JPH04501654A - Production method of natural HIV GP160 - 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] HIV GP160' The invention described in this specification is provided by the National Cancer Institute. e.

Department of Health and Human 5ervi ces Contract No.

It was done during research conducted under N0I-CP-67694.

Light j eye and LL The present invention is directed to human immunodeficiency virus, which is the pathogen of acquired immunodeficiency syndrome (AIDS). The company is involved in the production of native gp160 from the US (formerly known as V).

Human immunodeficiency virus (I) is now known as acquired immunodeficiency syndrome (AIDS). ) is a well-documented pathogen.

This virus exhibits tropism for cells that have the CD4 antigen and also has helper antibodies. 1 (IV envelope), which has a large cytopathic effect on inducer (T4) cells. The Rho1 gene product is synthesized as a gpieo precursor molecule, and this product is further Envelope protein gp120 and transmembrane protein gp41 becomes. between gp160 and the smaller proteins gp120 and gp41 The precursor/product relationship is similar to the amino acid sequences of these three proteins. It is now well described in the literature [^11an et al., 5science, 228 : 1091-1094 (1985) and Veronese et al., 5science, 229:1402-1405 (1985)], the lateral glycoprotein gp120 is The initial phase of virus cell fusion and the formation of giant cells induced by the virus. [Dalgleish et al., Na ture, 312 Near 63-766].

In addition to its role in cell surface receptor recognition and cell fusion, HIV gp120 and g941 is the main target for immune recognition in individuals infected with IIIV. the Therefore, these proteins are of particular interest in virus neutralization research and vaccine development. Another large segment of gp120 expressed by the recombinant DNA method that has attracted attention. Natural gp12G purified from infected cells or IIIV-infected cells is mainly found in animals. was found to generate type-specific neutralizing antibodies. ing. In addition, HI expressed in insect cells harboring a baculovirus vector The V-envelope precursor protein gp160 plays a role in strong type-specific immunity in goats. caused a reaction (Rusche et al., PNAS US^, 84:6924-6 928 (1987)].

Infecting cell lines of different species with HIV and continuously infecting the infected cells with the complete virus. A method for establishing a producer is disclosed in U.S. Pat. No. 4,652,599. The cell line infection method of the present invention and the old V variant strain have also been disclosed [Ge tchell et al., J, Cl1n, Microbiol, 23 Near 37-74 2 (1986)].

However, these factors alone do not allow the HIV glycoprotein gp180 to be synthesized. No method has been established that can produce it in 15 hours. Natural gp160 is normally g It is degraded into ptzo and gp41. Obtained from cell culture media or lysed virus The envelope proteins involved are gp120 and gp41. Therefore, gp16 It is truly amazing that 0 can be produced in its natural form.

Glycoprotein gp160 has hitherto been produced only by recombinant methods. deer However, recombinant gp160 differs from native gp160, especially with regard to glycosylation. different. These differences are particularly important for the development of neutralizing antibodies against viruses. Viral tropism and harbor epitopes are unique to the HIV envelope. HIV vaccine research because it is determined by the rope glycoprotein This will have extremely important meaning.

light m One of the objects of the present invention is that chronic infection with HTLV-III-s+ The unconventional HU releases the complete viral glycoprotein gptso into the extracellular medium. The objective is to provide a T78 cell clone.

Another object of the invention is to ensure that the cell line releases gp160 in its native form into the culture medium. The purpose of the present invention is to provide an infinitely proliferative cell line grown in a serum-free medium under the same conditions.

Yet another object of the present invention is to provide the complete gp160 of HIV in its native form. It is in.

These and other objects and advantages of the present invention are directed to the infectious cell line 8D5.3. No Isolate native gpiso grown in serum medium and released into the medium by cells. This is achieved by

1 riffl Figure 1 (lane 1) shows glycoproteins eluted from lentil lectin Sepharose. The E profile is shown in 5OS-PA.

Lane 2 shows purified gptao stained with Coomassie blue.

Figure 2 shows the release of IIIV-specific proteins by 605<s+ cells. . Panel A: Media from cells grown in FCS; Panel B: 1 (in BIOI) Lane 1: HIV-positive human serum; Zone 2: rabbit Anti-FITLV-IIIs gp41; L/-in 3: Rabbit anti-121 peptide (Centocore); Lane 4: Goat anti-HTLV-111-gp120 ; Lane 5: Normal h) serum; Lane 6 + normal rabbit serum; Lane 7: Normal goat serum.

Figure 3 shows HIV-induced syncytosis by HTLV-1114s + glycoprotein. It shows the damage caused by umbilical formation. CEM cells are Molt-3/[IT LV-IIl, cultured with cells. Cell photos were taken 48 hours later for 7 viruses. To investigate the effect of glycoproteins, CEM cells were tanned prior to the co-culture. It was pre-incubated with protein for 1 hour. Panel A: Untreated CEM cells: t < * Le B: CEM [cells + Not t-3/HTLV-IIIm cells; Panel C2 CEN cells pretreated with glycoproteins from uninfected 6D5 cell cultures. Cells; Panel D: OEM cells pretreated with 6D5<i>$8 protein. 4th The figure shows the binding of CD4 to gp, 120 and gp160.

6D545 a-identified with 3SS-methionine. Conditioned culture medium at 2000 x g This medium was clarified by centrifugation and then filtered through a 0.45u filter. /21 is CEMs with a total amount of 21 as described below.

incubated with cells. Immunoprecipitation of bound proteins with 0KT4 antibody Ta. Lane 1: 0.5x 10' cells; Lane 2: 1xlO' cells ; Lane 3: 2xlO' cells; Lane 4: 5xlO' cells; Lane 5 : 10x 10' cells; Lane 6: 20x 10' cells.

I want it, of;■ A single cell clone of 11UT78 cells was isolated from human immunodeficiency virus type 1 (formerly v-1). ), thereby making the infected cell line a continuous producer of virus. Black 6D5 was obtained by Getchell et al.

C, C! In, Mierobiol, 23 Near 37-742 (1986 111V-H:: susceptible to chronic infection, clone 6D5 was isolated from H The infected cell line 6D54 was infected with a specific strain of IV-1, HTLV-1114, l. 0 forming sl then 6D5. , pellet the cells and place them in serum-free medium (e.g. For example, resuspend in IIBIOI medium (commercially available from DuPont). In the practice of the present invention, the infected cell line is grown in serum-free medium. Serum-free medium HB104, also available from DuPont, may also be used. .

Glycoprotein gp160 is isolated from other proteins in the medium only when serum-free medium is used. It can be separated from texture. When using a serum-containing medium, gp160 It cannot be separated from other medium components.

In a preferred embodiment, the HBOI medium contains transferrin, insulin and To promote cell proliferation, including proliferative supplements such as serum albumin Cells were subcultured every 4 days. 605451 cells last for 2-3 generations I made it grow. The amount of old V protein released into the medium is determined by extracellular reverse transcriptase As measured by activity, the amount was nearly 4 times higher in the serum-free medium than in the serum-containing medium. Feeling Reverse transcriptase (RT) in the culture medium of stained cells was determined by Polesz et al., PNAS US A, 7f: 7415-7419 (1980). and analyzed using (dT)~15·(^)n.

Cell-free medium was used as the glycoprotein source. This medium was adjusted to pH 7.5. Sodium phosphate and 0.5% Triton X-100 and 0.1 mN fluoride Adjusted to 20mM with phenylmethylsulfonyl and 400d sodium chloride. . After incubation for 1 hour at room temperature, the medium was mixed with Millipore's commercially available It was concentrated to a 30-fold concentration using a Pelicon cassette system. This medium sable The foreign protein derived from the protein is added to the protein in a growth supplement of serum-free medium. Immunoaffinity using Sepharose conjugated with goat antibodies against proteins was removed from the concentrate by adsorption (-overnight). The tag bound to the goat antibody Proteins are removed and unbound material is transferred to a lectin affinity column, preferably a lectin affinity column. It was passed through a cutin-Sepharose column (Phar+Saccia). Preferably Use a lentil lectin column, but other lectins that recognize mannose, e.g. For example, concanavalin-^ may be used, washed with phosphate buffered saline (PBS). After that, the column was eluted with 400mM α-methylmannoside to remove viral glycoproteins. I took out the texture. Preferably use methylmannoside for column elution However, any mannose, pyranosine that competes with the lectin in the affinity column It is also possible to use saccharides. Figure 1 (lane 1) is a lentil 5DS-PAGE profile of glycoproteins eluted from Cutin-Sepharose It shows. The prominent glycoproteins in the sample were 120 and 160 kD proteins. Met. These proteins also tested positive for HIV-1 antibodies in immunoplots. Reacted strongly with human serum. HTLV-111451 glycoprotein immunopro The cut analysis can be carried out using known methods, such as Sarngadharan et al., 5science. 224:506-508 (1984). The cellulose was applied to a 7% 5OS-polyacrylamide gel, and a nitrocellulose strip was applied. Transfer this nitrocellulose strip to a commercially available strip. and the plots are further treated with a peroxidase-conjugated second antibody. Before Visualize the bands by reacting the strip with diaminobenzidine. Ru.

Immunotherapy using a monoclonal antibody against the [941 protein of 111V-1] Lentil lectin Sepharose scalar by affinity chromatography gp160 was purified from a mixture of glycoproteins eluted from the protein. in a standard way , monoku salmon using partially purified HTLV-I11ns+glycoprotein. A local antibody was created. The immunoglobulin fraction of this antibody (Pharmacia).

The eluate from the lentil lectin Sepharose column was % Triton X-100 and IN potassium chloride and 0.1ml PMSF 20-H containing pH 8.5)! J X - Anti-HIV-1gp4 in HCl Sepharose was equilibrated at 4°C using 1Sepharose. Wash with PBS and dissolve bound proteins with 100 MM sodium bicarbonate. I let go. HTLV-III4s+ pg160 is almost homogeneous from the column. eluted. Figure 1 (lane 2) was separated by 5D5-PAGE and coma Purified gP160 stained with sea blue is shown.

Glycoprotein gP160 and its derivatives produced by the method of the present invention are commonly used in may be used in immunotherapeutic and/or immunodiagnostic methods and compositions. In that case Treatment methods and amounts used will be clear to those skilled in the art and may be selected from existing methods. For example, gpteo produced by the method of the present invention can be used for appropriate diagnosis in ELIS^ assays. can be combined with a pharmaceutically acceptable adjuvant in an amount effective to Ru.

Although the preferred embodiments of the present invention have been described above, the present invention is not limited thereto. do not have.

In order that the invention may be better understood, the following examples are included. However, these fruits It is understood that the examples are non-limiting and do not define the scope of the invention. stomach.

EILL ! JLLL Twenty million 605<s+ cells were treated with 5% standard amount of methionine and 3sS of 1-C1. - HBI of 10-I containing methionine and 5% dialyzed ■101 supplement Labeling was performed in OI serum-free medium for 15 hours. 0.45 cell-free supernatant Pass through a micron filter, concentrate, 0.5% Triton X-100, Treated with 500 mM sodium chloride and 1 H phenylmethylsulfonyl fluoride. I understood. After standing at room temperature for 1 hour, the solubilized medium was treated with 0.5% Trit. on X-100 and 1% deoxycholate and 0.1% sodium dodecyl sulfate 1 ml of Shishiri and Kono mixture mixed with an equal volume of PBS (PBS-TDS) containing Tom. 101Jl anti-HIV serum and 15h + 10% protein-Sepharose It was incubated overnight with Sepharose was pelletized and PBS-TDS Wash 4 times with 1% SOS, 1% β-mercaptoethanol and 125MM) Boiled for 2 minutes with Lith-HCl < pH 6,8). This solubilized labeled protein Proteins were separated on 7.5% SOS polyacrylamide gels and Veronese et al. , 5science 229:1402-1405 (1985). subjected to radiography. Figure 2 shows distinct immunoreactive proteins in the culture medium. gp180 as a quality product is shown.

dog 1jLL Viral proteins in the extracellular medium of 605451 cells grown in serum-free medium. was analyzed by metabolic labeling with <35S-methionine as described above. release The released radioactive protein was collected from HIV-1 serologically positive human serum or HTLV. -III.

Immunoprecipitation was performed with either gp120 or gp41 specific antibodies. major Core protein (p24) and two types of proteins of approximately 120kD and 160kD In addition, HIV-1 positive human serum was precipitated.

Goat antibodies against HTLV-111-gp120G: 120kD and 160k Both D proteins were precipitated. This suggests that these proteins are similar to gp120. Suggests that it contains an immunoreactive region. On the other hand, rabbit anti-gp4t is a 160kD protein. Only protein was immunoprecipitated. These results indicate that the 160 kD protein has both gplZo and gp41 regions of HIV, whereas 120k This means that the D protein contains only the gp120 epitope of HIV.

Tue 11th grade The characteristics of the natural 120 and 160 kD glycoproteins produced by the method of the present invention were further investigated. HTLV-111s gp120 and gp41 specific antibodies It was analyzed by To this end, proteins were separated by 5OS-PAGE and nitrided. HTLV-IIIs gp120 and gp 41-specific antibody. 120kD protein and 160kD protein Both proteins reacted with [IIV-1 positive serum and goat anti-gp120]. HTL The 89160 kD protein reacted with the antibody against V-111, gp41. It was. Of the two monoclonal antibodies against gp120 used, the Only one reacted with both proteins. This is the monoclonal This means that the antibodies react type-specifically with different isolates of HIV.

U destination The main target of HIV-1 infection is T lymphocytes carrying the CD4 cell surface marker. The helper/inducer subset, which is a virus-prone target. The actual mechanism of infection of human cells is only beginning to be understood.

Monoclonal antibodies directed against a species epitope of the CD4 antigen inhibit viral infection. It was found that it was possible to block and immunoprecipitate the complex of CD4 and gptzo.

This suggests that the key interaction between gptzo and CD4 is involved in the HIV-1 infection process. This is considered to indicate the start of a process. One of the consequences of viral infection is , the formation of multinucleated giant cells resulting from cell fusion.

Clones of CEM cells rapidly develop syndithiae when mixed with an HIV-1 infected cell line. [Mathews et al., PNAS, US 84:5424-5428 (1987)], this type of syndithia formation is often , used as a measure of gptzo-CD4 interaction during viral infection. H TLV-II1, the glycoprotein is induced in CEM cells by [1TLV-II11 induced fusion. To measure the ability to interfere with Molt-3/HTL, target CEM cells were V-III - with partially purified glycoprotein preparations before mixing with cells Incubated for 36 hours. CEM cells were treated with glycoproteins from uninfected 6D5 cells. Pre-incubation with protein preparations had no effect on syndithia formation ( (Fig. 3C), whereas CEM cells were treated with HTLV-I114s + glycoprotein When pre-treated with the product, ■TLY-IIIs/Molt-3 cells induced Syndithia formation was completely blocked (Fig. 3D), which is due to viral sugar protein formation. The protein may selectively bind to the CD4 antigen on target III cells, resulting in This suggests that infection by -1-infected cells is inhibited.

Immunoprecipitation with human serum reveals viral glycoproteins after high-speed centrifugation of conditioned medium. More than 90% of the protein was found in soluble state. HTLV-I11=s+sugarta The interaction between proteins and CD4 molecules was further enhanced by labeling gp120 and CEM cells. This was investigated by specific binding of gp160. For this purpose, 3SS-methionine labeling The cell-free supernatant from 605451 was combined with CEM cells while increasing the number of cells. incubated together. After washing the cells with PBS, the bound 111V sugar on the cells Protein-C[14 complexes were solubilized with detergent alone as described above. soluble The extracted extracts were immunoprecipitated with two monoclonal antibodies against CD4 molecules. . Both of these HIV glycoproteins were precipitated by 0KT4. However, interest Deeply, limiting receptor density allows gptzo to become the dominant species that binds to cells. Ta. Increase cell density FIG, 3A FIo, 3B FIG. 4 Copy and translation of written amendment) Submission (Article 184-7, Paragraph 1 of the Patent Act) April 1, 1990 6th Yoshi, Commissioner of the Patent Office 1) Takeshi Moon, Yami 1. Indication of patent application: PCT/US 891034282, title of invention: Natural type Layer V GP16[1 manufacturing method 3, patent applicant Address: Netherlands, 68GO El Nis Arnhem, P.O. Box ・186, Fuelpelwech ・7G name: Akzo NV 4. Representative Yamada Building 5, Shinjuku 1-1-14, Shinjuku-ku, Tokyo, submission of amendment. Date of issue: December 11, 1989 6, List of attached documents Amendment of claims [Received by the International Bureau on December 11, 1989 (11,12,89).

Original claims 1 to 7 have been amended and claims 8 to 10 have been added. ] 1. (Correction) A method for producing natural human immunodeficiency virus gp160, comprising: Cells from the HUT78T cell line were stained with IITLV-111,4:l:ffi. Sacer, selecting infected HUT78 cells that produce native gp160; 0-producing cell lines were incubated in serum-free medium under conditions that promote cell proliferation. and isolating native gp160 from said culture medium.

2. (Correction) Infect the T cell line with the above virus and culture it under conditions that promote cell proliferation. incubating in the ground to secrete glycoproteins into said medium. In a method for producing envelope protein from human immunodeficiency virus, 6D5. which produces native gp160. S□ Cells from infected T cell line in serum-free medium and native HIV envelope glycoprotein g9. isolating a 160 kD protein corresponding to 160 from the medium. improved method.

3. (corrected) natural gpiso producing cells.

4. (Amendment) 605. of claim 3. s, naturally occurring human produced by cells Immunodeficiency virus glycoprotein gpteo.

5. (Correction) Natural gp160°6 purified by the method according to claim 1, (Correction) Natural gp160°7 purified by the method according to claim 2, (corrected) according to claim 1. Manufactured using the method described above. Contains p160, and in immunoassays gp160, gp It is effective as a diagnostic agent by reacting with antibodies against 120 or gp41. Certain reagent compositions.

8. Natural human immunity according to claim 4 in an amount effective to cause an (additional) immune response. comprising the failure viral glycoprotein gp160 and a pharmaceutically acceptable carrier. vaccine composition.

9. (Additional) Natural human immunodeficiency virus glycota according to claim 4 in a serum-free medium. Protein gp160°10, (additional) natural g according to claim 3 in a serum-free medium p160 producing cells.

international search report PCT1058910342B

Claims (7)

[Claims] 1. A method for producing natural human immunodeficiency virus gp160, comprising: Cells from the HUT78 cell line were infected with HTLV-III451 to produce 6D545. producing a single cell line; The 6D5451 cell line was incubated in serum-free medium under conditions that promote cell proliferation. incubating and isolating native gp160 from the medium. how to. 2. T cell lines are infected with the virus and incubated in culture under conditions that promote cell proliferation. The human immune system consists of incubating and secreting glycoproteins into the medium. 6D54 in a method for producing envelope protein from epizootic virus incubating the cells from the 51-infected T cell line in serum-free medium; and A 160kD protein corresponding to the native HIV envelope glycoprotein gp160. An improved method comprising isolating protein from said medium. 3. 6D5451 cells in serum-free medium. 4. Human immunodeficiency virus glycoprotein gp160 in serum-free medium. 5. gp160 purified by the method according to claim 1. 6. gp160 purified by the method according to claim 2. 7. Containing gp160 produced by the method according to claim 1, effective as a diagnostic agent pharmaceutical composition.