PT92477A - PROCESS FOR EXPRESSING HIV PROTEINS IN DROSOPHILA CELLS - Google Patents

Description

FIELD OF THE INVENTION The present invention relates generally to the expression of HIV proteins in Drosophila cells and the purification of expressed gene products. More specifically, the present invention relates to the production of the gp160 and gp-120 gene products of the novel mutant by this expression system.

BACKGROUND OF THE INVENTION Type 1 human immunodeficiency virus (HIV-1) is the etiological agent of the acquired immunodeficiency syndrome, also known as AIDS. This retrovirus has a complex genetic organization, including long terminal replicates (LTRs), the gag, pol, and env genes, and other genes. This retrovirus comprises a number of viral antigens which are potential candidates, either alone or in combination, as agents capable of inducing a protective immuno-response.

Among the most promising HIV-1 antigens are the viral envelope glycoprotein (gp110) or their specific fragments. The env gene encodes the 160 kilodalton precursor glycoprotein (kd) from the viral envelope. Gp160 is cleaved, after translation, into a 120 kD glycoprotein (gp120) and a 41 kD glycoprotein (gp41), which are present on the surface of the virus. Gpl20, a 5H amino acid glycoprotein, is located in two-thirds of the gp160 glycoprotein towards the amino-terminal, and is exposed outside the virus. Gp220 is cross-linked to the interaction of the virus with its cell receptor, the CD4 protein present on the surface of the helper lymphocytes, macrophages, and other cells of the immune system. Gp-120 thus determines the tissue selectivity of the viral infection and contributes to the cytopathogenicity of HIV by its involvement in the formation of the syncytium. Gp41, a 345 amino acid protein derived from the carboxyl terminus of gp160 is an integral membrane protein of HIV-1, gp4l contains a series of hydrophobic amino acids that attach to the protein in the lipid bilayer of the cell plasma membrane. The carboxyl terminus of gp41 is believed to be stressed on the viral particle -3- 70 291 SBC CAS1 14430. Gp41 or a portion thereof is believed to be responsible for the fusion between the HIV glycoproteins, which are expressed on the surface of the cell, with the cells having Tâ,, surface receptors. The portion of gp 41 believed to be responsible for this fusion is located at the amino terminus. It is believed that this fusion plays 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 projecting outwardly from the surface of the viral particle. It is believed that about 70 to 80 spikes are associated with each new, synthesized viral particle. As the viral particle ages, the spikes disappear, apparently due to the fact that the association between gp120 and gp4l is weak. Thus, for novel synthesized viral particles, it is believed that this viral glycoprotein spike is the most readily accessible target for the immuno system after infection. Virus neutralizing antibodies directed against the gp120 and gp41 epitopes have been described. It has been specifically noted that a target site for type-specific neutralizing antibodies is located on the 3 'half of the gp120 glycoprotein molecule. The HIV-1 env gene has thus been the target of numerous recent investigations, the expression of the glycosylated gp160 has previously been obtained in mammalian cells and in certain baculovirus insect cells by clusters which also described the induction of the immunoregulators humoral and cellular effects to these antigens. Gp120 was recombinantly expressed using heterologous promoters in various systems. See, e.g., S. Chakrabarti et al, Nature (London), 320: 535 (1986); S.I. Hu et al, Wature (London), 320? 537 (1986); and M.P. Kieny et al., Biotechnology, 4: 790 (1986). L.A. Laski et al., Science, 233: 209-212 (1986) constructed a number of plasmids containing mutant env genes for transfection into mammalian cells, specifically, in " hamster " ovary cells " Chinese (CHO). These researchers secreted a gene product encoded in a plasmid containing the first 50 amino acids of glycoprotein D (gD) bound in an amino acid sequence (# 61-531) of the env protein, a HBsAg polyA signal, a DHFR gene and the SV40 origin of replication. A recombinant envelope antigen containing 25 amino acids of gD was produced at its amino terminus and without residues of the mature antigen processed to from gp120, and also with a deletion of the gp41 sequence (about 20 amino acids from the carboxy terminus to the 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.

One last publication, L-A. Laski et al., Gell, 50: 975-986 (1987), discussed the interaction between gp120 glycoprotein and its cellular receptor, CD4. Removing 12 amino acids contained between amino acids 4410-421 of gp120 resulted in complete loss of binding. Similarly, simple substitution of the acid at position 417 resulted in a decrease in binding.

Kowalski et al., Supra, introduced deletion and insertion mutations in a plasmid encoding envelope glycoprotein derived from HIV-1 HTLV-IIIg strain. The plasmid also contained the gene art. The CD4 + and CD4 mammalian cell lines were expressed by expressing the tat gene product as transfection vessels to study the ability of the transfected cells to fuse.

Knight et al, supra, describes the expression of HIV art / trs transactivating protein in mammalian cells. The mammalian cell line used for the expression of these HIV proteins was the C0S-7 monkey cell line. These plasmids used HIV LTK as promoter and SV4o RNA processing signals to express the inserted DNA as a ABU functional messenger · To express gp120, a plasmid pEIV160 containing the entire env gene coding region fused to the HIV-SW ITR ITR was developed and Aids Research and Human Retroviruses, 3 (4): 387 ( 1987) describes the purification of glycoprotein gp120 > from H9 cell culture fluids infected with HIV, by immunoaffinity chromatography. -5 "70 291 SBC CASE 14430 U.S. Pat. 4,725,669 also describes glycoprotein with approximately 16o kd and 120kd obtained from the human H9 / HTLV-III cell line, each with a non-glycosylated group of approximately 90 kd.

Fox, Biotechnology, 6: 116 (1988) describes the HIV-1 vaccine, VAXSYN developed by MicroGeneSys. This publication does not describe any details about this vaccine. D. L. Lynn, et al., &Quot; Mechanisms of Gontrol of Gene Expression ", Eds. Allan R. Lisa Ine. pp. 359-368 (1988) describe the cloning of the entire gp160 gene behind the baculovirus polyaddition promoter Autographacalifornica. These in-sect cells infected with the recombinant virus express a protein that is released from the cell after lysis. This protein co-migrates with gp160 'is not cleaved into gp120 and gp41, and is glycosylated and associated with the cell membrane. When deglycosylated with N-glycine nase, the protein had a molecular weight of approximately 96 kd. The recombinant protein was immunoreactive with HIV-infected H9 cell proteins, with antisera against a recombinant gp120 fraction > with gpl20 itself > with a peptide fragment of gp4I and with human AIDS sera - R.L. Willey, et al., Proc. Hat'1 Acad. Sci., USA, 83: 5038 (1986) discusses hypervariable amino acid regions in the gp120 protein. A later publication RL Willey et al., J. Virol., 62 (1): 139 (1988), studied a region in the env gene required for infection. Specifically described herein are amino acid substitutions in the Asp codes of four N-linked glycosylation sites in the gp120 gene. W.R. Gallagher, Gell, 50, 327 (1987) discusses a fusion peptide sequence of the HIV transmembrane gp4I protein. S. Putney et al., Science, 234: 1392 (1986) describes the production of neutralizing antibodies to a fragment produced by E. coli of the HIV env gene. B.J. Bond et al, Mol. Cell. Blol., 6 (6): 2080 (1986) describes the structure of the Drosophila melanogaster actin gene 50. This publication discusses the two transcription initiation sites of the 5C actin gene and fusions between the promoter sequences and the bacterial chloramphenicolae ethyltransferase gene inserted -6- 70 291 SBC CASE 14430 in the host cells of p. melanogaster. P.J. Barr et al., Vaccine, 5: 90 (1987) describes the expression of HIV proteins in yeast Sacoharomyces cerevislae. H. Johansen et al, 28th Annual Drosophila Gonference, p. 41 (1987) is a summary published by the inventors of the present application which briefly describes the expression of E. coli gal K genes regulated by a Drosophila metallothionein promoter in Drosophila cell lines. A. Vanderatraten et al., Proceedings of the 7th International Conference on Invertebrate and Fish Tissue Gulture, Abstract, University of Tokyo Press, Japan, (1987) and A · Vanderatraten et al., &Quot; invertebrate and Fish Tissue Gulture ", Eds. Y. Iuroda et al., Japan Scientific Societies Press, Tokyo. 131-134, (1988) are also publications of the present inventors which discusses a hygromycin B selection system for use in expression of foreign genes in cultured D. melanogaster cells. The summary reports that the system was used to co-introduce and over-express the gal K gene of 1. coli and other genes of mammalian origin.

The need to produce HIV proteins with high levels of production for use as vaccine and diagnostic agents is maintained.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to an HIV env gene expression unit which includes a DNA coding sequence for the desired protein and the regulatory sequences necessary for the transcription of the protein coding sequence and subsequent translation into one Drosophila cell. one

In related aspects, the present invention describes a DNA construct comprising the gene expression unit of the present invention.

In yet another related aspect, the present invention relates to a Drosophila cell transfected with the DNA vector of the invention

In other related aspects, the present invention describes an HIV env protein, or a derivative thereof, produced by the transfected insect cells of the present invention. The GASE 14430 derivative comprises any HIV env protein such as those resulting from deletions, additions, substitutions or rearrangements of amino acids or of their chemical modifications, which retain their ability to be recognized by antibodies raised against the wild type HIV env protein.

In another aspect, the present invention relates to a vaccine for stimulating protection against HIV infection, which comprises a non-toxic immuno-protective amount of the HIV env protein produced in accordance with the present invention. The present invention provides also a diagnostic agent useful for detecting the presence of HIV infection in a sample of biological fluid containing an HIV protein produced by a Drosophila cell of the present invention. In addition, the env protein of the present invention may be used to identify . or isolate proteins or protein substances that bind to HIV, such as GD4 or derivatives thereof. The present invention also relates to a method of preparing an HIV env protein or an immunogenic derivative thereof. The method comprises culturing Drosophila cells transfected with an HIV env gene expression unit in a medium suitable for cell growth. The transfected cells, cultured in said suitable medium, are capable of expressing said protein of interest. The protein can then be harvested from the cells or from the culture medium of the cells. The present invention also describes a method of purifying HIV proteins or fragments thereof which bind to a reactive monoclonal antibody with an epitope present on the mature gp120 and also on the unprocessed intracellular protein gp110. Among the antibodies of this class The mouse monoclonal antibody designated 178.1.

Further aspects and details of the present invention are described in the following description.

DETAILED DESCRIPTION OF THE INVENTION The process and expression system of the present invention facilitates the production of HIV proteins with high levels of SBC CASE 14430 particularly of gp2020, gp120 and its derivatives in a cell structure of Drosophila. Drosophila cells are transfected using standard cloning techniques that allow introduction of the foreign DNA into a host cell without adversely affecting the foreign DNA or host cell. Recombinant Drosophila cells thus constructed produce HIV proteins.

In contrast to the prior art Baculovirus system, where only HIV protein is obtained by lysis of infected insect cells, the process of the present invention provides a continuous cellular expression system for HIV proteins. Upon secretion, the protein is obtained by purification from the culture medium using standard techniques. Alternatively, the protein may be produced intracellularly or ligated to the membrane. Protein can be extracted from the cells using standard techniques. Alternatively, membrane bound protein can be used in a variety of cell-associated assays.

A preferred lineage of Drosophila cells for use in the practice of the present invention is the D. melanogas ter strain. Sg / fSchneider cells, J. Eknbryol. Exp. Morph. 27, 353 (1972) are stable cell cultures of polyploid embryonic Droso-phila cells. The introduction of the cDNA coding sequence to the gp120, or its gp120 or gp41 subunits or derivatives thereof, into Drosophila cells 82. By DNA transfection techniques, it produces unexpectedly high amounts of glycoprotein. The use of the Drosophila S2 cells has many advantages which include, but are not limited to, its ability to grow to a high density at room temperature. The stable integration of the selection system produced up to 1000 copies of the gene expression unit transfected into the chromosomes of the cell.

Culture systems from other Drosophila cells may also be used in the present invention. Some useful cell lines may include, for example, the cell line of Drosophila melanogaster KC-0 which is a serum free cell line / Schulz et al., Pr. Nat. Acad. Know. TJSA, 83: 9428-9701, SBC CASE 14430 (1986)]. Preliminary studies using the KC-0 strain have suggested that transfection is more difficult than with S £ · cells

Another cell line that may be useful is the Drosophila hydei cell line. Expression of the protein can be obtained using the hydei cell line; however, transfection in this cell line can result in the expression of the transfected DNA with very little efficiency / Sinot et al,

Mol. Cell. Biol., 5: 3208 (1985). Other available Drosophila cell lines that can be used in the present invention include the strains and S ^

Drosophila cells selected for use in the present invention may be cultured in a variety of suitable culture media, including, e.g., the medium. The medium comprises a formulation of balanced salts and essential amino acids at a pH of 6.6. The medium is prepared essentially as described in Lindquist, DIS, 58: 163 (1982). Other conventional means for the growth of Drosophila cells may also be used.

A recombinant DNA molecule or vector containing the HIV protein gene expression unit may be used to transfect the Drosophila cells selected according to the invention. The gene expression unit contains a DNA coding sequence for a selected HIV protein or a derivative thereof. These derivatives can be obtained by manipulating the gene sequence using traditional genetic engineering techniques, e.g., mutagenesis, restriction endonuclease treatment, binding of other gene sequences including the synthetic sequences, and the like. See, e.g., T. Maniatis et al., Molecular Gloning, A Laboratory Manual., Cold Gpring Harbor Lab., Cold Spring Harbor. NY (1982).

The HIV coding sequence has recently been published. See Katner 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 the present invention can be obtained from HTLVt-III infected cells using known techniques (see, Hann et al, 10 10 70 291 SBG CASE 14430

Nature 312-166-169 (1984), or alternatively, may be synthesized by conventional oligonucleotide techniques. In addition, there are numerous recombinant host cells containing the cloned ADI coding sequences, which are usually available.

The derivatives may be prepared by conventional techniques including ADI synthesis. Such derivatives may include, for example, gp120 or gp160 molecules where one or more amino acids have been substituted, added or eliminated without significantly adversely affecting binding ability or biological characteristics of the protein. Derivatives of these proteins may also be prepared by conventional chemical modification techniques, e.g., acylation, methylation.

Also included in the gene expression unit are the necessary or desirable regulatory regions for the transcription of the HIV protein coding sequence and their subsequent translation and expression in the host cell. The regulatory region typically contains a promoter region that functions at the binding of ARI polymerase and at the start of ARI transcription. The promoter region is typically upstream of the HIV protein coding sequence.

Preferred promoters are from Drosophila, e.g., the Drosophila / ΓLastowski-Perry metallothionein promoter et al, J. Biol. Chem., 260: 1527 (1985) J7. This inducible promoter directs the high level transcription of the gene in the presence of metals, eg, CuSO4. The use of the Dro-sophila metallothionein promoter results in the expression system of the invention maintaining a complete regulation even for very high copy number .

This is in direct contrast to the use of the mammalian metallothionein promoter in mammalian cells, in which case the regulatory effect of the metal decreases as the number of copies increases. The Drosophila expression system, this induction effect which is maintained, increases the expression of the gene product in the Drosophila cell for a large number of copies. The Drosophila B.J. Bond et al., Mol. Cell. Biol., 6: 2080 (1986) .7 also constitutes a desirable promoter sequence. The actin promoter G is a constitutive promoter and does not require the addition of metal. It is therefore more suitable for use in a large-scale production system, such as a perfusion system, than the Drosophila metallothionein promoter. A further advantage is that the absence of a high concentration of copper in the medium maintains the cells in a healthier state for longer periods of time.

Examples of other known Drosophila promoters include, e.g., the heat shock inducible promoter (Hsp70) and the COPIA LTR promoter. The SV40 preterm promoter yields lower expression levels than with the Drosophila metallotoxin promoter. Promoters that are commonly used in cell expression vectors, including, e.g., avian Rous sarcoma LTR and simian virus (SV4o preterm promoter) demonstrate poor functioning and expression in the Drosophila system.

An expression unit or a gene expression vector desirable for the HIV protein can be constructed by fusing the coding sequence of the HIV protein with a desirable signal sequence. The signal sequence acts in order to direct the secretion of the protein from the host cell. This signal sequence can be obtained from the actin sequence. of tissue plasminogen (tPA). Other available sequences include, e.g., Those derived from the HSV-I gene of Herpes Simplex virus, Lasky et al., Science, supra. The HIV-DNA coding sequence may also be followed by a polyadenylation region (poly A), such as, for example, a SV40 premature poly A region. The poly A region that functions in the polyadenylation of the ARH transcripts appears to play a role in the stabilization of transcription A similar poly A region can be obtained from a variety of genes in which it is naturally present. This region may also be modified to alter its sequence provided that the polyadenylation and transcription stabilization functions are not significantly adversely affected. The recombinant DNA molecule may also comprise a genetic selection marker "as well as the functions of the HIV protein gene. The selection marker may be any gene or genes that cause easily detectable phenotypic change in a transfected host cell. This non-typical change may be, for example, resistance to drugs, for example, the hygromycin B resistance gene.

Alternatively, a selection system using the drug gene methotrexate, and the prokaryotic dihydrofolate reductase (BHFR) gene, can be used with Brosophil cells. The endogenous eukaryotic DHPR of the cells is inhibited by methotrexate In this way, by transfecting the common plasmid cells containing prokaryotic DHPR, which is insensitive to methotrexate, and selecting with methotrexate, only the cells transfected with, and expressing, the prokaryotic DHPR will survive. In contrast to methotrexate in the selection of mammalian and bacterial cells, in the Drosophila system, methotrexate can be used for transfectants with an initially high number of cells. Only cells that incorporate the protective prokaryotic DHPR gene will survive. Concomitantly, these cells have a gene expression unit of interest.

An exemplary plasmid produced in accordance with the present invention is pGPL6AA32, which contains a gp110 derivative subtitling the N-terminal amino acid sequence of gp110 by the first amino acid of tPA, the serine. Example 1 describes this plasmid in more detail.

Another of these plasmid vectors is pgpl20FA32 which has the sequence of gp160 with the first 52 amino acids serine substituted and containing a carboxyl terminus of 216 amino acids. Example 1 also describes this plasmid.

Yet another plasmid illustrating the proteins derived from the present invention is the plasmid pgp2AA32, which contains the entire coding sequence of gp120 less than the first 52 amino acids at the H-terminus which are replaced by serine. In addition, the plasmid Pgpl2oA2-4 contains a sequence of the gP120 protein whose first 274 amino acids of the amino terminus are replaced by the first amino acid of tPA, the serine, and containing the remaining amino acids of gp120 to to the gp160 processing site. In these examples vectors are described in more detail.

After a recombinant DNA molecule or an expression vector containing the HIV protein gene expression unit has been constructed, it can be transfected into Drosophila cells by selecting nothing using standard transfection techniques. Such techniques are known to those skilled in the art and include, for example, calcium phosphate co-precipitation, cell fusion, electroporation, microinjection and viral transfection.

In the present invention a two-vector system can be used to co-transfect, in the Drosophila cell, a gene expression unit for the desired HIV protein or derivative, and the coding region for the selection system a use·

A preferred exemplary embodiment of the present invention is the production of an HIV protein using a vector containing an HIV protein expression unit, eg, Pgp-12β / 32, and a vector containing a gene expression unit resistance to hygromycin B, e.g., pCOHVGRO · pgpl'2oA32 contains an expression unit comprising the Drosophila methionothionein promoter, a derivative of the gp120 gene, and the SV40 poly A site. This gp120 expression unit in combination with the pCOHTGRO vector system will produce a gp120 derivative on Drosophila cells maximizing the advantage of hygromycin resistance for selection. With this system the antibiotic hygromycin B can be used to select the cells containing the transfected vectors. A more complete description of this embodiment is given in Example 2

As another example, an expression system can be used using the DHFR / methotrexate gene selection system comprising the pgp120 /? And pHGCO vectors to select the methotrexate resistant cells expressing the gp120 or a derivative thereof. The vector pgpl2oA32 comprises a gp120 gene expression unit where the promoter is the Drosophila metallothionein promoter. The pHGCO vector comprises an expression unit of the DHFR gene and is co-transfected with the vector pgpl2oA32 -14-70 291 SBC CASE 14430 thereby providing the DHF! required for selection. These selectable markers in conjunction with the co-transfection of Drosophila cells are described in more detail by Johansen et al., In U.S. patent application Ser. Serial No. 07/047736, filed May 8, 1987, which is hereby incorporated by reference.

According to the invention, the two vectors are co-transfected into the Brosophila Sg cell using the method described by Wigler et al, Cell, 16: 777 (1979). Vectors are co-transfected in various proportions depending on The selected cells are then selected, for example, in serum containing medium and the appropriate selection agent, p * e, hygromycin B or methotrexate.

Once an appropriate vector is constructed and after it is transfected into the selected Drosophila cell line, the expression of gp120 is induced by the addition of a suitable inducing agent to the inducible promoter. For example, cadmium and copper are inducing agents for the metallothionein promoter. Heat is an inducing agent for the Hsp70 promoter. For constitutive promoters such as the actin 5 C promoter, no inducing agent is required for expression.

The evolution of transcription and expression of the HIV protein coding sequence in the above-referenced systems can be followed. For example, Southern blot analysis can be used to determine the copy number of the gp120 gene. Northrn spot analysis provides information regarding the size of the transcribed genetic sequence / " see, p, e. Maniatis et al., Supra, cited above. The level of transcription can also be quantified. Expression of the selected HIV protein in the recombinant cells can further be verified by Western blot analysis and activity tests on the resulting glycoprotein / quot; see Example 5_7 *

Brosophila cells are especially suitable for the production of the protein in a high yield according to the method of the present invention. The cells can be maintained in suspension cultures at room temperature (24 + 1eC). The culture medium is medium supplemented with between 5 and 10% (v / v) 70 291 SBC CASE 14430 fetal bovine serum (PBS). In the preferred embodiment of the invention, the culture medium contains 5% PBS. Following induction the cells are cultured in free-standing media. When the pGOHYGRO vector system is used, the medium is also supplemented with 300æg / ml hygromycin B. Sg cells can be cultured as suspension cultures For example in stirred flasks of 250 to 2000 ml with stirring at 50-60 rpm. The cell density is typically maintained between 10 Âμ and 10 cells per ml. In one embodiment of the present invention, the cells were culled prior to induction into shaken flasks of 1500ml in medium containing 5% serum.

After culturing the cells, the HIV protein can be isolated from the medium consumed, e.g., using a monoclonal antibody affinity column. To purify the HIV protein from the culture medium, other known protein purification steps, e.g., metal chelates, various affinity chromatography steps or absorption chromatography may be used. The use of the cell line S0 which secretes the gene product directly into the medium is an important aspect of the present invention. Direct secretion into the medium allows the use of an effective purification system in one step. Using a monoclonal antibody column directed against HIV protein, the culture medium consumed can be directly added to the column and the protein eluted using 1.5 M KSCN in phosphate buffered saline (PBS).

A preferred purification technique allowing the large-scale efficient production of HIV proteins of the present invention utilizes an immunoaffinity column containing a monoclonal antibody directed against an epitope present in the gland and present in the mature secreted gp120 proteins. This monoclonal system is vanish due to its ability to recognize the protein sequence. in more than one configuration. An antibody with these faces and useful in immunoaffinity columns for various HIV proteins, their derivatives or fragments, is designated 178.1. In Example 3 this monoclonal antibody is described in more detail. This column of the invention can be prepared by coupling a CAS-14450 antibody with the characteristics of 178.1 to a conventional absorbent carrier such as Sephadex under conditions of pH, temperature, and others, conventional and appropriate. This purification column and this procedure can be used to separate the HIV proteins and fragments thereof prepared in accordance with the present invention.

In this purification procedure other monoclonal antibodies may be used. Other monoclonal antibodies that are capable of binding to HIV proteins, particularly to gp010 or gp20 proteins, have been described in the art and are available. Other novel monoclonal antibodies useful in the present invention may be developed by conventional techniques.

The glycoproteins produced by the Brosophila cells according to the present invention are completely free of contaminating materials, e.g., mammalian cells, yeast, bacteria, and, most importantly, other viral HIV materials. It has also been shown that the Drosophila-produced HIV proteins have glycosylation patterns different from those evidenced in other systems, e.g., mammalian systems.

The HIV proteins and their derivatives, prepared in accordance with the present invention, may be useful in the preparation of a variety of products. For example, these recombinant proteins can 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 an HIV protein or derivative thereof of the invention blended with a pharmaceutically acceptable carrier. The composition may be administered systemically, parenterally, intravenously or subcutaneously. When administered systemically, the pharmaceutical composition is in the form of an aqueous, parenterally acceptable, pyrogen-free solution. It is known to those skilled in the art to prepare such a parenterally acceptable protein solution with respect to pH, isotonicity, stability and other parameters. The dosing regimen will be determined by the attending physician, considering various factors that modify the action of drugs, eg, condition, weight, sex and diet of the patient, severity of any infection, the time of administration and other clinical factors. The pharmaceutical carrier and other components of a pharmaceutical formulation will be selected by one skilled in the art.

Additionally, the recombinant proteins of the present invention can be used as components of vaccines to inoculate mammalian subjects against HIV infection. These proteins may be used alone or with other recombinant proteins or therapeutic vaccine agents. The components of this vaccine will be determined by one skilled in the art.

Finally, the proteins of the present invention may be useful as diagnostic agents for detecting the presence of HIV infection or the presence of antibodies to a non-effective HIV agent in biological fluids such as blood, serum, saliva and the like. These proteins of the invention may also be used in methods for identifying and / or isolating proteins that bind to HIV or other substances that bind to HIV in biological fluids and tissues, e.g. SCD4 or derivatives thereof. These substances may thus be components of diagnostic kits for carrying out these methods. To identify a substance which binds to HIV a protein according to the invention is used for contact with the substance or an impure mixture containing the substance under conditions which promote the connection between the protein and the substance which binds to the HIV. A conventional assay for detecting the occurrence of binding, p-e., Detection of radioactive labels or the like, is also part of the method. The presence of the binding between the protein and the binding substance is thus indicative of HIV binding.

Similarly, in a method for isolating a HIV binding substance from the blend, the binding step may be followed by a conventional procedure to purify the bound entity formed by the protein of the present invention and the HIV binding substance of the mixture . Other components of such systems and diagnostic assemblies may be conventional diagnostic assemblies and may be selected by those skilled in the art.

The following examples illustrate the construction of examples of vectors and transformants of the invention, the preferred purification system and assays for determining the level of production of the gp120 and gp60 proteins. These examples are not to be construed as limiting the spirit of the present invention-

Restriction enzymes and other reagents were used, substantially according to the instructions of the sellers.

EXAMPLES

Example 1. Vector Constructs

a) pMTtPA

As the basic vector for expression of the gene in Drosophila the tPA expression vector, pMTtPA (also called pDItPA) was used. This vector is a derivative of the pML1 vector, a small pBR322 vector containing the beta-lactamase gene which renders the venomous sequences / " Mellon et al., Cell, 271. 297 (1982). These sequences are inhibitors of vector amplification. This vector was digested with Sal 1 and Aat 2 which removed a small portion of pBR322, and the digested ends were filled. The missing portion of pBR322 is then replaced by a cassette containing the metallothionein Drosophila promoter in an EcoRl-Stu 1 fragment with filled ends, followed by a filled HindIII-Sac1 fragment from pDSPI / DS. Pfarr et al., DM, 4 (6): 461 (1985)] containing a tPA sequence containing the signal sequence, the prl-peptide and the entire coding region of tPA. In this fragment, the tPA gene is followed by a pre-mature polyadenylation center SV4o · b) pgpl6p / 32

A HindIII-Xba1 fragment containing the entire env gene was isolated from an HIV /? L isolate clone BH10. Ratner et al, supra; GenBankJ7. The entire gpl60 sequence was then inserted into an Nco 1-Xba 1 digested pDSPI vector. The resulting SU2 strain was digested with Mel followed by a " m-ung " bean nuclease treatment and subsequent digestion with Sac 1, thus isolating the gp160 gene. Digestion with Me1 cut the gp110 sequence at amino acid # 32. Digestion with Sac 1 -19- 70 291 SBC CASE 14430 cleaves the 3 'portion of the gp010 gene, including in the sequence, part of the original pBSP1 vector containing a poly-linker. This fragment was inserted into the above-mentioned expression vector, pMTtPA which had been digested with BglII, its filled ends, and subsequently cut with Sao 1, which eliminates the mature dotPA sequence. The BglII site is located in the first amino acid of tPA . Accordingly, the resulting pgp110A32 vector encodes a modified gp110 protein having the 32 N-terminal amino acids of gp160 substituted by serine. c) pgpl2QF / 32

Another vector containing a modified gene sequence encoding the HIV-1 surface glycoprotein, gp160, was digested with HindIII and Sac I, thereby removing the carboxyl terminus from gp06. this digestion approximately two-thirds of the sequence encoding gp41. Thus, in this sequence gpl6o lack 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 ligand sequence coding for a stop codon on a HindIII-Sac 1 fragment. The sequence of the 6 amino acid linker is as follows: 5'AGCTTTGACTCACTGAGCT 3 '' d) PgPl20A32

A further vector containing a gp60 mutant gene was further constructed by digesting pgp60 / 1 with Sty 1 and Xba 1, removing. the entire sequence of the gp41 protein and about 30 amino acids at the carboxyl terminus of the gp120 glycoprotein sequence is thus obtained. This fragment was replaced with a synthetic Sty 1-Xba 1 linker sequence encoding the correct carboxyl terminus from the Sty 1 site to the gp120-gp41 processing site. A stop codon was followed in this sequence. This sequence thus contained the entire gp120 coding sequence minus the first 32 amino acids and none of the gp41 coding sequence. e) ΡβΡΐ 2θΔ 2 4 4

Yet another example of a vector containing a mutated gp120 gene was constructed as follows: a -20-70 291 fragment was isolated

SBC CASE 14430 720 bp carboxyl terminus of the gp120 by partial digestion of pgp220 with Bgl II followed by Xba I digestion. This fragment was then inserted in place of the tPA gene in the vec- The resulting vector, pl2oA274 encodes a gp120 protein that has the first 274 amino acids of the amino terminus replaced by the first amino acid from tPA to serine. f) pGOHYGRQ A commercially available plasmid, PU018 / BRL1 / containing a BamHI site and a SmaI site was prepared. The LTR 5 'of an integrated COPIA element (357 base pairs) was cloned into the BamHI site of the pUC18 vector, resulting in a vector designated pTICOPIA. COPY is a representative member of the dispersed intermediate repeat sequences that are scattered throughout the genome of JDrosophila Uubin et al, in Colde Spring Harbor Symp. Quant. The pUCOPIA vector was cut at the Sma I site and the E. coli gene coding for hygromycin B phosphotransferase ("hygromycin B cassette") was cut out, in pUCOPIA, using standard cloning techniques. A " cassette " of the hygromycin B was isolated from a 1481 bp Hind III-Bam HI fragment of the BSP-hygro / Gertz et al., Gene, 25: 179 (1983) J7 · A " cassette " of hygromycin B contains the coding sequence of the hygromycin B phosphotransferase gene and the prepolymerized poly A region SV40. The Ηίηά III and Bam HI sites were filled using the AM T poli polymerase, and ligated to " hygromycin B cassette at the Sma I site of the pUCOPIA vector yielding the pCOHTGRO vector

Example 2. Transfection in Brosophila cells

PCOHYGRO was cotransfected into Brosophila Sg cells in conjunction with one of the vectors comprising a gp160 mutant gene under the control of the Brosophila metallothionein promoter as previously described. For purposes of this example, the vector used is pgpl2cA32. Transfected cells were screened in serum containing medium and 30æg / ml hygromycin B. After 2-3 days under identical conditions the untransfected cells stopped dividing and began to die. The time of selection in order to obtain stable cells resistant to the developing hygromycin B in the transfected cultures is approximately two to three weeks.

In order to obtain cultures with different copy numbers of the gp120 mutant on their chromosomes, the proportions of the two vectors were varied. The ratio in this example was 20: 1. Similar proportions were used for other gpl6G mutant vectors of the present invention. This ratio is the same when any of the gp160 mutant vectors are used.

Expression of the pgpl2β gene product was observed after induction of the metallothionein promoter with 500 Âμg of CuSO4. Western blot analysis of the supernatant consumed from the induced cell cultures revealed a single band with approximately 100 kd.

The content of the mutant gp160 gene product was measured in the supernatants prior to cell cultures using the gp120 ELISA assay, which is described in Example 4 and using as standard, purified viral gp120. 5 x 10 células cells / bxl were seeded in serum-free medium and induced for 5 to 4 days. The level of gp120 measured in the supernatant is approximately 1-2 mg / l.

Cells were maintained in suspension cultures in shaken flasks of 250 ml to 2000 ml. The culture medium was supplemented with 300 μg / ml of Mgramicin B. Cells were incubated at 24+ +120 and shaken at 50-60 rpm. The cell densities were typically maintained between 10 ° and 10 'cells per ml in medium supplemented with hexagon B. CuSO4 was added to a final concentration of 500 and the cultures were allowed to grow for 3 to 4 days in serum-free medium, prior to harvesting the modified glycoprotein gp120.

Proteins prepared according to this method had approximately a molecular weight of 100 and the level of expression was higher than in any other gp120 / gp160 expression system known in any eukaryotic cell system. In standard assays for biological activity, the purified, expressed modified gp120 as described above is capable of inhibiting virus infection in tissue culture, binds to and reacts with antibodies against gp120 ·

It will be expected that one skilled in the art will be able to express the gp160 and gp120 proteins and their fragments, which are described in the present invention, using substantially the same systems and procedures as exemplified above for the fragment of the protein encoded by pgp12 / 32.

Example 3 · Lonoclonal antibody 178.1

An affinity purification column using a novel monoclonal antibody was used in the purification scheme applied to the aforementioned gpl6o / gp120 mutant proteins. This monoclonal antibody can be characterized in that it is capable of reacting with non-denatured HIV glycoprotein products present in the cell lysate and with the mature gp120 as it is secreted into the supernatant of a yeast culture. This monoclonal antibody, specific for the epitope which is contained in both the unprocessed gploO recombinant molecule and the complete processed gp120 protein, is a mouse monoclonal antibody, 178.1.

An expression system, using the glucoamylase promoter and the signal peptide of G. albicans, was used to produce the partially purified recombinant yeast gland for the production of 178.1. In U.S. patent application Ser. 07/236699, co-owned, copending, filed August 25, 1988, describes the production of this yeast-derived gp160. This patent application is hereby incorporated by reference.

Balb / c mice, eight weeks old, were injected three times subcutaneously and intraperitoneally with the partially purified yeast recombination gland (1> 5% purity) in Freund's adjuvant at 4 g intervals. weeks. After a resting period of 3 months, a mouse was sacrificed, and its spleen cells were fused with myeloma cells (see, eg, RP Siraganian et al, Meth. Snz., 92: 17 (1983)). The myeloma cells used are a subclone of the SPg / O-Ag2 lineage, previously selected for optimal growth on agar medium and for a high melt efficiency Pranssen et al., Proc.

Golloq. Protids Biol. Fluids, 29: 645-649 (1981)]. After ten days, the supernatants were removed for analysis in a capture ELISA using a commercial monospecific anti-gp 2 2-reagent-23-70 291 SBC CASE 14430 / ; Biochorm, Seromed Ref. D7324J as capture antibody.

Briefly, Nunc I (n2,4-4,454) plates were coated overnight at 4Â ° C with 50 Âμl of a solution with 5% / ml of IgG of sheep anti-gp120 in BBS . The plates were washed with wash buffer (PBS, Tween 20, 0.1%) and saturated with 100æl saturation buffer /> PBS, 4% newborn calf serum, 1% bovine serum albumin (BSA), 0.1% Tween 20-7 for 1 hour at 37 ° C. As antigen, 50 ul of lysate of crude Kolt2 / HTLV-IIIg or Molt2 cells (1θ 'cells / ml in PBS, 1% Triton X-100) or the supernatant fraction (S2-30) from recombinant yeast gp160 (or a similarly treated negative control), and incubated on the plates for 3 to 5 hours at room temperature. The washers were washed extensively and added to each well, 50 μl of hybridoma supernatants, and incubated overnight at 4 ° C. After a washing step, the plates were incubated for 1 hour at 37Â ° C with 50 Âμl / well of a 1/500 dilution of biotinylated anti-mouse immunoglobulins (Amersham Ref. RPN 1021) in saturation buffer. The plates were reslurried, and 50 Âμg / well of a 1: 100 dilution of streptavidin-hinitinylated horseradish peroxidase complex (Amersham Ref. RPN 105 I) in saturation buffer was added to each well.

After a further washing step, 50/1 of a solution of 0.4 mg / ml orthophenylenediamine dihydrochloride (0PD, Sigma PI526) and 1 of H2O2 (30% in citric acid / 0.1 M sodium citrate pH 5) supplemented with 0.1% Tween 20. The plates were then incubated for 20 minutes at room temperature in the dark and quenched by the addition of 50/1 H 2 SO 4 M per well. The optical density was determined at a wavelength of 492 nm and 50 positive clones were selected for further subcloning into soft agarose, according to P. Herion et al., Proc. XXIX Golloq. Protids Blol λ Pluids, 29, 627 (1981) The cloned hybridomas were then cultured in vivo by injecting 2 to 5 x 10 6 hybridoma cells into the peritoneal cavity of Balb / c mice pretreated by intraperitoneal injection of pristane (2,6,10,14-tetramethyl) pentadecane.

Monoclonal antibodies selected according to the above procedure were characterized by Western blot analysis (WB), radioimmunoassay (RIPA) assay, purification, and biotin labeling and competition assays. The resulting monoclonal antibodies were further characterized by analyzing their reactivity to various antigens, recombinant and native.

By this procedure, a high yield of hi-bridomas was obtained. In more than 200 wells, more than 200 wells were positive. However, among the positive, only 50 wells were selected and, after cloning, the cells were expanded in ascitic acid. All ascites fluids were tested for WB and RIPA. Among the 39 monoclonal antibodies tested, 37 showed a gp120 band in RIPA. In the same assay, none reacted in the gp120 form. Monoclonal antibodies which showed only gp110 recognition in RIPA, yet were clearly reactive to gp120 in WB, were analyzed to determine the subclass. Three monoclonals that were IgC-2A were purified on a B-labeled protein A-Sepharose column. Competition assays were performed using vaccine formulation as antigen, and the result obtained defined at least different groups of epitope recognition with the monoclonal gp110.1 protein was selected for an epitope present on mature gp120 and gp120 unprocessed intracellular.

Western blot analysis (WB) was performed according to standard techniques to demonstrate that 178.1 is capable of binding to the HIV virus, isolated from human cells infected with HTLV-IIIg / llolt / lITLV-III ^ J7

Radioimmuno-precipitation (RIPA) assays were performed, as described in P.J. Ianki et al., Science, 228, 1199 (1985) to demonstrate that 178.1 could immunoprecipitate human cells infected with the HTLV-III virus strains.

The reactivity of the monoclonal antibodies, recognizing non-overlapping epitopes, against a large panel of antigens was investigated using a sandwich ELISA involving anti-25-70 291 SBC GASI 14430-sheep epsilon as capture reagent. monoclonal antibody 178.1 was negative in ELISA with divergent HIV isolates attolt / mv-IIIg, H9 / ETLV-IIIRF1 and Hut78 / ARV2, but was clearly positive when tested with HTLV-IILg in RIPA, WB, or ELISA using recombinant antigens. This monoclonal antibody recognizes an epitope that is apparently conserved between gp160 and gp120 and thus, when used in the purification technique described in Example 4 below, constitutes an additional advantage for the production of gp160 glycoproteins / gpl20 in various constructs.

Example 4. Purification of gp120 from Brosophila cell culture medium The recombinant gp120 protein from Example 2 was purified as follows: 30 liters of Dro-phila (CM) conditioned medium was prepared, containing gpl2oA52, 1 mM phenylmethylsulfonyl fluoride (PMSP), 10 mM ethylenediaminetetraacetic acid (EDTA), and 70 units of Kallikrein inhibitor. CM was filtered through a 0.45æm Durapore membrane using a Pellicon device (Millipore). The filtered CM was applied to fast-flow 3-Sepharose (Pharmacia) (5 liters; 25.2 cm x 11 cm) with a linear flow rate (LFR) of 37 ml / hr, equilibrated in buffer A, containing 20 ml of 2- [1-morpholino [ethanesulfonic acid (MES)] pH 6.0 After the application of all CM, the column was eluted in one step with buffer B containing 20 mM MES, pH 6.0, 0.4! was added to a column (60 ml, 3.2 cm x 6.5 cm) of Sepharose 4B-anti-gonad monoclonal antibody mouse with an RFI of 10 ml / cm3. This column was equilibrated in buffer B. After application of half the S-Sepharose eluate, the column was washed with 1 volume of buffer B column, 2 column volumes of 20 mM MES, pH 6.0, 0M in IaCl (Buffer C) and 2 column volumes of buffer A · gpl2pA32 was charged with 0.11 acetic acid, pH 2.8, and the fractions were immediately neutralized by the addition of 0.1 volumes of tris - (hydroxymethyl) aminomethane (Tris), pH 10.4. The mouse anti-mouse gp2020 mouse hybridomorphonuclear antibody 178.1 was produced according to Example 3 above. -26- 70 291 SBC CASE 14430

This hybridoma was seeded at 2 x 10 células cells / ml and cultured for four days in Eagle's Medium with Eulbecco's Modification " Hazelton Research Products " supplemented with 4.5 grams / liter glucose, 41 of glutamine and 1% of serum. The OM containing antibody 178.1 (membrane 0.2 μm) was loaded and applied to a protein A-Spharose (Pharmacia) column (17 ml) 1.5 cm × 10 cm) equilibrated in Tris 0.11, pH 8.2. The antibody was eluted with 0.1 M sodium citrate, pH 3.5, and neutralized immediately with Tris.

The purified anti-gp? 20 monoclonal antibody to CNBr activated Sepharose 4B (Pharmacia) was coupled according to the manufacturer's instructions at a density of 2 mg of resin-Âμg / ml of resin and with an efficiency of coupling of 98%, resulting in an anti-gp120-Sepharose affinity resin. This affinity resin will specifically bind the gp120 protein by the interaction of the antibody with a unique structural epitope on the gp120 · Δ purity of the gp120 protein end product, according to this purification technique, is 80-90%, in a yield estimated from 8.5 mg / 30 liters of conditioned medium. Recovery is estimated to be between $ 25- $ 50.

It is also appreciated that this purification technique, and this affinity residue, is effective with other HIV proteins, or their fragments, with this epitope.

EXAMPLE 5 Assay The assay described below is a non-isotopic assay using an enzyme and a substrate for the detection of gp120 or fragments thereof which has been used in the detection of gp120 proteins produced by the methods and by the compositions of the present invention

In the assay, the criteria for detecting gp120 are dependent on antibody specificity. To capture the protein in gp120 a single anti-gp? 20 /? -Supont, ne of Cat 9284J7 antibody is used, diluted in sodium carbonate buffer 0.1M (pH 9.5) to 2 Âμg / ml · 100 Âμl of this antibody dilution is added to each well in duplicate on an assay plate, except for wells designated as controls. Incubate the plates at 42 ° C overnight. The next day the antibody was removed by washing, and the plate was blocked by adding 300æ. of blocking buffer, consisting of 1% BSA in PBS, each well, for 1 hour at room temperature.

Viral gp120 standards were diluted at 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.1 μg / rul and 0.2 μg / ml in a wash buffer consisting of PBS and 0.05% Tween 20. 100/1 of the standard dilutions are added to each well in duplicate. The plates were incubated on a stirring plate for 2 hours at room temperature, and then, each dish was washed four times with wash buffer. To each well, 100 .mu.l of rabbit anti-gp120 antibody (described by BeBouck et al, U.S. Patent Application No. 07 / 056553f deposited at 29 ° C) was added, diluted 1/1000 in wash buffer, and each plate was incubated on a shaking plate for 1 hour. This second antibody causes gp120 to be between the two antibodies. The plates were then washed four times with wash buffer. To de-tect this complex, a third 10 μl goat anti-rabbit peroxidase-labeled goat antibody (P0B) (mostly IgG and IgI antibody) diluted in azide-free wash buffer is added to each well. The plates are then incubated for 2 hours on an agitator at room temperature.

After washing the plates four times, 100æg of a colorless substrate (1 mg / ml of 0PD) was added in 4% aqueous buffer with 4æg of 35% hydrogen peroxide per 10 ml of buffer ). The hydrogen peroxide was added just prior to the addition of the substrate to the wells. These wells were incubated for 8 minutes on a shaker and the reaction was quenched by the addition of 100 μl of 0.11 sodium fluoride in each well. In the presence. peroxidase conjugated antibodies, the substrate becomes yellow in color. The optical density, or color intensity, was read out which is proportional to the amount of gp-2 captured in a plateau at 450 manometers, and a standard curve was constructed with the concentrations of unknowns being calculated. The amount of gp120 in the culture supernatant was determined by comparison with this standard curve. The foregoing description and examples exhaustively describe the invention, including preferred embodiments thereof. Modifications of the methods described above, for example using other truncated gp120 / gp120 sequences are assumed to be > which are obvious to one skilled in the art of molecular genetics and related sciences, are encompassed by the field of the following claims.

Claims (5)

-29- 70 291 SBG GASE 14430 -REIVIIBITIONS-1e. A method of producing an HIV protein in Brosophila cells, characterized by culturing Brosophi-la cells transfected with an HIV protein gene expression unit, in a suitable medium, said unit having a coding sequence of AM of said protein and a regulatory element necessary for transcription of the coding sequence and translation into a Brosophila cell. A process according to claim 1, characterized in that said regulatory element comprises an actin promoter, or a Brosophila metallothionein promoter. 3-3S. 2. A method according to claim 2, characterized in that said gene expression unit comprises the DNA coding sequence of gp120 or gp110. 4. A method according to claim 2, characterized in that said gene expression unit comprises the HIV-AM coding sequence as present in pgp160 / 32, pgp120P / 32, pgp220 / 32 or pgp220A274. 53. A method according to claim 1, wherein said gene expression unit is present in a DNA vector. 6 & Brosophila cell, characterized in that it is transfected with the vector of claim 5. 7. A process for the production of an EIV gpl20 or gpl60 protein, characterized in that insect cells according to claim 6 are cultivated. 83. A method of preparing a vaccine for stimulating protection against HIV infection characterized in that it comprises associating an immuno-protective and non-toxic amount of the protein prepared according to claim 7 with a pharmaceutically acceptable carrier or diluent. 93. A method of preparing a diagnostic agent for the detection of HIV infection in a human subject comprising comprising associating the protein prepared in claim 7 with a pharmaceutically acceptable carrier or diluent. 70 291 SBG CASE 14430 10-. 2. A method according to claim 1, wherein said cells are transfected with a first vector containing the hygromycin B coding sequence and with a second vector containing the coding sequence of an expression unit of H1Î ± 11s protein gene. 5. A process according to claim 1, characterized in that said first vector is the ρΟΟΗΪΟΗΟ * 12s. 2. A method according to claim 1 wherein said second vector comprises a unit of expression of the HIV gene as present in pgp110A32, pgp120FA32 > pgp2o / 32 or pgp120A274. 13. A method according to claim 1, characterized in that Brosophila cells are D. melanogaster Sg cells. 14s. The process of claim 13 > characterized in that said cells 82 are co-transfected with the pCOHYGRO vector and with a vector comprising a gene expression unit as present in pGPL6AA32 > pgpl20FA32, pgpl20A32 or pgpl20A274. 15ê. A method for identifying an HIV binding substance comprising contacting said substance with the protein of claim 7 and determining the occurrence of binding between said substance and said protein, said binding being indicative of binding to HIV. 16 ^. A method of purifying the HIV protein of claim 7 comprising the use of an affinity resin containing a monoclonal antibody capable of reacting with the epitope present on the products of the non-denatured gp160 protein and the mature gp120 protein. 17-. A method of purifying the HIV protein produced according to the process of claim 1 > characterized in that it comprises the use of an affinity resin containing a monoclonal antibody capable of reacting with an epitope present on the products of the non-denatured gp160 protein and the gp120 ma dura protein. Iiisboa, / + r * λ 0 By SMHHKLIHE BEECHAI CORPORATION ^ --- ΊΓ? nETflTAT- - -