AU630649B2 - Expression of hiv proteins in drosophila cells - Google Patents

Description

Title

EXPRESSION OF HIV PROTEINS IN DROSOPHILA CELLS

Field of Invention

The present invention relates generally to expression of HIV proteins in Drosophila cells and purification of the expressed gene products. More specifically, this invention relates to the production of novel mutant gpl60 and gpl20 gene products by this expression system.

Background of the Invention

Human immunodeficiency virus type 1 (HIV-i) is the etiological agent of acquired immune deficiency syndrome, also known as AIDS. This retrovirus has a complex genetic organization, including the long terminal repeats (LTRs), the gag, pol, and env genes, and other- genes. This retrovirus carries a number of viral antigens which are potential candidates either alone or in concert: as vaccinal agents capable of inducing a protective immune response.

Among the more promising of the HIV-1 antigens is the viral envelope glycoprotein (gplβO) or specific fragments thereof. The env gene encodes the 160 kilodalton (kd) precursor glycoprotein of the viral Onvelope. gplβO is cleaved posttranslationally into a 120 kd glycoprotein (gpl20) and a 41 kd glycoprotein (gp41) , which are present at the virus surface. gpl20, a 511 amino acid glycoprotein, is located on the amino terminal two-thirds of the gplβO glycoprotein and is exposed on the outside of the virus. gpi20 is crucial to the interaction of the virus with its cellular receptor, the CD4 protein present on the surface of helper T₄ lymphocytes, macrophages, and other cells of the immune system. gpl20 thus determines the tissue selectivity of viral infection and contributes to the cytopathogenicity of HIV through its involvement in syncytium formation. gp41, a 345 amino acid protein derived from the carboxyl terminus of gplβO, is an integral membrane protein of HIV-l. gp41 contains a series of hydrophobic amino acids which anchor the protein in the lipid bilayer of the cellular plasma membrane. The carboxyl end of gp4l is believed to protrude into the viral particle. gp41 or a portion thereof is believed to be responsible for fusion between the HIV glycoproteins expressed at the surface of the cell with cells displaying surface T. receptors. The portion of gp41 which is believed to be responsible for this fusion is located at the amino terminal. Such fusion is believed to play a role in viral replication. See, e.g., M. Kowalski et al, Science, 237: 1351-55 (1987); D.M. Knight et al, Science, 236: 837-36 (1987).

These viral glycoproteins assume a tertiary structure as viral spikes protruding outwards from the surface of the viral particle. About, 70 to 80 spikes are believed to be associated with each newly synthesized viral particle. As the viral particle ages, the spikes disappear, apparently because the association between the gpl20 and gp41 is weak. Thus, for newly synthesized vϊral particles, this viral glycoprotein spike is believed to be the most immediate target accessible to the immune system following infection. Virus neutralizing antibodies have been reported directed against gpl20 and gp41 epitopes. It has been specifically noted that a target site for type specific neutralizing antibodies is located in the 3' half of the gpl20 glycoprotein molecule.

The env gene of HIV-l has thus been the target of numerous recent investigations. Expression of glycosylated gpl60 has previously been obtained in mammalian cells and certain baculovirus insect cells by groups which have also reported the induction of both humoral and cellular immune responses to these antigens. gpl20 has been expressed recombinantly with the use of heterologous promoters in several systems. See, e.g., S. Chakrabarti et al, Nature (London), 320: 535 (1986); S.I. Hu et al, Nature (London), 320: 537 (1986); and M.P. Kieny et al, Biotechnology, 4_: 790 (1986).

L.A. Lasky et al, Science, 233: 209-212 (1986) constructed a number of plasmids containing mutant env genes for tranfection into mammalian cells, specifically Chinese hamster ovary (CHO) cells. These researchers secreted a gene product encoded in a plasmid containing the first 50 .amino acids of the glycoprotein D (gD) protein joined in phase to an amino acid sequence (#61-#531) of the env protein, an HBsAg polyA signal, a DHFR gene and the SV40 origin of replication. A recombinant envelope antigen was produced containing 25 amino acids of gD at its amino terminus and lacking 30 residues from the mature processed from of gpl20, and also having a deletion of the gp41 sequence (about 20 amino acids of the carboxyl terminus to the actual 160 kd precursor processing site) . The resulting gene was 520 amino acids in length. When transfected into CHO cells, the cell-conditioned supernatants contained a 130 kd protein, called gpl30. A later report, L.A. Lasky et al, Cell, 50 :

975-986 (1987), discussed the interaction between the gpl20 glycoprotein and its cellular receptor, CD4. By deleting 12 amino acids contained within amino acids #410-421 of gpl20, a complete loss of binding resulted. Similarly, a single amino acid substitution at position 417 resulted in decreased binding.

Kowalski et al, cited above, introduced deletion and insertion mutations into a plasmid that encodes the envelope glycoprotein derived from the HTLV-III- strain of HIV-l. The plasmid also contained the art gene. 0 Mammalian CD4+ and CD4- cell lines expressing the tat gene product were used as transfection recipients to study ability of the transfected cells to fuse.

Knight et al, cited above, describe expression of the art/trs transactivator protein of HIV in mammalian 5 cells. The mammalian cell line used for expression of these HIV proteins was the COS-7 monkey cell line. These plasmids utilized the HIV LTR as a promoter and RNA processing signals from SV40 to express the inserted DNA as a functional messenger RNA. To express gpl20, a 0- plasmid pENV160 was developed which contains the entire coding region of the env gene fused to the HIV LTR.

S.W. Pyle, Aids Research and Human Retroviruses, 3_(4): 387 (1987) disclose the purification of the gpl20 glycoprotein from culture fluids of HIV infected H9 cells 5 by immunoaffinity chromatography.

U.S. Patent 4,725,669 also discloses glycoproteins of approximately 160 kd and 120 kd obtained - from the human H9/HTLV-III cell line, each having an - approximately 90 kd unglycosylated moiety. ⁰ Fox, Biotechnology, 6_,: 116 (1988) reports the

VAXSYN HIV-l vaccine developed by MicroGeneSys. This report does not disclose any details of this vaccine.

D.L. Lynn, et al, in "Mechanisms of Control of Gene Expression Eds. Allan R. Liss Inc., pp. 359-368 5 (1988) disclose the cloning of the entire gpl60 gene behind the polyhedron promoter of the baculovirus Autographacalifornica. These insect cells infected with the recombinant virus express a protein that is released from the cell upon lysis. This protein co-migrates with gpl60, is not cleaved into gpl20 and gp41, and is glycosylated and associated with the cell membrane. When deglycosylated with N-glycanase, the protein had a molecular weight of approximately 96 kd. The recombinant protein was immunoreactive with protein from HIV-infected H9 cells, with antisera to a recombinant fraction of gpi20, with gpl20 itself, with a peptide fragment of gp41, and with human AIDS sera.

R.L. Willey, et al, Proc. Nat' 1 Acad. Sci», USA, 83 : 5038 (1986) discusses hypervariable regions of amino acids in gpl20 protein. A later report, R.L. Willey et al, J. Virol. , 62(1): 139 (1988), studied a region within the env gene necessary for infectivity. Specifically disclosed herein are amino acid substitutions at the Asp codons of four N-linked glycosylation ^"sites within the gpl20 gene. w.R. Gallagher, Cell, 5_0: 327 (1987) discusses a fusion peptide sequence of the transmembrane protein gp41 of HIV.

S.D.^' Putney et al, Science, 23 : 1392 (1986) discloses the production of neutralizing antibodies to an E^ coli-produced fragment of the HIV env gene.

B.J. Bond et al, Mol. Cell. Biol., 6(6): 2080 (1986) disclose the structure of the Drosophila • melanogaster actin 5C gene. The report discusses the two transcription start sites of the actin 5C gene and fusions between the promoter sequences and bacterial chloramphenicol acetyltransferase gene inserted into D. melanogaster host cells.

P.J. Barr et al, Vaccine, 5_: 90 (1987) discloses the expression of HIV proteins in the yeast Saccharomvces cerevisiae. H. Johansen et al, 28th Annual Drosophila

Conference, p. 41 (1987) is an abstract by the inventors of the present application which briefly states that E. coli gal K genes regulated by a Drosophila metallothionein promoter were expressed in Drosophila cell lines.

A. Vanderstraten et al, Proceedings of the 7th International Conference on Invertebrate and Fish Tissue Culture, Abstract, University of Tokyo Press, Japan, (1987) and A. Vanderstraten et al, in "Invertebrate and Fish Tissue Culture", Eds. Y. Kuroda et al, Japan

Scientific Societies Press, Tokyo, pp. 131-134, (1988) are also publications by the present inventors which discuss a hygromycin B selection system for use in expressing foreign genes in D^ melanogaster cells in culture. The abstract notes that the system was used to co-introduce and overexpress the _ coli gal K gene and other genes of mammalian origin.

There thus remains need in the art for high-level production of HIV proteins for use as vaccinal and diagnostic agents. ^{' ■} .

Summary of Invention

in one aspect, the present invention is an HIV env gene expression unit which includes a DNA coding sequence for the desired protein and regulatory sequences necessary for transcription of the protein coding sequence and subsequent translation within a Drosophila cell. in related aspects, this invention is a DNA vector which comprises the gene expression unit of the present invention.

In yet another related aspect, this invention is a Drosophila cell transfected with the DNA vector of th s invention. In further related aspects, this invention is an

HIV env protein, or a derivative thereof produced by the transfected insect cells of this invention. The derivative encompasses any HIV env protein such as deletions, additions, substitutions or rearrangement of amino acids or chemical modifications thereof which retain the ability to be recognized by antibodies raised to the wild-type HIV env protein.

In another aspect, this invention is a vaccine for stimulating protection against HIV infection, which comprises an immunoprotective and non-toxic -quantity of the HIV env protein produced by this invention.

Also provided by this invention is a diagnostic agent useful in detecting presence of HIV infection in a sample of biological fluid which contains a Drosophila cell-produced HIV protein of the invention. Additionally, the env protein of the present invention may be employed to identify or isolate HIV binding proteins or proteinaceous substances, such as CD4 or derivatives thereof.

This invention also relates to a method for production of an HIV env protein, or an immunogenic derivative thereof. The method entails culturing Drosophila cells transfected with an HIV env gene expression unit in a medium suitable for growth of the cells. The transfected cells, cultured in said suitable medium, are capable of expressing said protein of interest. The protein may thereafter be collected from the cell or cell culture medium. T e present invention also provides a method for purifying HIV proteins or fragments thereof which bind to a monoclonal antibody reactive with an epitope present on mature gpl20 and also within the gpl60 unprocessed intracellular protein. Among antibodies of this class is the mouse monoclonal antibody designated 178.1. Other aspects and details of the present invention are disclosed in the following description:

Detailed Description of the Invention

The method and expression system of the present invention facilitate high-level production of HIV proteins, particularly gpi20, gpl60 and derivatives thereof, in a Drosophila cell structure. The Drosophila cells are transfected by using standard cloning techniques which permit introduction of foreign DNA into a host cell without adversely affecting the foreign DNA or the host cell. The recombinant Drosophila cells so constructed produce HIV proteins.

In contrast to the Baculovirus system of the prior art in which the HIV protein is provided only upon lysis of the infected insect cells, the method of this invention provides a. continuous, cell expression system for HIV proteins. Upon secretion, the protein is available by purification from the culture medium using conventional techniques. Alternatively, the protein may be produced intracellularly or membrane-bound. The protein may be extracted from the cells using conventional techniques. Alternatively, membrane-bound protein may be employed in a variety of cell-associated assays.

A preferred Drosophila cell line for use in the practice of the invention is the D. melanogaster S₂ line. S₂ cells [Schneider, J. Embryol. Exp. Morph. 27 : 353 (1972)] are stable cell cultures of polyploid embryonic Drosophila cells. Introduction of the cDNA coding sequence for gpieo, or its subunits gpl20 or gp4i or derivatives thereof into Drosophila S, cells by DNA transfection techniques produces unexpectedly large amounts of the glycoprotein. Use of the S, Drosoohila cell has many advantages, including, but not limited to. its ability to grow to a high density at room temperature. Stable integration of the selection system has produced up to 1000 copies of the transfected gene expression unit into the cell chromosomes. Other Drosophila cell culture systems may also useful in the present invention. Some possibly useful cells are, for example, the KC-0 Drosophila Melanogaster cell line which is a serum-free cell line [Schulz et al, Proc. NatA Acad. Sci ■ USA, 83: 9428 (1986)]. Preliminary studies using the KC-0 line have suggested that transfection is more difficult than with S₂ cells. Another cell line which may be useful is a cell line from Drosophila hydei■ Protein expression can be obtained using the hydei cell line; however, transfection into this cell line can result in the transfected DNA being expressed with very low efficiency [Sinclair et al, Mol . Cell. Biol. , 5_: 3208 (1985)]. Other available Drosophila cell lines which may be used in this invention include S, and S₃. The Drosophila cells selected for use in the present invention can be cultured in a variety of suitable culture media, including, e.g. , M_ medium. The _ medium consists of a formulation of balanced salts and essential amino acids at a pH of 6.6. Preparation of the media is substantially as described by Lindquist, DIS, 58 : 163 (1982). Other conventional media for growth of Drosophila cells may also be used.

A recombinant DNA molecule or vector containing an HIV protein gene expression unit can be used to transfect the selected Drosophila cells, according to the invention. The gene expression unit contains ^• a DNA coding sequence for 'a selected HIV protein or for a derivative thereof. Such derivatives may be obtained by manipulation of the gene sequence using traditional genetic engineering techniques, e.g., mutagenesis, restriction endonuclease treatment, ligation of other gene sequences including synthetic sequences and the like. See, e.g., T._Maniatis et al. Molecular Cloning, A Laboratory Manual., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). The HIV DNA coding sequence has been recently published. See, Ratner et al, Nature 313:277-284 (1985) or Wain-Hobson et al, Cell 40_:9-17 (1985). The nucleotide sequence is also available from GenBank (clone BH10, Ratner et al, supra) .

DNA molecules comprising the coding sequence of this invention can be derived from HTLV-Ill infected cells using known techniques (see, Hahn et al. Nature 312: 166-169 (1984)), or, in the alternative, can be synthesized by standard oligonucleotide techniques. Moreover, there are numerous recombinant host cells containing the cloned DNA coding sequences, which are widely available.

Derivatives can then be prepared by standard techniques, including DNA synthesis. Such derivatives may include, e.g., gpl20 or gpl60 molecules in which one -or more amino acids have been substituted, added or deleted without significantly adversely affecting the binding capacity or biological characteristics of the protein. Derivatives of these proteins may also be prepared by standard chemical modification techniques, e.g., acylation, methylation.

Also included in the gene expression unit are regulatory regions necessary or desirable for transcription of the HIV protein coding sequence and its subsequent translation and expression in the host ceil. The regulatory region typically contains a promoter region which functions in the binding of RNA polymerase and in the initiation of RNA transcription. The promoter region is typically found upstream from the HIV protein coding sequence. Preferred promoters are of Drosophila origin, e.g., the Drosophila metallothionein promoter [Lastowski-Perry et al, J. Biol . Chem. , 260: 1527 (1985)]. This inducible promoter directs high-level transcription of the-gene in the presence of metals, e.g., CuSO, . Use of the Drosophila metallothionein promoter results in the expression system of the invention retaining full regulation even at very high copy number. This is in direct contrast to the use of the mammalian metallothionein promoter in mammalian cells in which the regulatory effect of the metal is diminished as copy number increases. In the Drosophila expression system, this retained inducibility effect increases expression of the gene product in the Drosophila cell at high copy number.

The Drosophila actin 5C gene promoter [B.J. Bond et al, Mol. Cell. Biol., 6_: 2080 (1986)] is also a desirable promoter sequence. . The actin 5C promoter is a constitutive promoter and does not require addition of metal. Therefore, it is better-suited for use in a large scale production system, like a perfusion system, than is the Drosophila metallothionein promoter. An additional advantage is that the absence of a high concentration of copper in the media maintains the cells in a healthier state for longer periods of time.

Examples of other known Drosophila promoters include, e.g., the inducible heatshock (Hsp70) and COPIA LTR promoters. The SV40 early promoter gives lower levels of expression than the Drosophila metallothionein promoter. Promoters which are commonly employed in the cell expression vectors including, e.g., avian Rous sarcoma virus LTR and simian virus (SV40 early promoter) demonstrate poor function and expression in the Drosoohila system.

A desirable gene expression unit or expression- vector for the HIV protein may be constructed by fusing the HIV protein coding sequence to a desirable signal sequence. The signal sequence functions to direct secretion of the protein from the host cell. Such a signal sequence may be derived from the sequence of tissue plas inogen activator (tPA) . Other available signal sequences include, e.g., those derived from Herpes Simplex virus gene HSV-I gD [Lasky et al, Science, supra.].

The HIV DNA coding sequence may also be followed by a polyadenylation (poly A) region, such as an SV40 early poly A region. The poly A region which functions in the polyadenylation of RNA transcripts appears to play a role in stabilizing transcription. A similar poly A region can be derived from a variety of genes in which it is naturally present. This region can also be modified to alter its sequence provided that polyadenylation and transcript stabilization functions are not significantly adversely affected.

The recombinant DNA molecule may also carry a genetic selection marker, as well as the HIV protein gene functions. The selection marker can be any gene or genes which cause a readily detectable phenotypic change in a transfected host cell. Such phenotypic change can be, for example, drug resistance, such as the gene for hygromycin B resistance.

Alternatively, a selection system using the drug methotrexate, and prokaryotic dihydrofolate reductase (DHFR) gene, can be used with Drosophila cells. The endogenous eukaryotic DHFR of the cells is inhibited by methotrexate. Therefore, by transfecting the cells with a plasmid containing the prokaryotic DHFR which is insensitive to methotrexate and selecting with methotrexate, only cells transfected with and expressing the prokaryotic DHFR will survive. Unlike methotrexate, selection of transformed mammalian and bacterial cells, in the Drosophila system, methotrexate can be used to initially high-copy number transfectants. Only cells which have incorporated the-protective prokaryotic DKFR gene will survive. Concomitantly, these cells have the gene expression unit of interest. An illustrative plasmid produced, according to the present invention, is pgpl60Δ32, which contains a gpl60 derivative replacing the N-terminal 32 amino acid sequence of gplβO with the first amino acid of tPA, serine. This plasmid is further described in Example 1. Another such plasmid vector is pgpl20FΔ32 which contains gplδO sequence having the first 32 amino acids replaced with serine and containing a carboxyl deletion of 216 amino acids. This plasmid is also described in Example 1.

Still another plasmid which illustrates the derivative proteins of the present invention is pgpl20Δ32, which contains the entire coding sequence for gpl20 minus the first 32 .amino acids at the N-terminal which are replaced with serine. Additionally, plasmid pgpl20Δ274 contains a gpl20 protein sequence which has replaced the first 274 amino terminal amino acids with the first amino acid of tPA,^' serine, and containing the remaining amino acids of gpl20 up to the processing site of gpl60. These vector constructions ^"are described more completely in Example 1.

Once a recombinant DNA molecule or expression vector containing the HIV protein gene expression unit has been constructed, it can be transfected into the selected Drosophila cell using standard transfection techniques. Such techniques are known to those of skill in the art and include, for example, calcium phosphate co-precipitation, cell fusion, electroporation, icroinjection and viral transfection. A two-vector system can be used in the present invention to co-transfect into the Drosophila cell a gene expression unit for the desired HIV protein or derivative and the coding region for the selection system to be used. A preferred illustrative embodiment of this invention is the production of an HIV protein employing a vector containing an HIV protein expression unit, e.g., pgpl20Δ32, and a vector containing a hygromycin B gene expression unit, e.g., pCOHYGRO. pgpl20Δ32 contains an expression unit comprising the Drosophila metallothionein promoter, a derivative of the gpl20 gene, and the SV40 poly A site. This gpl20 expression unit in combination with the pCOHYGRO vector system will produce a gpl20 derivative in S- Drosophila cells by maximizing the advantage of hygromycin B resistance for selection. With this system, the antibiotic hygromycin B can be used to select for those cells containing the transfected vectors. A more complete description of this embodiment is described in Example 2.

As another example, an expression system employing the DHFR gene/methotrexate selection system, consisting of the vectors pgpl20Δ32 and pHGCO, can be used to select methotrexate-resistant cells expression gp!20 or a derivative thereof. The vector pgpl20Δ32 comprises a gpl20 gene expression unit in which the promoter is the' Drosophila metallothionein promoter. The pHGCO vector comprises a DHFR gene expression unit and is co-transfected with the pgpl20Δ32 vector, thereby providing the DHFR gene necessary for selection. These selectable markers along with cotransfection of Drosophila cells is further described by Johansen et al, U.S. Patent

Application Serial No. 07/047,736, filed May 8, 1987 and is incorporated by reference herein.

According to the invention, the two vectors are co-transfected into the S₂ Drosophila cell using the method as described by Wigler et al, Cell, 16: 777

(1979). The vectors are co-transfected in varying ratios depending upon the particular copy number desired. The transfected cells are then selected, such as in M_ medium containing serum and the appropriate selection agent, e.g. , hygromycin B or methotrexate.

Once an appropriate vector has been constructed and transfected into the selected Drosophila cell line. the expression of gpl20^" is induced by the addition of an appropriate inducing agent for the inducible promoter. For example, cadmium or copper are inducing agents for the metallothionein promoter. Heat is the inducing agent for the Hsp70 promoter. For constitutive promoters, such as the actin 5C promo^'ter, no inducing agent is required for expression.

Transcription and expression of the HIV protein coding sequence in the above-described systems can be monitored. For example, Southern blot analysis can be used to determine copy number of the gpl20 gene. Northern blot analysis provides information regarding the size of the transcribed gene sequence [see, e.g., Maniatis et al, cited above] . The level of transcription can also be quantitated. Expression of the selected HIV protein in the recombinant cells can be further verified through Western blot analysis and activity tests on the resulting glycoprotein [see Example 5].

Drosophila S₂ cells are especially suited to high-yield production of protein in the method of the present invention. The cells can be maintained in suspension cultures at room temperature (24+ι°C). Culture medium is M- supplemented with between 5 and 10% (v/v) heat-inactivated fetal bovine serum (FBS). In the preferred embodiment of the invention, the culture medium contains 5% FBS. After induction, the cells are cultured in serum-free media. When the pCOHYGRO vector system is used, the media is also supplemented with 300 μg/ml hygromycin B. In this media, the S, cells can be grown in suspension cultures, for example, in 250 ml to 2000 ml spinner flasks, with stirring at 50-60 rpm. Cell

6 7 densities are typically maintained between 10 and 10 cells per ml. In one embodiment of this invention, the cells are grown prior to induction in 1500 ml spinner flasks in media containing 5% serum. — —

Following cell culture, the HIV protein can be isolated from the spent media, e.g., by use of a monoclonal antibody affinity column. Other known^" protein purification steps, e.g., metal chelates, various affinity chromatography steps or absorption chromatography, can be used to purify the HIV protein from the culture media. The use of the cell line S, which secretes the gene product directly into the media is an important feature of the present invention. Direct secretion into the media ° allows utilization of an efficient one-step purification system. Using a monoclonal antibody column directed against the HIV protein, the spent culture media can be added directly to the column and the protein eluted using 1.5 M KSCN in phosphate-buffered saline (PBS). 5 A preferred purification technique enabling large-scale efficient production of the HIV proteins of the invention employs an immunoaffinity column containing a monoclonal antibody directed against an epitope present in gpl60 and present in mature secreted gpl20 proteins . 0 Such a monoclonal is advantageous because of its capacity to recognize the protein sequence in more than one configuration. An antibody having these characteristics and useful in immunoaffinity columns for various HIV proteins, derivatives or fragments thereof is designated 5 178.1. This monoclonal antibody is described in greater detail in Example 3. Such a column of the invention may be made by coupling an antibody with the characteristics of 178.1 to a conventional absorbant carrier, such as Sephadex, under appropriate conventional conditions of pK, 0 temperature and the like. Such a purification column and procedure may be utilized to separate the HIV proteins and fragments of the present invention.

Other monoclonal antibodies may be used in this purification procedure. A variety of monoclonal 5 antibodies which are capable of binding to HIV proteins, particularly gpl60 or gpl20, have been described in the art and are available. Other new monoclonal antibodies useful in this invention may be developed by now-conventional techniques.

The glycoproteins produced by Drosophila cells, according to this invention, are completely free of contaminating materials, e.g., mammalian, yeast, bacterial and more importantly, other HIV viral materials. Drosophila-produced HIV proteins have also been demonstrated to possess different pattern of glyσosylation than that reported by other systems, e.g., mammalian systems.

The HIV proteins and derivatives produced, according to the present invention, may be useful in a variety of products. For example, these recombinant proteins may be used in pharmaceutical compositions for the treatment of HIV-infected subjects. Such a pharmaceutical composition, according to the present invention, comprises a therapeutically effective amount of the HIV protein or derivative of the invention in admixture wit a pharmaceutically acceptable carrier. The composition can be syste ically administered either parenterally, intravenously or subcutaneously. When systemically administered, the therapeutic composition for use in this invention is in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such a parenterally ^"acceptable protein solution, having due regard to pH, isotonicity, stability and the like, is within the skill of the art.

The dosage regimen will be determined by the attending physician, considering various factors which modify the action of drugs, e.g., the condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. The pharmaceutical carrier and other components of a pharmaceutical formulation would be selected by one of- skill in the art. Additionally, the recombinant proteins of the

^"present invention may be used as components of vaccines to innoculate mammalian subjects against HIV infection. These proteins may be used alone or with other recombinant proteins or therapeutic vaccinal agents. Components of such a vaccine would be determined_,by one of skill in the art.

Finally, the proteins of the present invention may be useful as diagnostic agents for the detection of ° the presence of HIV infection or antibodies to an HIV infective agent in biological fluids, such as blood, serum, saliva and the like. These proteins of the invention may also be employed in methods to identify - and/or isolate HIV-binding proteins or other HIV-binding 5 substances in biological fluids and tissues, e.g., sCD4 or derivatives thereof. The proteins may thus be components in kits to perform such methods . To identify an HIV-binding substance, a protein, according to the invention, is employed to contact the substance or an 0 impure mixture containing the substance under conditions to promote binding between the protein and the HIV-binding substance. A conventional assay to detect the occurrence of binding, e.g., detection of radioactive labels or the like, is also part of the method. The presence of binding 5 between the protein and the binding substance is, therefore, indicative of HIV binding.

Similarly, in a method to isolate an HIV-binding substance from the mixture, the binding event could be followed by a conventional procedure to purify the bound 0 entity formed by the protein of the present invention and the HIV-binding substance from the mixture. Other components of such diagnostic systems and kits may be conventional components of diagnostic kits and may be selected by those of skill in the art. 5 The following examples illustrate the construction of exemplary vectors and transformants of the invention, the preferred purification system and assays for determination of the production level of the glycoproteins gpl20 and gplβO. These examples are not to be considered as limiting the scope of this invention.

Restriction enzymes and other reagents were used substantially in accordance with the vendors' instructions

Examples

Example 1. Vector Constructions

a) pMTtPA

As the basic vector for gene expression in

Drosophila, the tPA expression vector pMTtPA (also called pDMtPA) was used. This vector is a derivative of vector pMLl, a small pBR322 vector containing the beta-lactamase gene which has deleted the poison sequences [Mellon et al, Ceil, 27: 297 (1982)]. These sequences are inhibitory to amplification of the vector. This vector was digested with Sail and Aat2 which removes a small piece of pBR322, and the digested ends were filled in. The missing piece of pBR322 is then replaced with a cassette containing the Drosophila metallothionein promoter on an end-filled

EcoRl-Stul fragment, followed by a filled-in HindiII-Sacl fragment from pDSPI [D.S. Pfarr et al, DNA, 4(6): 461 ^'(19-85)] containing a tPA sequence containing the signal sequence, prepeptide and the entire coding region of tPA. The tPA gene on this fragment is followed by an SV40 early polyadenylation site.

b) pqpl60Δ32

A HindllI-Xbal fragment containing the entire env gene was isolated from an HIV-isolate clone BH10 [L. Ratner et al, cited above; GenBank] . The entire gplδO sequence was then inserted into a Ncol-Xbal digested vector pDSPl. -The resulting vector, SU2, was digested with Ndel, followed by treatment with mung bean nuclease and subsequently digested with Sacl, thus isolating the gplδO gene. The digestion with Ndel cut the gplβO sequence at amino acid #32. The Sacl digestion cuts 3" of the gpl60 gene, including in the sequence part of the original pDSPl vector containing a polylinker. This fragment was inserted into the above-described expression vector pMTtPA which had been digested with Bglll, end-filled, and subsequently cut with Sacl, which deletes the mature tPA sequence. The Bglll site is positioned at the first amino acid of tPA. Consequently, the resulting vector pgpi60Δ32 codes for a modified gplδO protein which has replaced the N-terminal 32 amino acids of gpl60 w.ith serine.

c) pqp!20FΔ32

Another vector containing a modified gene sequence coding for HIV-l surface glycoprotein gplδO was constructed by digesting pgpl60Δ32 with HindiII and Sacl, thereby removing the carboxyl terminal of gpl60. Approximately two-thirds of the sequence coding for gp41 is removed by this digestion. Thus, this gplδO sequence is missing the first 32 amino acids and the last 216 amino acids of the natural gpl60 sequence. The deleted sequence was replaced by a short synthetic linker sequence coding^" for a stop codon on an Hindlll-Sacl fragment. The 6-amino-acid linker sequence is as follows:

5' GCTTTGACTGACTGAGCT 3' .

d) pgpl2QΔ32

Yet another vector containing a mutant gplδO gene was constructed by digesting pgpl60Δ32 with Styl and Xbal, thereby deleting all of the sequence of protein gp41 and about 30 amino acids at the carboxyl terminus of the gpl20 glycoprotein sequence. This fragment was replaced by a synthetic Styl-Xbal linker sequence coding for the correct carboxyl terminus from the Styl site to the processing site of gpl20-gp41. This sequence was followed by a stop codon. This sequence thereby contained all of the coding sequence for gpl20 minus the first 32 ° amino acids and none of the gp4l coding sequence.

e) pqpl20Δ274

Still another exemplary vector containing a mutant gpl20 gene was constructed as follows: a 720-base pair carboxyl terminal fragment of gpl20 was isolated by partial digestion of pgpi20Δ32 with Bglll followed by Xbal digestion. This fragment was now inserted in place of the tPA gene into the Bglll-Xbal cut pMTtPA expression vector. The resulting vector pl20Δ274 codes for a gpl20 protein which has replaced the first 274 amino terminal amino acids with the first amino acid of tPA, serine.

f) pCOHYGRO

A commercially available plasmid, pUC18 [BRL] containing a BamHI and Smal site was used. The 5' LTR from an integrated COPIA element (357 base pairs) was cloned into the BamHI site of vector pUC18, resulting in the vector designated pUCOPIA COPIA is a representative member of the disperse middle repetition sequences found scattered through the Drosophila genome [Rubin et al , in Cold Spring Harbor Symp. Quant. Biol., 45: 619 (1980)]. The vector pUCOPIA was cut at the Smal site and the Ξ_^ coli gene coding for hygromycin B phosphotransferase (hygromycin B cassette) was cloned into pUCOPIA using standard cloning techniques. The hygrymycin B cassette was isolated on a Hindlll-BamHI fragment of 1481 base pairs from the vector DSP-hygro [Gertz et al, Gene, 25: 179 (1983)]. The hygromycin B cassette contains the sequence coding for the hygromycin B phosphotransferase gene and the SV40 early poly A region. The HindiII and

BamHI sites were filled in using T. DNA poly erase, and the hygromycin B cassette was ligated into the Smal site of the vector pUCOPIA producing vector pCOHYGRO.

Example 2. Transfection into Drosophila S₂ Cells

pCOHYGRO was co-transfected into S₂ Drosophila cells together with one of the vectors carrying a gpl60 mutant gene under the control of the Drosophila metallothionein promoter as described above. For purposes of this example, the vector employed is pgpl20Δ32. The transfected cells were selected in M₃ medium containing 5% serum and 300 μg/ml of hygromycin B. After 2_.to 3 days under identical conditions, the untransfected cells stop dividing and begin to die. The time of selection in order to obtain stable, growing hygromycin B-resistant cells in the transfected cultures is approximately two to three weeks. To obtain cultures having integrated into their chromosomes different copy numbers of the gpl20 mutant gene, the ratios of the two vectors were varied. The ratio in this example was 20:1. Similar ratios have been employed for other gplδO mutant vectors of this invention. This ratio is the same when any of the gpl60 mutant vectors are used.

Expression of the pgpl20Δ32 gene product was verified after induction of the metallothionein promoter with 500 μM CuSO.. Western blot analysis of the spent supernatant from the induced cell cultures revealed a single band at approximately 100 kd. The level of the mutant gplδO gene product in the cell supernatants was measured using the gpl20 ΞLISA assay, described in Example 4, and using purified viral gpl20 as standards. 5 x 10 cells/ml were seeded in ₃ medium without serum and induced for 3 to 4 days. The level of gpl20 measured in the supernatant is approximately 1-2 mg/1.

Cells were maintained as suspension cultures in 250 ml to 2000 ml spinner flasks. Culture medium was _ supplemented with 300 μg/ml hygromycin B. Cultures were incubated at 24+l°C and stirred at 50-60 rpm. Cell densities were typically maintained between 10 and 10 cells per ml in ₃ medium supplemented with hygromycin B. CuSO. was added to a final concentration of 500 μ , and the cultures were allowed to grow for 3 to 4 days- in serum-free media prior to harvesting the modified gpl20 glycoprotein.

The proteins, according to this method produced, were approximately 100 MW, and the level of expression was higher than any^' other reported gpl20/gpl60 expression in any eukaryotic cell system. In standard biological activity assays, the purified modified gpi20 expressed, as described above, is capable of inhibiting virus infection in tissue culture, binds T₄ and reacts to antibodies to gpl20.

It is expected that one of skill in the art- could express the other gplδO and gpl20 proteins and fragments thereof, described by the present invention, using substantially the same systems and procedures as exemplified above for the protein fragment encoded in pgpl20Δ32.

Example 3. Monoclonal Antibody 178.1

An affinity purification column employing a novel monoclonal antibody was used in the purification scheme applied to the above-described mutant gpl60/gpl20 proteins. This monoclonal antibody may be characterized as being capable of reacting with non-denatured HIV glycoprotein products present in cell lysate and with mature gpl20 as secreted into the supernatant of a yeast culture. One such monoclonal antibody specific for the epitope which is contained both in the unprocessed gplδO recombinant molecule and in the full-size processed gpl20 protein is a mouse monoclonal antibody 178.1. An expression system employing the C^ albicans glucoamylase promoter and signal peptide was employed to produce partially purified yeast-recombinant gplδO for production of 178.1. The production of this yeast-derived gpl60 is described in co-owned, co-pending Bruck et al, U.S. patent application SN 07/236,699, filed August 25, 1988. This application is incorporated herein by reference.

Eight-week-old Balb/c mice were injected three times subcutaneously and intraperitonally with the partially purified (1.5 - 3% purity) yeast-recombinant gpl60 in Freund's adjuvant at 4-week intervals. After a resting period of 3 months, one mouse was sacrificed, and its spleen cells were fuse with myeloma cells [see, e.g., R.P. Siraganian et al, Meth. Enz. , 9_2: 17 (1983); EMBO Course on Hybridoma Production, Basel Inst. for Immunol. (1980)]. The myeloma cells used are a subclone of the Sp₂/O-Agl4 line previously selected for optimal growth in agar medium and high fusion efficiency [J.D. Franssen et al, Proc. XXIX Colloq. Protids Biol. Fluids, 29 : 645-649 (1981)]. After about ten days, supernatants were withdrawn for screening in a capture ΞLISA, using a commercial monospecific anti-gpl20 reagent [Biochorm, Seromed Ref. D7324] as capture antibody.

Briefly, Nunc Immunoplate I (nr 4-39454) were coated overnight at 4^βC with 50 μl of a solution of 5 μg/ml of sheep anti-gpl20 IgGs in PBS. The plates ¹ were washed with washing buffer (PBS, Tween 20 0.1%) and saturated with 100 μl of saturation buffer [PBS, Newborn Calf Serum 4%, bovine serum albumen (BSA) 1%, Tween 20 0.1%] for 1 hour at 37°C. Fifty μl/well of crude

⁵ Molt₃/HTLV-III_B or Molt₃ cell lysate (10⁷ cells/ml in PBS, Triton X-100 1%) or of the supernatant fraction (S2-30) of the recombinant-yeast gplδO (or similarly- treated negative control) were used as antigen and incubated in the plates for 3 to 5 hours at room

¹⁰ temperature. The plates were washed extensively, and 50 μl of hybridoma supernatans were added to each well and incubated overnight at 4^βC. After a washing step, 50 μl/well of a 1/500 dilution of biotinylated anti-mouse immunoglobulms (Igs) (Amersham Ref. RPN 1021)

I⁵ in saturation buffer were incubated in the plates for 1 hour at 37°C. The plates were washed again, and 50 μl/well of a 1/1000 dilution of streptavidin biotinylated horseradish peroxidase complex (Amersham Ref. RPN 1051) in saturation buffer were added to each well.

²⁰ After an additional washing step, 50 μl of a solution of 0.4 mg/ml of orthophenylene diamine •dihydrochloride (OPD, Sigma P1526) and 1 μl/ml of H,0₂ (30% in citric/Na citrate 0.1 M pH 5) supplemented with 0.1% Tween 20 were added to each well.

25 The plates were then incubated for 20 minutes at room temperature in the dark, and the reaction was stopped by addition of 50 μl/well of 2M H₂SO.. The optical density at lambda = 492 n was monitored, and 50 positive clones were selected for further subcloning in soft ⁰ agarose, according to P. Herion et al, Proc. XXIX Colioo:. Protids Biol. Fluids, 29: 627 (1981). The cloned hybridomas were then grown in vivo by injecting 2 to 5 x 10 hybridoma cells in the peritoneal cavity of Balb/c mice pretreated by intraperitoneal injection of ⁵ pristane (2, 6, 10, 14-tetramethyl pentadecane) . 1 The monoclonal antibodies selected from the above procedure were characterized by Western blot analysis (WB), radioimmuno precipitation assay (RIPA) , purification, biotin-labeling and competition assays.

⁵ Resulting monoclonal antibodies were further characterized by analysis of their reactivity toward various recombinant and native antigens.

A high yield of hybridomas was obtained by this procedure. More than 200 wells were positive in the

10 screening assays. However, among them, only 50 wells were selected and after cloning, the cells were expanded in ascitic acid. All the ascitic fluids were tested in WB and RIPA. Among the 39 monoclonals tested, 37 showed a gplδO band in RIPA. None reacted with the gpl20 form in

I⁵ the s-ame assay. Those monoclonal antibodies that displayed only gplδO recognition, in RIPA, while being clearly reactive to gpi20 in WB, were analyzed by subclass. Three monoclonals that were IgG2A were purified on a protein A-sepharose column and biotin-labeled.

2⁰ Competition assays using vaccinia gplδO as antigen were performed, and the obtained result defined at least five different groups of epitope recognition with the gpl60 protein. Monoclonal 178.1 was selected for an epitope present on mature gpi20 and unprocessed intracellular

25 gplδO.

A Western blot (WB) analysis was performed according to conventional techniques to demonstrate that 178.1 is capable of binding HIV virus isolated from human cells infected with HTLV-III_B [Moltg/HT V-HIg] .

³⁰ Radio immuno precipitation assays (RIPA) were performed, as described in P.J. Kanki et al, Science, 228: 1199 (1985) to demonstrate that 178.1 could immunoprecipitate the human cells infected with HTLV-Ill virus strains.

35 The reactivity of the monoclonal antibodies recognizing non-overlapping epitopes towards a large panel of antigens was assessed using a sandwich ELISA involving sheep anti-gpl20 as capture reagant. Monoclonal antibody 178.1 was negative in ELISA on divergent HIV isolates Molt₃/HTLV-III_B, Hg/HTLV-III^¹ and Hut78/ARV₂, while clearly positive when tested on HTLV-III- in RIPA, WB, or ELISA using recombinant antigens. This monoclonal recognizes an epitope that is apparently conserved between gplδO and gpl20 and thus, when used in the purification technique described in Example 4 below, provides an added ° advantage for the production of gpl60/gpl20 glycoproteins in various constructs.

Example 4. Purification of gp!20Δ32 from Drosophila- conditioned Cell Culture Medium 5

The recombinant gpl20 protein from Example 2 was purified as follows: 30 liters of Drosophi1a-conditioned media (CM) containing gpl20Δ32 was made with 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM ethylenedi mine tetraacetic acid (EDTA) and 70 Kallikrein inhibitor units. CM was filtered through a .45 μm Durapore membrane using a pellicon (Millipore) device. Filtered CM was applied to S-sepharose fast flow (Pharmacia) (5 liters; 25.2 cm x 11 cm) at a linear flow rate (LFR) of 37 ml/cm h 'equilibrated in Buffer A, containing 20 mM 2-[N-morpholino]ethanesulfonic acid (MES), pH 6.0. After application of all CM, the column was eluted in one step with Buffer B, containing 20 mM MES, pH- 6.0, 0.4M NaCL. The S-sepharose-eluted gpl20Δ32 was applied to an anti-gpl20 mouse monoclonal-sepharose 4B column (60 ml;

₂ 3.2 cm x 6.5 cm) at a LFR of 10 ml/cm hr. This column was equilibrated in Buffer B. After application of one-half S-sepharose pool, the column was washed with l column volume of Buffer B, 2 column volumes of 20 mM MES, pH 6.0, 1.0M NaCL (Buffer C) , and 2 column volumes of 1 Buffer A. gpi20Δ32 was eluted with 0.1M acetic acid, pH 2.8, and fractions were immediately neutralized by addition of 0.1 volumes of 1M Tris (hydroxymethyl)aminomethane (Tris) , pH 10. .

5 Mouse anti-gpl20 monoclonal antibody hybridoma

178.1 was produced according to Example 3 above. This hybridoma was seeded at 2 x 10 cells/ml and cultured for four days in Dulbecco's Modified Eagle Medium [Hazelton Research Products] supplemented with ° 4.5 grams/liter glucose, 2 μM gluta ine and 10% serum. CM containing 178.1 antibody was filtered (0.2 μm membrane) and applied to a protein A-sepharose (Pharmacia) (17 ml; 1.5 cm x 10 cm) equilibrated in 0.1M Tris, pH 8.2. Antibody was eluted with 0.1M sodium citrate, pH 3.5 and

15 immediately neutralized with Tris.

Purified anti-gpl20 monoclonal antibody was coupled to CNBr-activated sepharose 4B (Pharmacia) , according to manufacturer's instructions at a density of 2 mg antibody/ml resin and with a coupling efficiency of

2 98%, resulting in an anti-gpl20-sepharose-affinity resin. This affinity resin will specifically bind gpl20 protein through the interaction of the antibody with a unique structural epitope on gpl20.

The purity of the final gpl20 protein product,

25 according to this purification technique, is 80-90% with an estimated yield of 8.5 mg/30 liters conditioned media. Recovery is estimated at between 25-50%.

This purification technique .and affinity resin is also believed to be effective with other HIV proteins

³⁰ or fragments thereof having this epitope.

Example 5. Assay

The assay described below is a non-isotopic 35 assay utilizing an enzyme and a substrate for the detection of gpl20 or fragments thereof, which was employed in detecting the gpl20 proteins produced by the methods and compositions of the present invention.

In the assay, the criteria for detecting gpl20 is dependent on antibody specificity. An anti-gpl20

⁵ monoclonal antibody [DuPont, Cat. No. 9284], diluted in 0.1M sodium carbonate buffer (pH 9.5) to two μgs/ml, is used to capture the gpl20 protein. 100 μl of this antibody dilution is added to each well in duplicate in an assay plate, except for those wells designated as

¹⁰ controls. The plates were incubated at 4°C overnight. The antibody was washed-out the following day and the plate blocked by adding 300 μl of blocking buffer consisting of 1% BSA in PBS to each well for 1 hour at room temperature.

I⁵ The viral gpl20 standards were diluted to

1 μg/ml, 0.5 μg/ml, 0.25 μg/ml, 0.1 μg/ml, and 0.2 μg/ml in a washing buffer consisting of PBS and 0.05% Tween 20. 100 μl of the diluted standards are added to each well in duplicate. The plates were

²⁰ incubated on a plate shaker for 2 hours at room temperature, and thereafter, each plate was washed four times with washing buffer.

To each well, 100 μl of rabbit anti-gpl20 antibody (described by DeBouck et al, U.S. Patent ⁵ Application Serial No. 07/056,553, filed May 29, 1987) diluted 1/1000 in washing buffer was added and each plated incubated on a shaker for 1 hour. This second antibody sandwiches th gpl20 between the two antibodies. The plates were, thereafter, washed four times with washing ⁰ buffer. To detect this complex, a third antibody, 100 μl of peroxidase (POD) labeled goat anti-rabbit antibody (mostly IgG and IgM antibody) diluted in washing buffer with no azide, is added to each well. The plates were .then incubated for 2 hours on a shaker at room temperature ⁵ After the plates were washed four times,

100 μl of a colorless substrate (1 mg/ml of OPD in citrate buffer with 4 μl of 35% hydrogen peroxide per 10 ml of buffer) was added. The hydrogen peroxide was added just prior to adding substrate to the wells. These plates were incubated for 8 minutes on a shaker and the reaction stopped by adding 100 μl of 0.1 M sodium fluoride to each well. In the presence of peroxidase-conjugated antibodies, the substrate turns deep yellow. Optical density, or intensity of the color, which is proportional to the amount of gpl20 captured, was read on a plate reader at 450 nanometers, and a standard curve was constructed with concentrations of unknowns calculated. The amount of gpl20 in the supernatant culture was determined by comparison to this standard curve. The above description and examples fully disclose the invention, including preferred embodiments thereof. Modifications of the methods described, e.g, employing other truncated gpl60/gpl20 sequences that are obvious to one of ordinary .skill in the art of molecular genetics and related sciences, are intended to fall within the scope of the following claims:

Claims (18)

1 What is claimed is:

1. An HIV protein gene expression unit comprising a DNA coding sequence for said protein and a regulatory element necessary for transcription of the

5 coding sequence and translation within a Drosophila cell.

2. The gene expression unit of claim 1 wherein said regulatory element comprises an actin 5C promoter or a Drosophila metallothionein promoter.

3. The gene expression unit of claim 2 which 10 comprises the gpl20 or gplδO DNA coding sequence.

4. The gene expression unit of claim 3 comprising the HIV DNA coding sequence as present in pgpl60Δ32, pgpl20FΔ32, pgpl20Δ32, or pgpl20Δ274.

5. A DNA vector comprising the gene expression 15 unit of claim 1.

6. A Drosophila cell transfected with the vector of claim 5.

7. An HIV gpl20 or gplδO protein produced in a culture of insect cells of claim 6.

20 8. A vaccine for stimulating protection against

HIV infection wherein such vaccine comprises an immunoprotective and non-toxic quantity of the protein of claim 7.

9. A diagnostic agent for detecting HIV

25 infection in a human subject comprising the protein of ^• claim 7.

10. A method for production of an HIV protein in Drosophila cells comprising culturing in a suitable medium Drosophila cells transfected with an HIV protein gene

30 expression unit, said cells being capable of expressing said protein.

11. The method of claim 10 wherein said cells are transfected with a first vector containing the coding sequence for hygromycin B phosphotransferase and a second

^'35 vector containing the coding sequence for an HIV protein gene expression unit.

12. The method of claim 11 wherein said first vector is pCOHYGRO.

13. The method of claim 11 wherein said second vector comprises an HIV gene expression unit as present in pgpl60Δ32, pgpl20FΔ32, pgpl20Δ32 or pgpl20Δ27 .

14. The method of claim 10 wherein the Drosophila cells are P. melanogaster S₂ cells.

15. The method of claim 14 wherein said S- cells are co-transfected with the vector pCOHYGRO and a vector comprising a gene expression unit as present in pgpl60Δ32, pgpl20FΔ32, pgpl20Δ32 or pgpl20Δ274.

16. A method for identifying an HIV-binding substance comprising contacting said substance with the protein of claim 7 and assaying for the occurrence of binding between said substance and said protein, such binding being indicative of HIV binding.

17. A method for purifying the HIV protein of claim 7 comprising employing an affinity resin containing a monoclonal antibody capable of reacting with an epitope present in non-denatured gpi60 protein products and in mature gpl20 protein.

18. A method for purifying the HIV protein produced by the method of claim 10 comprising employing an affinity resin containing a monoclonal antibody capable of reacting with an epitope present in non-denatured gpi60 protein products and in mature gp!20 protein.