JP2810179B2 - Method for producing natural HIV GP160 - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The invention described herein is based on the National Cancer Institut
e, Department of Health and Human Services Contract
This was done during a study conducted under No. NO1-CP-67694.

BACKGROUND OF THE INVENTION The present invention relates to the native gp1 of the human immunodeficiency virus (HIV), a pathogen of acquired immunodeficiency syndrome (AIDS).
Involved in the manufacture of 60.

Human immunodeficiency virus (HIV) is now well established to be the pathogen of acquired immunodeficiency syndrome (AIDS). This virus is tropic for cells bearing the CD4 antigen and has a significant cytopathic effect on helper-inducer (T4) cells. The HIV envelope gene product is synthesized as a gp160 precursor molecule, which further becomes the outer envelope protein gp120 and the transmembrane protein gp41. The precursor / product relationship between gp160 and the smaller proteins gp120 and gp41, as well as the amino acid sequences of these three proteins, is now well described in the literature [Allan
Et al., Science , 228: 1091-1094 (1985) and Veronese et al.
Science , 229: 1402-1405 (1985)]. Outer glycoprotein
gp120 binds to the CD4 molecule of susceptible cells at the initial stage of virus cell fusion and giant cell formation induced by the virus [Dalgleish et al., Nature , 312: 7.
63-766].

In addition to their role in cell surface receptor recognition and cell fusion, HIV gpl20 and gp41 are major targets for immune recognition in HIV infected individuals. As such, these proteins have received special attention in virus neutralization studies and vaccine development. Large segments of gp120 expressed by recombinant DNA methods or native gp120 purified from HIV-infected cells are primarily type-specific (type-spe
cific) has been found to generate neutralizing antibodies. In addition, the HIV envelope precursor protein gp160 expressed in insect cells harboring the baculovirus vector elicited a strong type-specific immune response in goats [Rusc
hs et al., PNAS, USA , 84: 6924-6928 (1987)].

Methods of infecting certain cell lines with HIV and establishing the infected cells as a producer that continuously produces the whole virus are disclosed in US Pat. No. 4,652,599. Cell line infection methods and HIV variants of the invention have also been disclosed [Getchell et al . , J. Clin. Microbiol. 23: 737-742 (198
6)].

However, these items alone did not establish a method by which the HIV glycoprotein gp160 could be produced in its native form. Native gp160 is normally degraded to gp120 and gp41. The envelope proteins obtained from cell culture media or lysed virus are gp120 and gp41. Therefore, gp1
The fact that 60 can be produced in its native form is quite surprising.

Glycoprotein gp160 has so far been produced only by recombinant methods. However, recombinant gp160 differs from native gp160, particularly with respect to glycosylation. These differences are crucial in the study of HIV vaccines, especially because the viral tropism and harbor epitopes that are essential for the development of neutralizing antibodies to the virus are determined by the HIV envelope glycoprotein. Will have meaning.

SUMMARY OF THE INVENTION One object of the present invention is to provide a unique HUT78 cell clone that releases a functionally complete viral glycoprotein gp160 into the extracellular medium upon chronic infection with HTLV-III _451. is there.

It is another object of the present invention to provide an infinitely proliferating cell line grown in a serum-free medium under conditions such that the cell line releases gp160 in its native form into the medium.

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

These and other objects, as well as the advantages of the present invention, are achieved by growing the infected cell line 6D5 ₄₅₁ in a serum-free medium and isolating native gp160 released into the medium by the cells.

DESCRIPTION OF THE FIGURES FIG. 1 (lane 1) shows the SDS-PAGE profile of the glycoprotein eluted from lentil lectin Sepharose. Lane 2 is purified stained with Coomassie blue
gp160 is shown.

FIG. 2 shows the release of HIV-specific proteins by 6D5 ₄₅₁ cells. Panel A: medium from cells grown in FCS; Panel B: medium from cells grown in HB101; lane 1: HIV-positive human sera; lane 2: rabbit anti HTLV-III _B
Lane 4: rabbit anti-121 peptide (Centocore); lane 4: goat anti-HTLV-III _B gp120; lane 5: normal human serum; lane 6: normal rabbit serum; lane 7: normal goat serum.

FIG. 3 shows the inhibition of HIV-induced syncytia formation by HTLV-III ₄₅₁ glycoprotein. CEM cells were cultured with Molt-3 / HTLV-III _B cells as described below. Photos of the cells were taken 48 hours later. To examine the effect of the viral glycoprotein, CEM cells were pre-incubated with the protein for 1 hour prior to the co-culture. Panel A:
Untreated CEM cells; Panel B: CEM cells + Molt-3 / HTLV-III _B
Cells; Panel C: CEM cells pretreated with glycoprotein from uninfected 6D5 cell culture; Panel D: CEM cells pretreated with 6D5 ₄₅₁ glycoprotein. Figure 4 shows CD4 and gp120 and gp1
The bond with 60 is shown. 5 labeled with ³⁵ S-methionine
The conditioned media of D5 ₄₅₁ cultures were clarified by centrifugation at 2000 × g, then spent in 0.45u filter. 1 / 2ml of this medium
Was incubated with CEM ₅₀ cells in a total volume of 2 ml as described below. Bound proteins were immunoprecipitated with OKT4 antibody.
Lane 1: 0.5 × 10 ⁶ cells; lane 2: 1x10 ⁶ cells; lane 3: 2x10 ⁶ cells; lane 4: 5x10 ⁶ cells; lane
5: 10x10 ⁶ cells; lane 6: 20x10 ⁶ cells.

DESCRIPTION OF THE PREFERRED EMBODIMENTS A single cell clone of HUT78 cells was infected with human immunodeficiency virus type 1 (HIV-1), thereby rendering the infected cell line a continuous producer of the virus. Clone 6D5
Getchell et al., J. Clin . Microbiol ., 23: 737-742 (198
As described in 6), it is chronically susceptible to HIV-1 infection. Clones 6D5 to form infectious cell line 6D5 ₄₅₁ by infecting specific strains of _{HIV-1, HTLV-III 451} . The infected cell line is then grown in serum-free medium by pelleting and resuspending 6D5 ₄₅₁ cells in serum-free medium (eg, HB101 medium commercially available from Du Pont). In the practice of the present invention, a serum-free medium HB104, also commercially available from Du Pont, may be used.

Glycoprotein gp160 can be separated from other proteins in the medium only when using serum-free medium.
If one goes beyond the serum-containing medium, gp160 cannot be separated from other medium components.

In a preferred embodiment, the HB101 medium also contains growth supplements such as transferrin, insulin and bovine serum albumin. 6D5 ₄₅₁ cells were grown for 2-3 generations, with cell subculturing occurring every 4 days to promote cell growth. The amount of HIV protein released into the medium, as measured by extracellular reversal enzyme activity, was nearly four times higher in the serum-free medium than in the serum-containing medium. Reverse transcriptase (RT) in the culture medium of infected cells was determined by Poiesz et al.
As described in PNAS, USA , 77: 7415-7419 (1980), analysis was performed using (dT) -15 · (A) n as a primer template.

A cell-free medium was used as a glycoprotein source.
This medium is made up of pH 7.5 sodium phosphate and 0.5% Triton.
X-100 and 0.1 mM phenylmethylsulfonyl fluoride and 40
Adjusted to 20 mM with 0 mM sodium chloride. After one hour incubation with room sound, the medium was concentrated to a 30-fold concentration with a Pellicon cassette system available from Millipore.
Exogenous protein derived from this media supplement was removed from the concentrate by immunoaffinity adsorption (overnight) using sepharose conjugated to a goat antibody to the protein in a serum-free media growth supplement. Removing the protein bound to the goat antibody,
Unbound material was passed through a lectin affinity column, preferably a lectin-Sepharose column (Pharmacia). Preferably use a lentil lectin column,
Other lectins that recognize mannose, such as concanavalin-A, may be used. After washing with phosphate buffered saline (PBS), the column was eluted with 400 mM α-methyl mannoside to remove the viral glycoprotein. It is preferred to use methyl mannoside for column elution,
Any mannose, pyranoside or saccharide that competes with the lectin in the affinity column can also be used. FIG. 1 (lane 1) shows the SDS-PAGE profile of the glycoprotein eluted from lentil lectin-Sepharose. 12 prominent glycoproteins in the sample
0 and 160 kD protein. These proteins also reacted strongly with HIV-1 antibody positive human sera in immunoblots. Immunoblot analysis of HTLV-III ₄₅₁ glycoprotein can be performed by known methods, for example, as described in Sarngadharan et al., Scienc.
e 224: 506-508 (1984). Basically, proteins are run on a 7% SDS-polyacrylamide gel and transferred to Nirocellulose strips (commercially available). The nitrocellulose strip is then treated with the appropriate antibody and the blot is treated with peroxidase-conjugated secondary
Further treatment with antibodies. The bands are visualized by reacting the strip with diaminobenzidine.

Gp160 was purified from a mixture of glycoproteins eluted from a lentil lectin Sepharose column by immunoaffinity chromatography using a monoclonal antibody against the HIV-1 gp41 protein. Monoclonal antibodies were made using partially purified HTLV-III ₄₅₁ glycoprotein by standard methods. The immunoglobulin fraction of this antibody was bound to Sepharose in the manner indicated by the manufacturer (Pharmacia). Eluate from lentil lectin Sepharose column (elua
te) with 0.5% Triton X-100, 1M potassium chloride and 0.1%.
Anti-HIV in 20 mM pH 8.5 Tris-HCl with 2 mM PMSF
Equilibrated at 4 ° C. using -1 gp41 Sepharose. The Sepharose was then packed into a column, washed with PBS, and the bound protein was eluted with 100 mM sodium bicarbonate. HTLV
-III ₄₅₁ pg160 eluted from the column in a nearly homogeneous state. FIG. 1 (lane 2) shows purified gP160 separated by SDS-PAGE and stained with Coomassie blue.

Glycoprotein gP160 and its derivatives produced by the method of the invention can be used in a general manner in immunotherapy and / or immunodiagnosis methods and compositions. The treatment method and the amount used in that case will be apparent to those skilled in the art, and may be selected from existing methods. For example, gp160 produced by the method of the present invention
Can be combined with a pharmaceutically acceptable adjuvant in an amount effective to make a suitable diagnosis in an ELISA assay.

The preferred embodiments of the present invention have been described above.
The present invention is not limited to this.

The following examples are provided so that the invention might be better understood. It should be understood, however, that these examples are non-limiting and do not define the scope of the invention.

EXAMPLES Example 1 Twenty million 6D5 ₄₅₁ cells were cultured for 15 hours in 10 ml of HB101 serum-free medium containing 5% standard amount of methionine, 1 mCi of ³⁵ S-methionine and 5% of dialyzed HB101 supplement. Processed and labeled. The cell-free supernatant is passed through a 0.45 micron filter, concentrated, and 0.5% Triton X-100, 50%
Treated with 0 mM sodium chloride and 1 mM phenylmethylsulfonyl fluoride. After standing at room temperature for 1 hour, the solubilized medium was mixed with an equal volume of PBS (PBS-TDS) containing 0.5% Triton X-100, 1% deoxycholate and 0.1% sodium dodecyl sulfate. . 1 ml of this mixture
With 10 μl of anti-HIV serum and 150 μl of 10% protein-A
Incubated with Sepharose overnight. Pellet Sepharose, wash 4 times with PBS-TDS, 1% SD
S, 1% β-mercaptoethanol and 125 mM Tris-HC
1 (pH 6.8) and boiled for 2 minutes. The solubilized labeled protein was separated on a 7.5% SDS polyacrylamide gel and subjected to autoradiography as described in Veronese et al., Science 229: 1402-1405 (1985). FIG. 2 shows the results as separate immunoreactive protein products in the culture medium.
gp160 is shown.

Example 2 Viral proteins in extracellular medium of 6D5 ₄₅₁ cells grown in serum-free medium were analyzed by metabolic labeling with ³⁵ S-methionine as described above. The released radioprotein was purified from HIV-1 serologically positive human serum or H
TLV-III _B was immunoprecipitated with either gp120 or gp41 specific antibodies. Major core protein (p24) and about 1
In addition to the two proteins, 20 kD and 160 kD, HIV-1 positive human serum precipitated. Goat antibody to HTLV-III _B gp120 precipitated both 120 kD and 160 kD proteins.
This suggests that these proteins contain the immunoreactive region of gp120. On the other hand, rabbit anti-gp41 immunoprecipitated only the 160 kD protein. These results indicate that the 160 kD protein has both the gp120 and gp41 regions of HIV, whereas the 120 kD protein has
It means that it contains only epitopes.

Example 3 The properties of native 120 and 160 kD glycoproteins produced by the method of the invention were further analyzed by reaction to antibodies specific for HTLV-III _B gp120 and gp41. for that reason,
Proteins were separated by SDS-PAGE, transferred to nitrocellulose strips and treated with antibodies specific for HTLV-III _B gp120 and gp41. Both 120 kD and 160 kD proteins reacted with HIV-1 positive serum and goat anti-gp120. It was gp1 that reacted with the antibody against HTLV-III _B gp41.
Only the 60 kD protein. Only one of the two monoclonal antibodies to gpl20 used reacted with both of the proteins. This means that the monoclonal antibodies react type-specifically with different isolates of HIV.

Example 4 The primary target of HIV-1 infection is a helper / inducer subset of T lymphocytes with CD4 cell surface markers, but the actual mechanism by which the virus infects susceptible target cells has only just begun to elucidate. It is. Monoclonal antibodies against certain epitopes of the CD4 antigen block viral infection and CD4 and gp120
Was found to be capable of immunoprecipitation. this is,
It is believed that the major interaction between gp120 and CD4 initiates the HIV-1 infection process.
One consequence of viral infection is the formation of multinucleated giant cells resulting from cell fusion. The clone of CEM cells is HIV-
One infected cell line was found to show rapid and quantitative formation of syncytia [Mathews et al., PNAS , USA , 8
4: 5424-5428 (1987)]. This type of syncytial formation is often used as a measure of gp120-CD4 interaction during viral infection. To determine the ability of the HTLV-III ₄₅₁ glycoprotein to prevent HTLV-III _B induced fusion of CEM cells, target CEM cells were partially purified before mixing with Molt-3 / HTLV-III _B cells. 36 with glycoprotein preparations
Incubated for hours. Preincubation of CEM cells with a glycoprotein preparation from uninfected 6D5 cells did not affect syncytial formation (FIG. 3C). In contrast, pretreatment of CEM cells with the HTLV-III ₄₅₁ glycoprotein preparation completely abolished syncytia formation induced by HTLV-III _B / Molt-3 cells (FIG. 3D). This is because the viral glycoprotein is
It can bind selectively to antigens, suggesting that it blocks infection by HIV-1 infected cells.

Upon immunoprecipitation with human serum, more than 90% of the viral glycoproteins were found in soluble form after high speed centrifugation of the conditioned medium. The interaction of the HTLV-III ₄₅₁ glycoprotein with the CD4 molecule was further enhanced by labeling gp120 and gp160 on CEM cells.
Was examined by specific binding. Therefore, the cell-free supernatants from ³⁵ S- methionine labeled 6D5 _451, while increasing the number of cells were incubated with CEM cells. After washing the cells with PBS, the bound HIV glycoprotein on the cells-
The CD4 complex was solubilized with the surfactant alone as described above. The solubilized extract was immunoprecipitated with two monoclonal antibodies against the CD4 molecule. Both of these HIV glycoproteins were precipitated by OKT4. Interestingly, however, limiting receptor density caused gp120 to become the predominant species and bind to cells. Increasing cell densities increased the binding sites, revealing both the presence of gp160 and gp120 in the CD4 complex. The CD4-glycoprotein complex could not be sedimented by the monoclonal OKT4A. This is on the CD4 molecule
This supports the previous observation that the binding site of HIV gp120 is an OKT4A epitope. Gp1 when limiting CD4 concentration
Since gp160 from a mixture of 20 and gp160 is hardly bound, the relative affinity of the receptor site is g
It is thought to act more strongly on p120. How close the topological constraints of the larger glycoprotein gp160 are to this binding difficulty in accessing cell surface CD4 has not yet been elucidated.

While a specific embodiment of the present invention has been described above, the present invention is not limited to this, and various changes and modifications can be made within the scope of the appended claims.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12P 21/00 C12R 1:91) (72) Inventor Karyana Raman, Banyan Badez-Es Jiaman, Maryland 20874, United States of America Town, Spinning Wheel Place, 20821 (56) References JP-A-61-233700 (JP, A) SCIENCE, Vol. 228, (1985); 593-595 SCIENCE, Vol. 228, (1985); 1091-1094 (58) Fields investigated (Int.Cl. ⁶ , DB name) C12N 5/00-7/08 C12P 21/00-21/08 C07K 14/00-14/825 A61K 38/00-38 / 58 BIOSIS (DIALOG) WPI (DIALOG)

Claims (12)

(57) [Claims] 1. A single cell clone of the HUT78T cell line
III ₄₅₁ , isolating an infected cell line that produces the native human immunodeficiency virus glycoprotein gp160, incubating the infected cell line in a serum-free medium under conditions that promote cell growth, And isolating native gp160 from the serum-free medium. 2. The method of claim 1, wherein said single cell clone is clone 6D5. 3. The process as claimed in claim 1 or 2 wherein infected cell line is 6D5 _451. 4. The method according to claim 1, wherein said serum-free medium contains a protein growth supplement. 5. The method of claim 4, wherein said protein growth supplement is selected from the group consisting of transferrin, insulin and bovine serum albumin. 6. The method according to claim 1, wherein the isolation of natural gp160 is performed by immunoaffinity adsorption, lectin affinity adsorption, or both immunoaffinity adsorption and lectin affinity adsorption. 7. The method according to claim 6, wherein the lectin affinity adsorption is lentil lectin or concanavalin-A affinity adsorption. 8. A single cell clone of the HUT78T cell line is expressed as HTLV-
III ₄₅₁ and isolating an infected cell line that produces the native human immunodeficiency virus glycoprotein gp160, wherein the infected cell line is incubated in serum-free medium under conditions that promote cell growth. A cell line that is capable of producing native gp160 when produced. 9. The cell line according to claim 8, which is contained in a serum-free medium. 10. The cell line according to claim 8, wherein the single cell clone is clone 6D5. 11. 6D5 with no claim 8 ₄₅₁ to cell lines of any one of 10. 12. A culture obtained by incubating the cell line according to claim 8 in a serum-free medium under conditions that promote cell growth.