodies. Therefore, the group recombinant HrV proteins (i.e. rgp41, rgp120, rgp160 and mixtures thereof) have been effectively administered in strategies to treat patients infected with HIV. You could say that.

In the embodiments of the present invention, the effective amount of HIV protein is determined based on the known techniques shown below. determined. Generally, such effective amount is 1 kg body weight (Kg) of the infected patient. It is within the range of μg to 100 μg. The number of doses for administration also depends on the known It will be determined by the method. Preferably, a person having ordinary knowledge in the field of the present invention As is well known, parenteral administration, i.e. intravenous, intraperitoneal It is administered by intramuscular or subcutaneous administration.

A. Applicant selection Among the HIV-infected patients, 30 volunteers were recruited. Early stage of HIV infection Seropositive volunteers at the Walter Reed stage defined as Tier 1 or Tier 2, these persons are eligible for registration. It was. What is Walter Reed Stage 1 or Stage 2? , lymphoma (1ympbader+.

Regardless of whether pathogenesis occurs or not, the number of CD4 cells will increase to 400 after 3 months or more. (Redfield et al. ), New England Journal Medicine, 314:131-132 (1986)). Additional applicant criteria is limited to adults between the ages of 18 and 50. , normal blood cell counts, no end organ disease, and no active status for 12 months prior to the study. He was not a alcoholic or drug addict. All patients were treated using random sampling. They underwent a 2-month baseline evaluation before being included in the treatment group. Retrospective VV during the trial administration of anti-retroviral drugs or slow-acting drugs. No one received it.

Of the 30 applicants, 26 were male and 4 were female. Of these, 14 people 13 were white (Caucasian), 13 were black, and 3 were Spanish. The average age is , was 29 years old (age range 18 to 49 years). Eight of the registered candidates are Walter Reed is in stage 1, and the other 22 are in Walter Reed stage 1. I was in Luther Reed Stage 2. The basic mean CD4 count was 668 (3 88 to 1639). Average time taken from first diagnosis to becoming a research subject The period was 24 months (range 3 to 49 months).

B. Vaccine production and immunization plan As already explained, the test vaccine is a recombinant protein expressed by rod-shaped viruses. It consists of a non-infectious subunit glycoprotein derived from gp160. Immune proteins are Manufactured from lepidopteran insect cells and biologically purified, the final vaccine formulation Therefore, it was adsorbed on aluminum phosphate.

Three dosing regimens of gp160 were used. i.e. 40 per milliliter μg, 160 μg per milliliter, 320 μg per milliliter It was μg. Injection volume was 1 ml for both 40 μg and 160 μg. For the 640 μg dose, 2 ml of 320 μg per milliliter was used.

Thirty volunteers were divided into six groups of five people each. Two immunization plans: plan A and plan B. was planned. In Plan A, vaccine doses are divided into 0130 and 120 days. Ta. Plan B has vaccine doses of 0.30.60.120.150 and 18 It was divided into 0 days. Within each immunization regimen (A or B), as shown in Table 7 below. , three groups received different doses of the vaccine. All vaccines are It was administered by intramuscular injection into the deltoid muscle. The experiment duration was 10 months. the first Two months are baseline evaluations and the next eight months are supplementary evaluations from the first dose.

Table 7 Immunization plan gpieo dosage (μg) Dose 0 30 60 120 150 180 Plan A 1st group 40 40 40 3rd group 160 160 160 5th group 640 640 640 Plan B 2nd group 40 40 40 160 160 160 4th group 160 160 160 640 640 640 6th group 640 640 640 640 6 40 640C8 Safety and Toxicity Evaluation Each volunteer was tested on days 0, 1, 2, 3, 15, and 30 after administration. I met with each patient on the same day and examined them. For each of these applicants, we will ask whether or not they have a fever and if they are cold. Qi, stiffness, nausea, arthralgia (pain in the joints), myalgia (skinny muscles), discomfort , hives (temporary skin symptoms), coughing, glare, or headaches. did. Dosage via vaccine injection! Tests evaluating local reactions for erythema , swelling, itching, pain, hypersensitivity, skin discoloration, rough skin, local changes in lymphoma, Functional changes at the entry end and formation of a subcutaneous layer at injection site I were investigated. Monthly Blood Reach Ball count, serum chemistry, coagulation surface, and urine analysis were also evaluated.

Cellular immune function in vitro is characterized by T cell-phenotype (total lymphocytes, CD4, and CD8). Phenotype) was evaluated by (Rickman et al. ), Clinical Immunology 52: 85-95.1989; Birx et al. t al,), Journal of Acquired Immune Deficiency Syndrome 4:188-196, 1991) . Mitogens (Pokeweed and Concanavalin A) ~aliX1A) (mitotic lectin produced from jack beans)) and Proliferation of T cells against control antigens (Candida albicans and tetanus doctor) physical responses were also assessed (Birx et al., supra). Control antigens (Rubella rubella, tetanus toxoi Albicans albicans and trichophyt Delayed skin hypersensitivity test to It was.

For quantitative virus culture of peripheral blood mononuclear cells (PBMC) and plasma For more information, see Burke et al. and Acquired Immune Deficiency Syndrome. 3:1159-1167.1991. DNA Polymerase chain reaction (Wages et al.), medical Eels, 33:58-63.1991) and serum p24 antigen levels. It was evaluated by observing the HIV virus load in the body.

Although no evidence of systematic toxicity was found, local reactive activity was observed in test subjects. It was observed in 87% (13 people in the vaccine group). Local reactive activity is associated with tissue induration. , hypersensitivity, temporary subcutaneous layer formation at the injection site, and worsening of local secretion disorder. Almost nothing was discovered. None of the subjects refused the booster dose.

The frequency of local reactions is determined by the initial immunization, booster doses, or multiple doses. No difference occurred.

Mitogenic and antigen-specific proliferative responses in vitro and delayed-form skin hypersensitivity in humans Adverse effects of the immune system, as measured by test response or accelerated quantitative C4 cell loss. The basis for this was not found. Baseline average CD4 cell count indicates vaccine response The number was 716 in those who did not respond to the vaccine, and 605 in those who did not respond to the vaccine. The average CD4 cell count is , 180 days and 240 days from the experiment date, vaccine responders and vaccine non-responders. The numbers were 714 and 561, respectively. During the 240-day experiment, the average CD4 The net change in cell count was minus 0.0 in vaccine responders. 2%, and the vaccine There was a 7.3% decrease in non-responders (see Figure 11). induced by vaccine HIV immunity caused by accelerated reduction of CD4 cells in the subjects during all steps of the experiment. It was not related to the observed phenomenon.

As a result of vaccination, HIV virus proliferation and viral load in subjects In assessing the increase in viral activity in the body, plasma quantification, P BMC virus culture, PBMC-DNA polymerase chain reaction and p24 anti- This was done by measuring the serum levels of the original. This virus culture and DN In the A polymerase chain reaction experiment, no changes were observed during the experiment. serum p24 No antigen was detected in the subject.

D. Evaluation of immunity Recombinantly produced viral genes gp160, p66, p24 and HIV Using both the original and variant MN whole lysed viruses, antibodies against whole HIV proteins were detected. was measured. Toubin et al. Published in Academy of Science Bulletin USA 76:4350-4354 (1979) dot blot and western A plot method (Western blot) was used. specific epitope Antibody responses were also measured (see Figure 7).

Figure 7 shows epitopes 88 (amino acids 88-98 in gp120) and 4. 48C (amino acids 448-514 in gp120) was selected. This is g It has been reported that antibodies against this region of p120 are correlated with early HIV infection. This is because there is.

Epitope 106 (amino acids 106-121 in gp120), 241C Amino acids 241-272) in gp120), 254 (amino acids in gp120 acids 254-272), 300 (amino acids 300-340 in gp120) , 308 (amino acids 308-322 in gp120), 422 (gp12 0 (amino acids 422-454) and 735 (amino acids 73 in gp120) 5-752) was selected. This was chosen for its putative functional importance. It is. Epitopes 106 and 422 are involved in CD4 binding. Epito 241.254 and 735 are involved in group-specific neutralization and are associated with epitope 300 and 308 are involved in type-specific neutralization.

Epitope 582 (amino acids 582-602) was selected as a control.

This is because it relates to the immune-dominated region in natural infection with HIV. the study Additional epitopes of interest include 49 (amino acids 49-128) and 342 (amino acids 49-128). Contains amino acids 342-405).

Figure 7 shows that the shaded boxes indicate changes in the HIV envelope-directed immune response. is recorded. The shaded box marked with (=) is the first body fluid reaction; The shaded box with +) is the second humoral response. Also, the (-) sign indicates exemption. Indicates negative antibody against specific epitope before and after epidemic, marked with (+) sign. indicates antibody positivity to specific epitopes before and after immunization. in this case , it is assumed that there is no quantitative change. The shaded box with (,) indicates gp1 associated with immunity. 60 shows the neo-T cell proliferation response to 60. On the other hand, only the mark (1) indicates gp160. Hb showed high background d) J (uninterpretable), nd means [not done J] do.

The neutralization activity was published by Nara (Nara) in Nature magazine (333: 469-470゜ As described in (1988), three types of protoplasmic isolates ( HIV-II IIB, RF and MN). HIV-specific cells Reactions were performed using gp160, p24 and rod virus expression system control proteins (described above). It was measured using a known lymphocyte proliferation sample using the method described by Kuss et al.

E. Cells directed against HIV envelope-specific epitopes in vaccine responders and vaccine non-responders. Reproduction-selective increases in both the and humoral immune responses are associated with serial vaccination. In some cases, subjects were classified as vaccine responders only (see Figure 7). vaccine Humoral immunization induced by seroconversion to HIV envelope-specific epitopes, Alternatively, it is defined as a second, enhanced immune response to the envelope-specific epitope. Wa Cellular immunization induced by cutin induces a new regenerative, viral response to gp160. It is defined as the development of a proliferative response associated with cutin inoculation. For vaccine responders The definition of From the ground up, it is extremely restrictive. Subjects with no humoral or cell proliferative response; Alternatively, subjects who show only a humoral response or only a cell proliferation response may not receive the vaccine. Classified as a responder.

F. Vaccine-induced body fluid responders As shown in Figure 7, 30 subjects Of these, 19 (63%) showed both gp160-specific and cellular immune responses. did. These 19 subjects were classified as vaccine responders. 11 people Four of the non-responders showed only a humoral response or only a cell proliferation response. won. For all seven patients in whom no vaccine-induced reactions were detected, 3 They received only one vaccination (see Plan A). HIV polymera (p66), structural (p24) genetic material, or non-HIV control antigens. No changes in antibody binding to Clostridium tetani were detected. any subject However, no antibodies against the rod virus lepidoid cell control protein were observed. .

An increase in envelope antibody (gp160) was observed in mice using HrV-MN with total virus lysis. It was detected in 13 subjects using the Estern plot method. This change is caused by immune In Plan A, 3 out of 15 subjects (20%) In the other cases, 10 out of 15 subjects (67%). In plan B, the envelope protein (P = 0.025. Fisher's two tails) (according to the law precision test). All 13 subjects underwent seroconversion to specific epitopes. Indicated.

On the other hand, 10 of the subjects showed no serovariance for any specific epitope. The western plot method also showed an increase in antibodies against the encapsulated antibody. Didn't show it. The remaining 7 subjects who developed seroconversion to specific epitopes The Western plot method did not show any changes in the overall viral envelope antibody. Ta. No changes in antibodies to non-enveloped HIV proteins were observed in any of the subjects.

As with Plan B, 6 doses, 14 of 15 subjects (93%) showed an increase in antibodies against gp160. On the other hand, plan A's 3 pitches In the given case, only 7 of 15 subjects (47%) showed showed an increase in antibodies (P = 0.01, by Fisher's two-tailed test). ) (see Figure 7).

As shown in Figure 8, gp160 specificity after vaccination compared to before vaccination. The incidence of epitopes is as follows. Epitope 49 is 27-70% cents, 28-52 percent for epitope 88 and 50 percent for epitope 106. ~87% for epitope 214, 0-14% for epitope 25 For epitope 4, O-13%; for epitope 300, 47-77%; Tope 308: 42-69%, Epitope 342: 0-27% 3% to 10% for epitope 422 and 73% for epitope 448C. -87% and 17-33% for epitope 735. Waku The seroconversion induced by Chin induced all specific epitopes except 582 (Figure 7). I was seen by a friend. Antibodies against epitope 241.254 or 342 (seroconversion) ) were detected in the following cases.

Secondary immune responses were detected for the following epitopes. That is, 88.1 06.300.448C and 582. Antibody development against epitope 582 The survival rate was 100% before vaccination, but only one subject (3%) survived the second vaccination. showed a secondary immune response.

The form of vaccine-induced HIV antibodies directed against the enveloped epitope is shown in Figure 7. are diverse. Initial antibody response against at least one epitope (seroconversion) ) occurred in 20 subjects. Regarding this breakdown, in Plan A, out of 15 people, 14 people, and 6 out of 15 people in Plan B (P = 0.005 (according to Tussier's two-tailed test). For subjects in Plan A, seroconversion The cases were among 110 people who did not have antibodies against the epitope before immunization. Of these, only 15 (14%) responded. For all subjects in Plan A, seroconversion was performed. Only 60 (47%) of the 129 people experienced this (P < 0.00 01 Fisher's two-tailed test). 3 or more enveloped epitoes Seroconversion to Plan A occurred in 9 patients (60%) enrolled in Plan B; This occurred in only 2 patients (13%) (P = 0.02, Fisher's (by two-tailed test).

Serum neutrality against three different types (HIV-I IIB, MN and RF) The activation activity was based on 7 subjects on days 0, 90 and 195 after vaccination. Determined by Four of the five vaccine responders had severe or severe isolation. It showed strong neutralizing activity against substances. These vaccine responders are vaccine non-responders. It was found that it has the ability to strongly suppress multinucleate formation compared to .

G. Vaccine-induced cellular responses Changes in cellular immune responses Using Jilcoxon rank sum test + Jilcoxon rank sum test) Average pre-vaccination lymphocyte stimulation index (basic value LS and average post-vaccination lymphocyte stimulation index) Based on a comparison with the parabulbar stimulation index (LSI).

Of the 30 subjects, 21 (70%) received a new T after immunization with gp160. A cell proliferation reaction was occurring (see Figure 7).

Figure 9 shows gp160, p24 and rod VE in a typical vaccine responder. Figure 3 shows a proliferative response to the virus control protein. In all subjects, gp160 Proliferation was promoted, and the average prevaccination lymphocyte stimulation index (basic value LSI) was 3. (Following the final immunization below, the 4-value average Calculate the average). In contrast, against HIV p24 or the control rod virus protein, No changes were observed in the proliferative response.

All subjects, subjects divided into subgroups by vaccine response, and immunization regimen Vaccine-induced changes in mean LSI values in subjects grouped by are shown in FIG. 10, respectively.

Proliferative responses to gp160 are distinct in vaccine responders and vaccine non-responders. There was a difference in opinion (<0.001. Wilcochran method, two-tailed test). plan The proliferative response to gp160 induced in Plan A (6 doses) was (3 doses) than the proliferative response to gp160 induced in (<0.10, Wilcochran method, two-tailed test).

Nineteen of the 21 subjects developed a proliferative response to gp160 and also They also showed humoral reactions (vaccine responders). Maximum average lymph for gp160 The spherical index (LSI) was observed in all subjects and was recorded as 50.1. but However, regarding the reactions of each vaccine responder, as shown in Figure 7, the top of the LSI The cell values for gp160 vary, ranging from 3 to 171. The magnitude and duration of the response to the vaccine seem to be in a temporary relationship (see Figure 9). (see).

Discussion about H1 results In this experiment, although the sample size was limited, several elements were It has something to do with immunity. In Plan A, 6 out of 15 subjects (40%) In Plan B, 13 out of 15 subjects (87%) were vaccine responders (P= 0, 02 Fisher method, two-tailed test) (see Figure 7). per liter Thirteen of the 16 subjects (8 1%) were vaccine responders. On the other hand, less than 600 per liter Of the 14 subjects with an average CD4 count of The patient was a chin responder (P=0.07, Fisher's method, two-tailed test). Table 8 Subjects with an average number of CDJs of 600 or less per liter, as summarized in It can be seen that a multifaceted immunization method improves immune competence even in patients with severe disease. example For example, in Plan B, 5 out of 6 subjects (6 doses administered) were vaccine responders. In contrast, in Plan A, only 1 out of 8 subjects was vaccinated after 3 doses. It was only a chin responder (P=0.03 Fisher method, two-tailed test) (See Table 8).

Table 8 Based on basic value CD4 < GP160 vaccine immune reactivity and immunization schedule CD 4 numbers N #Responses (%) #Non-responses (%) Plan A >600 7 5 (71% > 2 (29%) 500-600 5 1 (2ON ) 4 (8ON) <500 3 0 (OX) 3 (100%) Total 15 6 (40%> 9 (60%) Plan B >600 9 8 (89%) 1 (11%) 500-600 2 2 (100% )0 (0%) <500 4 3 (75%) 1 (25%) Total 15 13 (87%) 2 (13%) Total 30 19 (63%) 11 (37%) Waku The therapeutic use of chin began in the 19th century as a treatment for acute rabies infections. Introduced by Le. However, for treatment of other infections, this Research using this method has not been carried out extensively. For hepatitis A and B infections Example of treatment for post-infection mitigation of virus-specific immunity, such as treatment for virus-specific immunity Although there are various types of virus infection, immunization is effective against confirmed or chronic virus infection. There have been no systematic studies on its use in humans.

The present invention provides virus-specific immunization therapy by active immunization after infection. Ru. In particular, vaccines against gp160 derived from the HIV gene envelope Accordingly, 19 out of 30 patients with initial HIV infection were infected with viruses specific to humans. Increased humoral and cellular responses.

This study demonstrated that the specific HIV epitopes during natural infection and post-infectious immunization were Differences in antibody responses between these two groups could be measured both qualitatively and quantitatively. In this way, the feeling Accurate determination of vaccine-induced humoral immunity in individuals infected with 70% of subjects were systematically recorded. For example, 20 subjects (19 were vaccine responders, One patient (a vaccine non-responder) was seroconverted to a specific enveloped epitope. Waku Seroconversion involving only chin (epitopes 241, 254 and 342) This occurred in 0 subjects.

Furthermore, changes in humoral responses to this vaccine are characterized by epitope mapping. This study provides a promising perspective on the causal relationship of specific antibody responses, as provides a unique opportunity to characterize immunoregulatory mechanisms not derived from natural infection. will give.

The in-vivo incidence of serum neutralization activity is currently unknown, but the vaccine For each of the four responders, each epitope species (II IBSRF, MN) The observation of strong neutralizing activity suggests that post-infection immunization induces functional changes in antibodies. It would suggest that. Vaccines under investigation are targeted against individual epitope species. Increases neutralizing capacity and helps define neutralizing epitopes specific to group classification. There is a possibility that it will stand.

A proliferative response to the HIV envelope protein rarely occurs in natural HIV infection. stomach. However, after immunization with gplso, the specific T-cell proliferation response A response was recorded in 21 subjects (70 percent).

The reason for such a difference is not clear. one possibility and As a result, new proliferative responses may occur against vaccine-specific epitopes. It can be pointed out that (as a result of vaccine production method or alternative to internal antigen processing) ). Alternatively, the protein used in the growth reagent is a homologous “wild type” of the natural virus. It is possible that the epitope does not stimulate the initial T-cell proliferative response to the epitope. .

However, there is additional evidence that vaccination stimulates cellular immune responses in humans. It is known that progress will be made. According to selected vaccine responders, after immune enhancement HIV-IITB type-specific cytotoxin T-cell responses were demonstrated.

The factors governing vaccine responsiveness in HIV-infected individuals remain unclear. This is something that should be made clear. Considerable control measures even in early stage HIV infection responds moderately to various vaccines compared to This high reactivity is due to the initial It is associated with cell suppression and T-cell dysfunction. Vaccine immune reactivity is It is involved in the basic CD4 cell count, and this means that the human immune status affects the vaccine response. This is consistent with the hypothesis that it is an important determining factor. However, specific T-cells Immunization regimen within a range of numerical changes affected vaccine responses. Plan B (6 (doses administered twice) were excellent. Immunization of the subject if the CD4 cell count is low The responsiveness decreases, but this decrease can be improved by increasing the number of vaccinations. There was potential. This may also include the number of vaccinations, regimens, adjuvants or This suggests that human immunity can be improved by improving the formulation.

Safety for HIV-specific vaccine products when actively immunizing HIV-infected individuals Although there are concerns about the toxicity, there was no evidence of immune-specific toxicity.

According to quantitative cultures, DNA polymerase chain reaction samples, and serum antibody samples, We saw an increase in the HIV burden in the body. Excellent in-vivo labeling for H V regeneration and CD4 cell The rate of attrition makes it more effective, especially among subjects classified as vaccine responders. There was a significant difference in the influence. The average CD4 cell count change for vaccine responders is , -0.2%, and -7.3% in vaccine non-responders. These materials are Post-infection immunization reactivity does not contribute to a decrease in CD4 cell numbers, but reduces HIV reproduction in the body. suggests that it is related to.

Vaccination results in this study were compared to age, race group, and basal CD4 cells. The numbers were compared to a database of 10 infected, untreated humans. This comparison An 8.7% decrease in mean CD4 cell count in the group This corresponds to a decrease of 7.2% for subjects enrolled in Plan B and 0% for subjects enrolled in Plan B. ．． This corresponds to a decrease of 6%. These results suggest that recombinant HIV envelope protein is not effective. Post-infection vaccines are practical, and there are generally no concerns regarding their use for preventing infection. It can be seen that this shows promise.

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Claims (36)

[Claims] 1. Human acquired immunity characterized by administering recombinant HIV envelope protein to an infected person How to treat people infected with the virus. 2. The recombinant HIV envelope protein is approximately 1 microgram to 1 microgram per kilogram of body weight. 00 micrograms is administered as a single dose. A method for treating a human acquired immunodeficiency virus infected person according to item 1. 3. The recombinant HIV envelope protein is administered in a single dose of approximately 10 μg to approximately 4000 μg. The human acquired immune system according to claim 1, characterized in that the human acquired immunity is administered as Treatment method for people infected with HIV infection. 4. The recombinant HIV envelope protein is administered in a single dose of approximately 40 μg to approximately 1280 μg. The human acquired immune system according to claim 1, characterized in that the human acquired immunity is administered as Treatment method for people infected with the epidemic virus. 5. Claim 3, characterized in that at least three doses are administered. A method for treating a person infected with a human acquired immunodeficiency virus as described in . 6. Claim 4, characterized in that at least 6 doses are administered. A method for treating a person infected with a human acquired immunodeficiency virus as described in . 7. At least each dose should be administered 30 to 60 days apart. Human acquired immunodeficiency virus infection according to claim 5, characterized in that How to treat dyers. 8. At least each dose should be administered 30 to 60 days apart. A person infected with human acquired immunodeficiency syndrome according to claim 6, characterized in that treatment method. 9. sufficient to elicit an HIV-specific cellular response or an increased humoral immune response. A method for post-human therapy, characterized in that it comprises administering HIV envelope protein to an infected person. Treatment method for people with congenital immunodeficiency virus infection. 10. The recombinant protein is produced from a rod virus insect cell expression system. Human acquired immunodeficiency virus infected person according to claim 1 characterized in treatment method. 11. The recombinant protein is produced from a rod virus insect cell expression system. Human acquired immunodeficiency virus infected person according to claim 3 characterized by: treatment method. 12. The recombinant protein is produced from a rod virus insect cell expression system. Human acquired immunodeficiency virus infected person according to claim 5 characterized by: treatment method. 13. The recombinant protein is characterized in that it has a molecular weight of approximately 145,000. Treatment of a person infected with a human acquired immunodeficiency virus according to claim 1 Method. 14. The recombinant protein is characterized in that it has a molecular weight of approximately 145,000. Treatment of a person infected with a human acquired immunodeficiency virus according to claim 3 Method. 15. The recombinant protein is characterized in that it has a molecular weight of approximately 145,000. Treatment of a person infected with a human acquired immunodeficiency virus according to claim 5 Method. 16. The HIV envelope protein is one of gp160, gp120 and gp41. Acquired human disease according to claim 1, characterized in that there is at least one Treatment method for immunocompromised virus-infected patients. 17. The HIV envelope protein is one of gp160, gp120 and gp41. Acquired human disease according to claim 3, characterized in that there is at least one Treatment method for immunocompromised virus-infected patients. 18. The HIV envelope protein is one of gp160, gp120 and gp41. Acquired human disease according to claim 5, characterized in that there is at least one Treatment method for immunocompromised virus-infected patients. 19. The recombinant protein was obtained by the rod virus insect cell vector Ac3046. The human body according to claim 1, which is adapted to be expressed. Treatment method for acquired immunodeficiency virus infected patients. 20. The recombinant protein was obtained by the rod virus insect cell vector Ac3046. The human body according to claim 3, which is adapted to be expressed. Treatment method for acquired immunodeficiency virus infected patients. 21. The recombinant protein was obtained by the rod virus insect cell vector Ac3046. The human body according to claim 5, which is adapted to be expressed. Treatment method for acquired immunodeficiency virus infected patients. 22. The recombinant protein has a molecular weight of at least about 2,000,000. The human acquired immunity according to claim 1, characterized in that it is aggregated in children. Treatment method for people infected with the epidemic virus. 23. The recombinant protein has a molecular weight of at least about 2,000,000. The human acquired immune system according to claim 3, characterized in that it is aggregated in children. Treatment method for people infected with the epidemic virus. 24. The recombinant protein has a molecular weight of at least about 2,000,000. The human acquired immune system according to claim 5, characterized in that it is aggregated in children. Treatment method for people infected with the epidemic virus. 25. The patent claim is characterized in that the recombinant protein is combined with an adjuvant. A method for treating a human acquired immunodeficiency virus infected person according to item 1. 26. The patent claim is characterized in that the recombinant protein is combined with an adjuvant. A method for treating a human acquired immunodeficiency virus infected person according to item 3. 27. The patent claim is characterized in that the recombinant protein is combined with an adjuvant. A method for treating a human acquired immunodeficiency virus infected person according to item 5. 28. recombinant particles formed into particles having a molecular weight of at least approximately 2,000,000 A composition comprising an HIV envelope protein and an aluminum adjuvant is administered to an infected person. A method for treating a human acquired immunodeficiency virus-infected person characterized by: 29. The recombinant protein is produced from a rod virus insect cell expression system. Human acquired immunodeficiency virus infection according to claim 28 characterized in How to treat people. 30. The recombinant proteins include recombinant gp160, recombinant gp120 and recombinant gp120. p41, the recombinant HIV envelope protein has a molecular weight of approximately 145,000. , the recombinant protein is expressed by the rod virus insect cell vector Ac3046. The human acquired disease according to claim 28, characterized in that: Treatment methods for immunocompromised virus-infected patients. 31. The recombinant protein comprises approximately 757 concatenated amino acids of gp160; A patent claim characterized in that approximately 40 terminal chain amino acids of 160 are substantially excluded. The method for treating a human acquired immunodeficiency virus infected person according to item 28. 32. The recombinant protein is administered in a single dose of approximately 10 μg to approximately 4000 μg. Human acquired immunodeficiency according to claim 28, characterized in that Treatment method for people infected with virus (HIV). 33. The recombinant protein contains a recombinant HIV envelope protein and an aluminum adjuvant. characterized by being formed into particles having a molecular weight of at least approximately 2,000,000. A therapeutic HIV vaccine composition. 34. The recombinant HIV envelope protein is approximately 10 μg to 4000 μg per dose. Therapeutic H according to claim 33, characterized in that the amount of μg is adjusted. IV vaccine composition. 35. The recombinant protein is produced by a rod-shaped virus insect cell expression system. 35. The therapeutic HIV vaccine composition of claim 34, characterized in that: 36. The recombinant protein comprises approximately 757 concatenated amino acids of gp160; Claims characterized in that approximately 40 terminal amino acids of 160 are substantially excluded. 35. The therapeutic HIV vaccine composition according to paragraph 34.