CA1340548C - Method for producing native hiv gp160 - Google Patents

Abstract

Human Immunodeficiency Virus (HIV) glycoprotein gp 160 is produced in its native form using a clone of HUT78 cells chronically infected with HTLV-III451, and grown in serum-free medium.

Description

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METHOD FO~ P~ODUCING NATIVE HIV GP160 The invention described herein was made during the course of work performed under National Cancer Institute, Department of Health and Human Services CGn~act No. NOl-CP-67694.

Backqround of the Tnvention The present invention relates to the production of native gpl60 of human immunodeficiency virus (HIV), the etiologic agent of Acquired Immune Deficiency Syndrome (AIDS).
Human Immunodeficiency Virus (HIV) is now well established as the etiological agent of acquired immunodeficiency syndrome (AIDS). The virus is tropic for cells bearing the CD4 antigen and is highly cytopathic for helper-inducer (T4) cells. The envelope gene product of HIV is ~ynthesized as a gpl60 precursor molecule, which is ~ubsequently processed into the external envelope pr~otein gpl2p and the transmembrane protein gp41. The precursor/product relationship between gpl60 and the smaller proteins, gpl20 and gp41, has now been well documented, as well as the amino cid sequences of all three tAllan, et al., Science, 228:1091-1094 (1985) ~nd Veronese, et al, Science, 229:1402-1405 (1985)]. The external glyco~LoLein gpl20 binds to the CD4 molecule on susceptible cells in the initial phase of viral cell fusion and giant cell formation induced by the virus tDalglei~h, et al., Nature, 312:763-766].

In addition to their role in cell surface receptor recognition and cell fu~ion, HIV gpl20 and gp41 ~re the primary targets for immune recognition in individual6 infected with HIV.
Hence, the~e protein~ have received ~peci~l ~ttention ln virus neutralization ~tudies and vaccine development. It h~s been ob~erved that l~rge Regment6 of gpl20 espressed by recombinant DNA techniques, or native gpl20 purified from HIV-infected cell~, elicit mostly type-~pecific neutralizing ant~hoAie~ in ~nimal~.
In addition, the HIV envelope precur~or protein gpl60 expres~ed in insect cells with baculovirus vector~ produced a ~trong type-6pecific immune respon6e in goats tRu6che, et al., PNAS, USA
84:6924-6928 (1987)~.
The ability to infect certain cell line6 with HIV, ~nd to establish the infected cell6 into a continuou6 producer of intact virus has been de6cribed in U.S. Patent 4,652,~99. Even the ability to infect the cell line and the HIV variant of the pre6ent invention have been previou61y de6cribed lGetchell, et al., J. Clin. Microbiol. 23:737-742 (19B6)].
However, neither of the6e event6 alone permitted the establi6hment of a process capable of producing the HIV
gly~o~ otein gpl60 in its native form. Normally, native gpl60 break6 down into gpl20 ~nd gp41. ro~e~uently, the envelope protein obt~i~e~ from cell culture media or from lysed virus is gpl20 ~nd gp41. It is therefore most ~urpri~ing that gpl60 may be obt~ineA in it~ native fosm.

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Glycoprotein gpl60 has only been produced through recom~inant meAnR. However, recombinant gp160 i8 different th~n the n~tive ~p160, particularly in regard to glycosylation. These diff~e~ s become crit$cAl in the ~earch for an HIV vaccine, particularly ~ince the envelope glycoproteins of HIV determine viral tropi~m and harbor epitopes which are es6ential for the development of neutralizing ant~ho~ie~ against the ~irus.

Summary of the Invention It is an ob~ect of the present invention to provide a unique clone of HUT7B cellR which, when chronically infected with HTLV-III4sl, releases functionslly intact viral glycoprotein gpl60 into the extracellular medium.
Another ob~ect of the present invention is to provide an immortalized cell line grown in a serum-free medium under such conditions that the cell line releases gpl60 in its native form into the medium.
Yet another ob~ect of the present invention is to provide intact HIV gpl60 in its native form.
The~e and other ob~ect~ and advantages of the invention are accomplished by growing infected cell line 6D5451 in a ~erum-free medium, and iRolat~ng the n~tive gp160 released by the cells into the medium.

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-3a-Accordlngly, the present lnventlon provldes a method for the productlon of natlve human lmmunodeflciency virus gpl60 comprlslng:
lnfectlng cells from a HUT78 cell line with HTLV-III451 to incubating sald 6D5451 cell llne ln serum-free medlum under condltlon whlch promote cell growth; and lsolating natlve gpl60 from sald medlum.
The present lnventlon also provldes a process for the productlon of envelope protelns from human lmmunodeflclency vlrus wherein a T-cell line is infected wlth sald vlrus, lncubated ln a medlum under condltlons whlch promote cell growth, and glyco-protelns are secreted lnto sald medlum, the lmprovement whlch comprlses:
incubatlng cells from the 6D5451 lnfected T-cell llne ln serum-free medlum, and isolatlng from sald medlum a 160 kD proteln whlch corresponds to natlve HIV envelope glycoproteln gpl60.

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Description of the Fiqures Figure 1 (lane 1) show~ the SDS-PAGE profile of the ql~oy oLein eluted from the lentil lectin Sepharose. L~ne 2 fihows the purified gpl60 st~in~ with Coomas~ie blue.
Figure 2 show~ the relea~e of ~IV-specific proteins by 6D5451 cells. Panel As medium from cells grown in FCS; Panel B:
medium from cells grown in B 101. Lane ls ~IV-positive human serum; ~ane 2: rabbit anti-HTLV-IIIB gp41; ~ane 3s rabbit anti-121 peptide (Centocore); ~ane 4: goat anti-HTLV-IIIg gpl20;
Lane 5s normal human cerum; Lane 6 normal rabbit serum; Lane 7s normal goat serum.
Figure 3 shows the inhibition of HIV-induced syncytium formation by HTLV-III4sl glyocoproteins. CEN cell~ were cocultivated with Molt-3/HTLV-IIIg cells a~ descr~bed below. The cells were photographed after 48 hours. To study the effects of the viral glycoprotein~, CEM cells were pre-incub~ted for 1 hour with the protein~ before cocultivation. Panel As untreated CEM
cells; Panel B: CEM cells plu6 Molt-3/HTLV-IIIg cells; Panel C:
CEM cells pretreated with glycoproteins from uninfected 6D5 cell culture; Panel D: CEM cells pretreated with 6D54sl glycoproteins.
Figure 4 shows the bin~inq of gp120 and gp160 by CD4.
Conditioned medium from a 35S-meth~on~e labeled 6D54sl culture was clarified by centrifugation at 2000xg followed by filtration through a 0.45 u filter. One-half milliliter of the medium was incubated with CEMso cell~ in a total volume of 2 ml, a~

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1340~48 descrlbed below. The bound protelns were lmmunopreclpltated wlth OKT4 antlbody. Lane 1 0.5 x 106 cells; Lane 2 1 x 106 cells; Lane 3: 2 x 106 cells; Lane 4: 5 x 106 cells;
Lane 5 10 x 106 cells; Lane 6 20 x 106 cells.
Descript lon of the Preferred Embodiments A slngle cell clone of HUT78 cells has been lnfected wlth Human Immunodeflciency Vlrus Type 1 (HIV-l), whereby the infected cell llne became a contlnuous producer of vlrus.
Clone 6D5 ls susceptlble to chronlc lnfectlon wlth HIV-l, as descrlbed ln Getchell, et al., J. Clln. Mlcroblol., 23:737-742 (1986). Clone 6D5 ls lnfected wlth a speclflc straln of HIV-l, HTLV-III45l, to produce the lnfected cell llne 6D5451.
A deposlt of such cell llne was made on January 9, 1989 at the Amerlcan Type Culture Collectlon and referenced as 6D5/HTLV-III451 under ATCC Deslgnatlon CRL 9946. The lnfected cell llne ls then grown ln serum-free medlum, by pelletlng 6D5451 cells and resuspendlng them ln serum-free medlum (such as HB101 medlum, commerclally avallable from Du Pont). Serum-free medlum HB104, also avallable from Du Pont, may also be used in the practlce of thls lnventlon.
Only when serum-free medlum ls used can glycoproteln gpl60 be separated from other protelns ln the medla. gpl60 cannot be excluded from other medla components when serum-contalnlng medla ls used.
In the preferred embodlment, the HB101 medlum also contalns growth supplements such as transferrln, lnsulln, and bovlne serum albumln. To asslst ln the growth of cells, the cells were ..

1340 5~8 subcultured every four days. The 6D5451 cells were grown for 2 to 3 generations. The amount of HIV proteins released into the media, as measured by extracellular reverse transcriptase activity, was nearly five-fold greater in serum-free medium than in serum-cont~in;ng medium. Reverse transcriptase (RT) in the culture medium of the infected cells was analyzed with (dT)-15-(A)n as primer template, as described in Poiesz, et al., PNAS, USA, 77:7415-7419 (1980).
The cell-free medium was used as the source of the glycoprotein. The medium was adjusted to 20 mM with sodium phosphate, pH 7.5, 0.5% Triton* X-100, 0.1 mM
phenylmethylsulfonyl fluoride, and 400 mM sodium chloride.
After incubation at room temperature for one hour, the medium was concentrated 30-fold with a Pellicon* cassette system, commercially available from Millipore. Extraneous proteins derived from the media supplement were removed from the concentrate by immunoaffinity absorption (overnight) with a Sepharose*-bound goat antibody raised against the proteins in the growth supplement in the serum-free medium. Proteins which bound to the goat antibody were removed, and the unbound material was then passed through a lectin affinity column, preferably a lectin-Sepharose* column (Pharmacia). Although the use of a lentil lectin column is preferred, other lectins which will recognize mannose, such as concanavalin-A, may also be employed. After washing with phosphate buffered saline *Trade-mark ~l ' . . . . .

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(PBS), the column was eluted with 400 mM alpha-methylmannoside in order to recover the viral glycoprotein. Although the use of methylmannoside to elute the column is preferred, any mannose, pyranoside, or saccharide which competes with the lectin in the affinity column may be used. Figure 1 (lane 1) shows the SDS-PAGE profile of the glycoprotein eluted from the lentil lectin-Sepharose*. The prominent glycoproteins in the samples were the 120 and 160 kD proteins. These proteins also reacted strongly in immunoblots with HIV-1 antibody-positive human serum. The immunoblot analysis of HTLV-III451 glycoprotein is carried out by a well known procedure, such as described in Sarngadharan, et al., Science 224:506-508 (1984).
Essentially, the proteins are run on 7% SDS-polyacrylamide gels and transferred to nitrocellulose strips (commercially available). The nitrocellulose strips are then treated with the appropriate antibodies, and the blots are developed with peroxidase-conjugated secondary antibodiest the bands are visualized by reacting the strips with diaminobenzidine.
gpl60 was purified from the mixture of glycoproteins eluted from lentil-lectin Sepharose* column by immunoaffinity chromatography using a monoclonal antibody to HIV-l gp41 protein. The monoclonal antibody was developed using partially purified HTLV-III451 glycoproteins by standard techniques. The immunoglobulin fraction of the antibody was coupled to Sepharose* according to the method described by the manufacturer (Pharmacia). The eluate from the lentil-lectin *Trade-mark 1340 5ii 3 Sepharose* column was equilibrated at 4~C with the anti-HIV-1 gp41 Sepharose* in 20 mM Tris-HCl, pH 8.5 containing 0.5%
Triton* X-100, lM potassium *Trade-mark 7a 13~0548 chloride, and 0.1 mM P~SF. The Sepharo~e ~a~ then packed in a column, washed with PBS and the bound protein w~ eluted with 100 mM 60dium bicarbonate. The HTLV-III451 gp160 eluted from the column in ~ nearly homogenous state. Figure 1 (lane 2) ~how6 the purified gpl60, separated by SDS-PAGE and ~ta~ne~ with Cooma~sie blue.
Gl~op~oLein gp160 ~nd it~ deriv~t$ves, prepared ~ccording to the present invent~on, may be employed in a c~ cllL~onAl m~nner in immunotherapeutic and/or immunodiagno_tic methods and compositions. Such methods of treatment and quantitie_ employed are well-recognized in the art, and may be cho6en by tho~e of 6kill in the ~rt from avsilable methods and techniquee. For example, gp160, as produced in accordance with the present invention, may be combined with a phsrmaceutically acceptable ad~uvsnt in sn amount effective to provide diagnostic util$ty in ~n ELISA ~_fiay.
Although the ~bove description of the invention includes A
recitstion of preferred embodiment_, this i6 not inten~ to limit the $nvention.
In order that the invention herein de_cribed may be more fully under_tood, the following Example~ ~re ~et forth. It 6hould be under_tood that the~e ex~mple~ are for illu~trati~e purpo~e_ only, and ~re not to be con_trued a~ l$miting the invention in any manner.

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1340~.~48 Examples EXAMPLE 1.
Twenty million 6D5451 cells were labeled for 15 hours in 10 ml of the HB101 serum-free medium, contA;n;ng 5% of the normal amount of methionine, 1 mCi of 35S-methionine, and 5%
dialyzed HB101 supplement. The cell-free supernatant was filtered through a 0.45 micron filter, concentrated, and treated with 0.5% Triton* X-100, 500 mM sodium chloride, and 1 mM phenylmethylsulfonyl fluoride. After one hour at room temperature, the solubilized medium was mixed with an equal volume of PBS containing 0.5% Triton* X-100, 1% deoxycholate, and 0.1% sodium dodecyl sulfate (PBS-TDS). One milliliter of the mixture was incubated overnight with 10 ul of an anti-HIV
serum and 150 ul of 10% protein-A Sepharose*. The Sepharose*
was pelleted, washed four times with PBS-TDS and boiled for 2 minutes with 1% SDS, 1% betamercaptoethanol, and 125 mM
Tris-HCl (pH 6.8). The solubilized labeled proteins were separated on 7.5% SDS polyacrylamide gel and autoradiographed as described in Veronese, et al., Science 229:1402-1405 (1985). Figure 2 shows gpl60 as a distinct immunoreactive protein product in the culture medium.

EXAMPLE 2.
The viral proteins in the extracellular medium of 6D5 cells grown in serum-free medium were analyzed by metabolic *Trade-mark Xj .

13~0~48 labeling with 35S-methionine, as described above. The released radioactive proteins were immunoprecipitated with either HIV-l 9a X~ I

134~8 6eropo~itive human ~erum or ~nt~ho~ie~ ~pecific to HT~V-IIIg gpl20 or gp41. HIV-l positive human ~erum precipitated, in --~ddition to the ma~or core protein (p24), two protein~ of approximately 120 kD ~nd 160 kD. A ~oat ~ntibody to ~TLV-IIIB
gpl20 precipitated both the 120 kD and the 160 kD protein~, 6ugge~ting that they contain immunoreactive domains of gpl20. On the other h~nd, r~bbit anti-gp41 immu..op~cipitated only the 160 kD protein. These result~ indicate that the 160 kD protein ha~
both gpl20 ~nd gp41 dom~ins of HIV, while the 120 kD protein ha~
only the gpl20 epitopes of HIV.

EXAMPLE 3.
~ he native 120 ~nd 160 kD glycoprotein~ produced in accordance with thi~ invention were further characterized by their reactivities with anti~odies ~pecific for HTLV-IIIg gp120 and gp41. For thi~ purpo~e, the protein~ were ~eparated by SDS-PAGE, transferred to nitrocellulo~e strip~, and treated with antibodies ~pecific to HTLV-IIIg gpl20 and gp41. Both the 120 and 160 kD protein~ reacted with the HIV-l po~itive serum, ~nd with the goat anti-gp120. Only the gpl60 kD protein reacted with the antihoA~e~ to HTLV-IIIg gp41. Of the two monoclonsl antihodies to gpl20 u~ed, only one reacted with both, showing type-~pecific reactivities of the monoclonal ant~ho~ with different i~olate~ of HIV.

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EXANPLE 4.
Although the prim~ry target of HIV-l infection i~ the helper/inducer sub~et of T lymphocytes bearing CD4 cell surface markers, the ~ctuAl mechani~m by which the virus infects ~usceptible target cellB iB only begin~ng to be under~tood.
~onoclonal antibodies to certain epitopes of CD4 antigen~ were found to block viral infection ~nd could immunoprecipit~te complexes of CD4 ~nd gpl20. Thi~ tends to ~uggest that the key interaction between the gpl20 and CD4 initiate~ the infection process of HIV-l. One consequence of the virus infection iB the formation of multinucleated giant cells re~ulting from cell fusion events. A clone of CEM cells has been shown to exhibit rapid ~nd quantitative ~yncytia formation when mixed with HIV-l-infected cell lines tMathews, et al., PNAS, USA 84:5424-5428(1987)~ (Fig. 3B). This ty-pe of syncytia formation often serves as a measure of gpl20-CD4 interaction during virus infection. The ability of HTLV-III4sl glycG~ oLein to interfere with HTLV-IIIB-induced fusion of C~ cell~ was as~ayed by incubation of the target CEM cells with a partially purified preparation of glycoprotein before mixing with Molt-3/HTLV-IIIg cell~ for 36 hour6. Preincubation of rF~ cell~ with glyco~oLein preparation~ from uninfected 6D5 cell~ had no effect on the syncytia formation (Fig. 3C). In contrast, pretreatment of the CEM cell~ with the HTLV-III4s1 gl~cG~oLein preparations completely blocked the 6yncytia format$on ~ ceA by 8TLV-IIIg/~olt-3 cell~ (Fig. 3D). Thi6 ~ugge6t~ that the ~ir~l 5~
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glycoprotein could selectively bind with CD4 antigen~ on t~rget cell~, thereby blocking infection by the HIV-l $nfected cell6. --By immunoprecipitation with a human ~erum, more than 904 ofthe viral gllcopro~ein~ ~ere found in the ~oluble form after high speed centrifugation of the conditioned medium. The inter~ction of HTLV-III4sl glyco~oLein ~ith the CD4 molecule ~a8 further examined by the specific binding of l~beled gpl20 and gpl60 to CEM cells. For this purpose, cell-free ~F-r~tant form 35S-methionine labeled 6D5451 w~ incubated with incressing numbers of CEM cells. After washing the cells with PBS, the bound HIV
glycoprotein-CD4 complex on the cell~ w~ ~olubilized with detergents as described alone. The solubilized extract was immunoprecipitated with two monoclonal ant1boA ies to the CD4 molecule. Both HIV glycoprotein6 were precipitated by OKT4. It i6, however, interesting that when the receptor density ~8S
limiting, gpl20 was the predominant species that wa~ bound to the cells. At higher cell density, when ~ g site~ were more sbundant, both gpl60 and gpl20 were clearly evident in the CD4 complex. The CD4-glycoprotein complex could not be precipitated with the monoclonal ORT4A. This i~ consistent with the previous ob~ervation~ th~t the site of attachment of HIV gpl20 on the CD4 molecule is the ORT~A epitope. The relatlve affinity of the ~ece~or site appsars to favor gpl20 over gp160 ba~ed on the ob~ervation that virtually no gpl60 was bound from a mi~ture of gpl20 ~nd gp160 at limiting CD4 concentrations. How much of thi~
difficulty of b~n~ng i~ dictated by topological con~$raints on .. ...

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the larger gpl60 in appro~chming the cell ~urface CD4 i6 yet to be determined.
While p~rticular embod~ments of the invention have been de~cribed, it ~ill be understood, of cour~e, that the ~nvention i~ not limited thereto, _nd that many obv~ous modifications _nd variation~ thereof can be made, _nd that such modification~ ~re inten~e~ to fall ~ithin the ~cope of the app~nAe~ claim~.

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Claims (4)

1. A method for the production of native human immunodeficiency virus gp160 comprising:
infecting cells from a HUT78 cell line with HTLV-III451 to produce a 6D5 451 cell line;
incubating said 6D5 451 cell line in serum-free medium under conditions which promote cell growth; and isolating native gp160 from said medium.

2. In a process for the production of envelope proteins from human immunodeficiency virus wherein a T-cell line is infected with said virus, incubated in a medium under conditions which promote cell growth, and glycoproteins are secreted into said medium, the improvement which comprises:
incubating cells from the 6D5 451 infected T-cell line in serum-free medium, and isolating from said medium a 160 kD protein which corresponds to native HIV envelope glycoprotein gp160.

3. 6D5 451 cells in a serum-free medium.

4. A composition comprising HUT78 T-cell line infected with HTLV-III451 in a serum-free medium having ATCC
designation CRL 9946 and having the characteristic of producing HIV glycoprotein gp160 in its native form in serum free medium.