NO314090B1 - HIV protein gene expression unit, DNA vector comprising it, Drosophila cell transfected with the vector, HIV gp 120 and HIV gp 160 protein produced in a culture of said cells, vaccine and diagnostic agent comprising the protein, - Google Patents

Description

The present invention mainly relates to the expression of HIV proteins in Drosop/w/a cells and purification of the expressed gene products. More specifically, this invention relates to HIV protein gene expression unit, DNA vector comprising it, Drosoptø/a cell transfected with the vector, HIV gp120 and HIV gp160 protein produced in a culture of said cells, vaccine and diagnostic agent comprising the protein, as well as method for the production of HIV protein in DrosopMa cells and methods for identifying HIV binding material, and purification of the HIV protein.

Human immunodeficiency virus type 1 (HIV-1) is the etiological gene for acquired immunodeficiency syndrome, also known as AIDS. This retrovirus has a complex genetic organization, including the long terminal repeats (LTRs) <g>a<g>, pol, and env genes, and other genes. This retrovirus carries a number of viral antigens that are potential candidates, either alone or together as vaccine agents, capable of inducing a protective immune response.

Among the more promising of the HIV-1 antigens is the viral envelope glycoprotein (gp160) or specific fragments thereof, the eny gene encoding the 160 kilodalton (kd) precursor glycoprotein of the viral envelope. gp160 is cleaved post-translationally into a 120 kd glycoprotein (gp120) and a 41 kd glycoprotein (gp41), which are present on the virus surface.

gp120, a 511 amino acid glycoprotein, is located at the amino terminus two-thirds from the gp160 glycoprotein and is exposed on the outside of the virus. gp120 is essential for the interaction between the virus and its cellular receptor, the CD4 protein present on the surface of helper T-lymphocytes, macrophages and other cells in the immune system. Thus, gp120 determines the tissue selectivity for virus infection and contributes to the cytopathogenicity of HIV through its involvement in syncytium formation.

gp41, a 345 amino acid protein formed from the carboxyl terminus of gp160, is an integral membrane protein of HIV-1. gp41 contains a number of hydrophobic amino acids that anchor the protein in the lipid bilayer in the cellular plasma membrane. The carboxyl terminus of gp41 is thought to protrude into the virus particle. gp41 or part of it is believed to be responsible for fusion between the HIV glycoproteins expressed on the surface of the cell and cells that have surface T4 receptors.

The part of gp41 thought to be responsible for this fusion is located at the amino terminus. Such a fusion is thought to play a role in virus 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 virus spikes protrude from the surface of the virus particle. About 70 to 80 spikes are thought to be associated with each newly synthesized virus particle. As the virus particle ages, the spikes disappear, apparently because the connection between gp120 and gp41 is weak. Thus, for newly synthesized virus particles, this viral glycoprotein spike is thought to be the most immediate target available to the immune system after infection.

Virus-neutralizing antibodies have been reported directed against gp120 and gp41 epitoms. It has been specifically noted that the target site for type-specific neutralizing antibodies is located in the 3' half of the gp120 glycoprotein molecule.

The env gene in HIV-1 has thus been the target of numerous recent investigations. Expression of glycosylated gp160 has previously been achieved in mammalian cells and certain baculovirus insect cells by groups that have also reported induction of both

humoral and cellular immune responses against these antigens. gp120 has been expressed recombinantly using heterologous promoters in several systems. See e.g. S. Chakrabarti et al., Nature (London), 320: 537 (1986); and M.P. Kieny et al., Biotechnology, 4: 790 (1986).

LET. Lasky et al., Science, 233: 209-212 (1986) constructed a series of plasmids containing mutant env genes for transfection into mammalian cells, specifically Chinese hamster ovary (CHO) cells. These researchers secreted a gene product coded for in a plasmid containing the first 50 amino acids

the glycoprotein D (gD) protein aligned in phase with an amino acid sequence (no. 61 - no. 531) in the env protein. an HPsAG polyA signal, a DHFR gene and the SV40 origin of replication. A recombinant coat antigen was produced which contained 25 amino acids of gD at the amino terminus and lacked 30 residues from the mature produced from gp120 and also had a deletion of the gp41 sequence (approximately 20 amino acids from the carboxyl terminus to the relevant 160 kd precursor production site) . The resulting gene was 520 amino acids in length. When transfected into CHO cells, the cell conditioned supernatants contained a 130 kd protein, named gp130.

A later report, L.A. Lasky et al., Cell, 50:975-986 (1987), discussed the interaction between the gp120 glycoprotein and its cellular receptor, CD4. By deleting 12 amino acids contained in amino acids #410-421 of gp120, a complete loss of binding resulted. Likewise, a single amino acid substitution at position 417 resulted in decreased binding.

Kowalski et al., mentioned above, introduced deletion and insertion mutations in a plasmid encoding the envelope glycoprotein formed from the HTLV-IIIB strain of HIV-1. The plasmid also contained the art-<q>ene. Mammalian CD4+ and CD4- cell lines that expressed this gene product. were used as transfection recipients to study the ability of the transfected cells to fuse.

Knight et al, cited above, describe expression of the art/trs transactivator protein of HIV in mammalian cells. The mammalian cell line used for expression of these HIV proteins was the COS-7 monkey cell line. These plasmids used the HIV LTR as a promoter and RNA production signals from SV40 to express the inserted DNA as a functional messenger RNA. To express gp120, a plasmid pENV160 containing the entire coding region of the eny gene fused to the HIV LTR was developed.

S.W. Pyle, Aids Research and Human Retroviruses, 3(4): 387 (1987) describes purification of the gp120 glycoprotein from culture fluids of HIV-infected H9 cells by immunoaffinity chromatography.

U.S. patent 4,725,669 also describes glycoproteins of approximately 160 kd and 120 kd obtained from the human H9/HTLV-III cell line, each having an approximately 90 kd unglycosylated portion.

Fox, Biotechnology, 6:116 (1988) reports the VAXSYN HIV-1 vaccine developed by MicroGeneSys. This report does not describe 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 (1988) describe the cloning of the entire gp160 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 by lysis. This protein travels together with gp160, is not cleaved into gp120 and gp41, and is glycolysed and associated with the cell membrane. When deglycolyzed 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 against a recombinant fraction of gp120, with gp120 itself, with a peptide fragment of gp41 and with human AIDS sera.

R.L. Willey et al., Proe. Nat'1 Acad. Sci., USA, 83: 5083 (1986) describes hypervariable regions of amino acids in gp120 protein. A later report, R.L. Willey et al, J. Virol., 62(1): 139 (1988) studied a region of the envelope required for infectivity. Specifically described here are amino acid substitutions in Asp codons in four N-linked glycosylation sites in the gp120 gene.

W. R. Gallagher, Cell, 50*: 327 (1987) discusses a fusion peptide sequence in transmembrane protein gp41 of HIV. S. D. Putney et al., Science, 234:1392 (1986) describe the production of neutralizing antibodies against an E. coli -produced fragment of the HIV env gene. B. J. Bond agency, Mol. Cell. Biol., 6(6): 2080 (1986) describes the structure of the Drosophila melanogaster actin 5C gene. The report discusses the two transcription start sites in the actin 5C gene and fusions between the promoter sequences and the bacterial chloramphenicol acetyltransferase gene inserted into D. melanogaster host cells. P. J. Barr et al., Vaccine, 5:90 (1987) describe the expression of HIV proteins in the yeast cell Saccharomyces cerevisiae. H. Johansen et al., 28th Annual Drosophila Conference, p. 41 (1987) is a summary of the present application which briefly confirms that E. coli gal K genes regulated by Drøsop/7//a-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 the expression of foreign genes in D. melanogasterceWer in culture. The abstract notes that the system was used to simultaneously introduce and overexpress the E. coli gal K gene and other genes of mammalian origin. Thus, there is again a need in the field for high-level production of HIV proteins for use as a vaccine and diagnostic agent. ;The applicant's Drosophila system has the ability to produce gene products in active form and in high yields (typically 5 mg/l without any optimization and as high as 50 mg/l with optimization) without the need for time-consuming concentration steps. In addition, this system provides stable cell lines, which is particularly important because access to certain proteins needs to be secured over years. Furthermore, the ability to grow these cells in large-scale cell cultures in inexpensive media has made this system a favorite of fermentation researchers and protein biochemists because large amounts of virus are not needed (as in the baculovirus system), and the expressed proteins are soluble and biologically active in contrast to E. co// expression which typically yields insoluble inactive proteins that often need re-folding. The use of the applicant's Drosophila system to produce viral antigens such as HIV gp120 and gp160 is described in the present application. A number of other viral antigens such as HSV-VP16, HCV protease, RSV F protein, etc, have been successfully expressed as well. Expression of RSV F protein is particularly noteworthy because no other system to date has been able to produce milligram quantities of this protein in an active (or functional) form. Expression of this protein using the baculovirus system results in the production of an incorrectly processed protein that is not functional. ;In one aspect, the present invention is an HlV protein gene expression unit for use in the production of high-level active HIV proteins, characterized in that it comprises a DNA selected from the group encoding gp120 or gp160, and a regulatory element necessary for transcription of the coding DNA sequence and translation in a Drøsopr>//a cell. ;In related aspects, this invention is a DNA vector comprising the gene expression unit according to the present invention. In yet another related aspect, this invention is a Drosophila cell transfected with the DNA vector of this invention. In further related aspects, this invention is an HIV gp120 or gp160 protein, which is or gives rise to active proteins produced in a culture of insect cells according to the invention. In another aspect, this invention is a vaccine for stimulating protection against HIV infection, comprising an immunoprotective and non-toxic amount of the protein produced by this invention. Also provided by this invention is a diagnostic agent useful in detecting the presence of HIV infection in a sample of biological fluid containing a Drosoprw/a cell-produced HIV protein according to the invention. In addition, the protein according to the present invention can be used to identify or isolate HIV binding proteins or proteinaceous substances such as CD4 or derivatives thereof. ;This invention also relates to a method for the production of an HIV protein which comprises the cultivation of Drosoprw/a cells transfected with an HIV protein gene expression unit according to claim 1, 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 can then be collected from the cell or the cell culture medium. The present invention also provides a method for purifying HIV proteins according to the invention by using an affinity resin containing a monoclonal antibody reactive with an epitope present in non-denatured gp160 protein products and in mature gp120 protein. Among antibodies in this class is the mouse monoclonal antibody designated 178.1. Other aspects and details of the present invention are discussed in the following description. The method and expression system according to the present invention facilitate high-level production of HIV proteins, especially gp120, gp160, in a Drosophila cell structure. The Drosop/7/7a cells are transfected using standard cloning techniques that allow the introduction of foreign DNA into a host cell without adversely affecting the foreign DNA or the host cell. The recombinant DrosopAw/a cells thus constructed produce HIV proteins. In contrast to the Baculovirus system from the prior art, where the HIV protein is provided only by lysis of the infected insect cells, the method according to this invention provides a continuous cell expression system for HIV proteins. In case of secretion, the protein is available by purification from the culture medium using conventional techniques. Alternatively, the protein can be produced intracellularly or membrane-bound. The protein can be extracted from the cells using conventional techniques. Alternatively, membrane-bound protein can be used in a number of cell-associated measurements. A preferred Drosopr»7a cell line for use in the practice of the invention is the D. melanogaster SHinje. Stem cells [Schneider, J. Embryol. Exp. Morph. 27: 353 (1972)] are stable cell cultures of polyploid embryonic Drosopr»/a cells. Introduction of the cDNA coding sequence for gp160 or its subunit gp120 or gp41, or derivatives thereof into Drosophila S2 cells by DNA transfection techniques produces unexpectedly large amounts of glycoproteins. Use of the S2 DrosopMa cell has many advantages, including, but not limited to, its ability to grow at 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 Drosop/7/Va cell culture systems may also be useful in the present invention. Some possible applicable cells are e.g. The KC-0 Drosophila Me/anogasfer cell line which is a serum-free cell line [Schulz et al., "Proe Nati. Acad. Sei. 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 that may be useful is a Drosophila hydei cell line. Protein expression can be achieved using the hydeÅ cell line; however, transfection into this cell line may result in the transfected DNA being expressed at very low efficiency [Sinclair et al., Mol. Cell. Biol., 5: 3208 (1985)]. Other available DrosopMa cell lines that can be used in this invention include Si and S3. ;Drosopr)//a- the cells selected for use in the present invention can be cultured in a variety of suitable culture media, including, for example, M3 medium. M3 medium consists of a formulation of balanced salts and essential amino acids at a pH of 6.6. Preparation of the media is essentially as described by Lindquist, DIS, 58:163 (1982). Other conventional media for the growth of Drosop/7//a cells can also be used. A recombinant DNA molecule or a vector containing an H1V 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 can be obtained by manipulating 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 recently been published. See Råtner 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 BH 10, Råtner et al., supra). DNA molecules comprising the coding sequence of this invention can be generated from HTLV-III infected cells using known techniques (see Hahn et al., Nature 312:166-169 (1984), or, alternatively, can be synthesized by standard oligonucleotide techniques. Furthermore, 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, for example, gp120 or gp160 molecules where one or more amino acids have been substituted, added or deleted without significantly adversely affecting the binding capacity or biological properties of the protein. Derivatives of these proteins can also be prepared by standard chemical modification techniques, eg acylation, methylation. ;Also included in the gene expression unit, regulatory regions are necessary or desirable for transcription of the HIV protein coding sequence and its following translation and expression in the host cell. The regulatory region typically contains a promoter region that functions in the binding of RNA polymerase and the initiation of RNA transcription. The promoter region is typically found upstream from the HIV protein encoding sequence. Preferred promoters are of Drosoprw/a 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. CuSO4. Use of the Drosoptø/α-metallothionein promoter results in the expression system according to the invention and preserves full regulation even at a very high copy number. This is in direct contrast to the use of the mammalian metallothionein promoter in mammalian cells where the regulatory effect of the metal is reduced as the copy number is increased. In the Drosophila expression system, this conserved inducibility effect increases expression of the gene product in the Drøsop/w/a cell at high copy number. ;The Drosophila actin 5C gene protomother [B.J. Bond et al., Mol. Cell. Biol., 6: 2080 (1986)] is also a desired promoter sequence. The actin 5C promoter is an intrinsic promoter and does not require the addition of metal. Therefore, it is better suited for use in a large-scale production system, such as a perfusion system, than the Drøsopri/7a-metallothionein promoter. An additional 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 Drosoptø/a promoters include e.g. the inducible heat shock (Hsp70) and COPIA LTR promoters. The SV40 early promoter gives lower levels of expression than the DrosopMa metallothionein promoter. Promoters commonly used in the cell expression vectors, including e.g. avian Rous sarcoma virus LTR and simian virus (SV40 early promoter) show poor function and expression in the Drosophila system. A desired gene expression unit or expression vector for the HIV protein can be constructed by joining the HIV protein coding sequence with a desired signal sequence. The signal sequence acts to direct the secretion of the protein from the host cell. Such a signal sequence can be formed from the tissue plasminogen activator (tPA) sequence. Other available signal sequences include e.g. those formed from the Herpes Simplex virus gene HSV-I gD [Lasky et al., Science supra]; The HIV DNA coding sequence may also be followed by a poly-adenylation (poly A) region, such as an SV40 early poly A- area. The poly-A region that functions in polyadenylation of RNA transcripts appears to play a role in stabilizing transcription. A similar poly-A region can be formed from a number of genes where they are naturally present. This region can also be modified to change the sequence, provided that polyadenylation and transcriptional 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 selectable marker can be any gene or genes that cause a readily detectable phenotypic change in a transfected host cell. Such a phenotypic change can e.g. be drug resistance, such as the gene for hygromycin B resistance. Alternatively, a selection system using the drug methotrexate and the prokaryotic dihydrofolate reductase (DHFR) gene can be used with Drosop/w/a cells. The endogenous eukaryotic DHFR in 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 survive. Unlike methotrexate in selection of transformed mammalian and bacterial cells, methotrexate can be used in the Drosoprw/a system to initially high-copy number transfectants. Only cells that have incorporated the protective prokaryotic DHFR gene will survive. At the same time, these cells have the gene expression unit of interest. An illustrative plasmid produced according to the present invention is pgp160A32, which contains a gp160 derivative that replaces the N-terminal 32 amino acid sequence of gp160 with the first amino acid of tPA, serine. This plasmid is further described in example 1. Another such plasmid vector is pgp120FA32 which contains the gp160 sequence which has the first 32 amino acids replaced by serine and which contains a carboxyl deletion of 216 amino acids. This plasmid is also described in Example 1. Yet another plasmid that illustrates the derivative proteins according to the present invention is pgp120A32, which contains the entire coding sequence for gp120 minus the first 32 amino acids in the N-terminus, which is replaced by serine . In addition, plasmid pgp120A274 contains a gp120 protein sequence that has replaced the first 274 amino-terminal amino acids with the first amino acid of tPA, serine, and which contains the remaining amino acids of pg120 up to the production site of gp160. These vector constructions are described more fully in Example 1. Once a recombinant DNA molecule or an 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 skilled in the art and include e.g. calcium phosphate co-precipitation, cell fusion, electroporation, microinjection and virus transfection. A two-vector system can be used in the present invention to co-transfect into the DrosopMa cell a gene expression unit for the desired HIV protein or a 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 using a vector containing an HIV protein expression unit, e.g. pgp120A32, and a vector containing a hygromycin B gene expression unit, e.g. pCOHYGRO. pgp120A32 contains an expression unit comprising the Drosophila metallothionein promoter, a derivative of the gp120 gene, and the SV40 poly A site. This gp120 expression unit in combination with the pCOHYGRO vector system will produce a pg120 derivative in S2 Drøsoptø/a cells by maximizing the advantage of hygromycin B resistance for selection. With this system, the antibiotic hygromycin B can be used to select for the cells that contain the transfected vectors. A more complete description of this embodiment is described in example 2. As another example, an expression system can be used where one uses the DHFR gene/methotrexate selection system, which consists of the vectors pgp120A32 and pHGCO, to select methotrexate-resistant cells for expression of gp120 or a derivative thereof. The vector pgp120A32 comprises a gp120 gene expression unit where the promoter is the Drosophila metallothionein promoter. The pHGCO vector comprises a DHFR gene expression unit and is co-transfected with the pgp120A32 vector, thereby providing the DHFR genes required for selection. These selectable markers together with co-transfection of DrøsopMa cells are further described by Johansn et al., US Patent Application Serial No. 07/047,736, filed May 8, 1987 and are incorporated by reference herein. According to the invention, the two vectors are co-transfected into the S2 Drosophila cell by using the method described in Wigler et al., Cell, 16: 777 (1979). The vectors are co-transfected in varying proportions, depending on the particular desired copy number. The transfected cells are then selected, such as in M3 medium containing serum and in a suitable selection agent, e.g. hygromycin-B or methotrexate. Once an appropriate vector has been constructed and transfected into the selected Drøsop/w/a cell line, the expression of gp120 is induced by the addition of an appropriate induction agent for the inducible promoter. For example, cadmium or copper are inducers of the metallothionein promoter. Heat is the induction agent for the Hsp70 promoter. For intrinsic promoters such as the actin 5C promoter, no inducer is required for expression. Transcription and expression of the HIV protein coding sequence in the systems described above can be measured. For example, Southern blot analysis can be used to determine the copy number of the gp120 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 quantified. Expression of the selected HIV protein in the recombinant cells can further be verified through Western blot analysis and activity tests on the resulting glycoprotein [see example 5]. Drosophila S^ cells are particularly suitable for production with a high yield of protein in the method according to the present invention. The cells can be maintained in suspension cultures at room temperature (24+1 °C). Culture medium is M3 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 medium. When the pCOHYGRO vector system is used, the media is also supplemented with 300 ug/ml hygromycin B. In this medium, the S2 cells can be grown in suspension cultures, e.g. in 250 ml to 2000 ml spinner flasks, with stirring at 50-60 rpm. Cell densities are typically maintained between 10<6> and 10<7> cells per ml. In one embodiment of this invention, the cells are cultured prior to induction in 1500 ml spinner flasks in medium containing 5% serum. ;After cell culture, the HIV protein can be isolated from the media used, e.g. using 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 medium. Use of the cell line S2, 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 efficient one-step purification system. By using a monoclonal antibody column directed against the HIV protein, the culture medium used can be added directly to the column, and the protein eluted by using 1.5 M KSCN in phosphate buffered saline (PBS). ;A preferred purification technique that enables efficient large-scale production of the HIV proteins according to the invention uses an immunoaffinity column containing a monoclonal antibody directed against an epitope present in gp160 and present in mature secreted gp120 proteins. Such a monoclonal antibody is advantageous because of its capacity to recover the protein sequence in more than one configuration. An antibody that has these properties and is applicable in immunoaffinity columns for various HIV proteins, derivatives or fragments thereof is designated 178.1. This monoclonal antibody is described in greater detail in Example 3. Such a column according to the invention can be made by binding an antibody with the properties of 178.1 to a conventional absorbent support, such as Sephadex, under suitable conventional conditions of pH, temperature and the like . Such a purification column and method can be used to separate the HIV proteins and fragments of the present invention. Other monoclonal antibodies can be used in this purification procedure. A number of monoclonal antibodies capable of binding to HIV proteins, particularly gp160 or gp120, have been described in the art and are available. Other new monoclonal antibodies useful in this invention may be developed by new conventional techniques. The glycoproteins produced by Drosophila cells, according to this invention, are completely free of contaminating materials, e.g. mammalian, yeast cell, bacterial and more importantly, other HlV virus materials. Drasop/j/7a-produced HIV proteins have also been shown to have different glycosylation patterns than those reported by other systems, e.g. mammalian systems. The HIV proteins and the produced derivatives according to the present invention can be used in a number of products. For example, these recombinant proteins can be used in pharmaceutical compositions for the treatment of HIV-infected individuals. Such a pharmaceutical composition, according to the present invention, comprises a therapeutically effective amount of the HIV protein or a derivative of the invention in mixture with a pharmaceutically acceptable carrier. The mixture can be administered systemically, either parenterally, intravenously or subcutaneously. When administered systemically, the therapeutic composition for use according to this invention is in the form of a pyrogen-free, parenterally acceptable aqueous solution. The production of such a parenterally acceptable protein solution, which is suitable in terms of pH, isotonicity, stability, etc., is within the knowledge of the field. The dosage form will be determined by the practitioner, taking into account various factors that modify the effect of drugs, e.g. the patient's condition, body weight, gender and diet, the severity of any infection, the time of administration and other clinical factors. The pharmaceutical carrier and other components of a pharmaceutical formulation will be chosen by one skilled in the art. In addition, the recombinant proteins according to the present invention can be used as components for vaccines to inoculate mammalian individuals against HIV infection. These proteins can be used alone or together with other recombinant proteins or therapeutic vaccines. Components for such a vaccine will be determined by a specialist in the field. Finally, the proteins according to the present invention can be used as diagnostic agents for detecting the presence of HIV infection or antibodies against an HIV infectious agent in biological fluids, such as blood, serum, saliva, etc. These proteins according to the invention can also be used in methods for identifying and/or isolating HIV-binding proteins or other HIV-binding substances in biological fluids and tissues, e.g. CD4 or derivatives thereof. The proteins can thus be components of test kits for carrying out such methods. In order to identify an HIV-binding substance, a protein according to the invention is used to bring the substance or an impure mixture containing the substance into contact, under conditions to promote binding between the protein and the HIV-binding substance. A conventional measurement to detect the occurrence of binding, e.g. detection of radioactive markers or the like is also part of the procedure. Presence of binding between the protein and the binding substance is therefore indicative of HIV binding. Similarly, in a method for isolating an HIV-binding substance from the mixture, the binding occurrence can be followed by a conventional method to purify the bound unit formed by the protein according to the present invention and the HIV-binding substance from the mixture. Other components of such diagnostic systems and test kits may be conventional components of the diagnostic test kit and may be selected by those skilled in the art. The following examples illustrate the construction of exemplary vectors and transformants according to the invention, the preferred purification system and measurements for determining the production level of glycoproteins gp120 and gp160. These examples are not to be construed as limiting the scope of this invention. Restriction enzymes and other reagents were used essentially according to the inventors' instructions. ;Examples ;Example 1. Vector constructions ;a) pMTtPA ;As the basic vector for gene expression in Drosophila, tPA expression vector pMTtPA (also called pDMtPA) was used. This vector is a derivative of vector pML1, a small pBR322 vector containing the beta-lactamase gene that has deleted the toxin sequences [Mellon et al., Cell, 27: 297 (1982)]. These sequences are inhibitory against propagation of the vector. This vector was cleaved with SalI and Aat2 which removes a small piece of pBR322 and the cleaved ends were filled in. The missing piece of pBR322 is then replaced with a cassette containing the Drosophila metallothionein promoter on an end-filled EcoR1-Stu1 fragment, followed by a filled-in HindIII-Sac1 fragment from dDSPI [D.S. Pfarr et al., DNA, 4* (6): 461 (1985)] containing a tPA sequence containing the signal sequence, the prepeptide and the entire coding region of tPA. The tPA gene on this fragment is followed by an SV40 early polyadenylation site.

b) pgp160A32

A Hind 111-Xba1 fragment containing the entire env gene is isolated from a

HIV isolate clone BH10 [L. Råtner et al., mentioned above; GenBank]. The entire gp160 sequence was then inserted into the Nco1-Xba1 cleaved vector pDSP1. The resulting vector, SU2, was digested with Nde1, followed by treatment with mung bean nuclease and further digested with Sac1, thus isolating the gp160 gene. The cleavage with Nde1 cut the gp160 sequence at amino acid no. 32. The Sac1 cleavage cuts 3' in the gp160 gene, including in the sequence a part of the original pDSP1 vector containing a polylinker. This fragment was inserted into the above-described expression vector pMTtPA which had been cleaved with Bgl II, end filled, and further cut with Sac1, which deletes the mature tPA sequence. The BgJ.ll site is located in the first amino acid of tPA. Accordingly, the resulting vector pgp160A32 encodes a modified gp160 protein that has replaced the N-terminal 32 amino acids of gp160 with serine.

c) pgp120A32

Another vector containing a modified gene sequence encoding

HIV-1 surface glycoprotein gp160, was constructed by cleaving pgp160A32 with Hindi 11 and Sac1, thereby removing the carboxyl terminus of gp160. About two-thirds of the sequence coding for gp41 is removed by this cleavage. Thus, this gp160 sequence lacks the first 32 amino acids and the last 216 amino acids of the natural gp160 sequence. The deleted sequence was replaced by a short synthetic linker sequence encoding a stop codon on a Hinlll-Sac1 fragment. The 6-amino acid linker sequence is as follows:

d) pgp120A32

Yet another vector containing a mutant gp160 gene was constructed

by cleaving pgp160A32 with Sty1 and Xba1. thereby cleaving the entire sequence in protein gp41 and approximately 30 amino acids in the carboxyl terminus of the gp120 glycoprotein sequence. This fragment was replaced by a synthetic Stv1-Xba1 linker sequence encoding the correct carboxyl terminus from the Sty1 site to the preparation site in gp120-gp41. This sequence was followed by a stop codon. This sequence thus contained the entire coding sequence for gp120 minus the first 32 amino acids and none of the gp41 coding sequence.

e) pgp120A274

Yet another exemplary vector containing a mutant gp120 gene,

was constructed as follows: a 720-bp carboxyl-terminal fragment of gp120 was isolated by partial digestion of pgp120A32 with Bgjll followed by XbaI digestion. This fragment was now inserted instead of the tPA gene into the Bqllll-Xba1 truncated pMTtPA expression vector. The resulting vector p120A274 encodes a gp120 protein that 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 seat. was applied. The 5'-LTR of 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 scattered central repeat sequences found scattered throughout the DrosopMa genome [Rubin et al, in Cold Spring Harbor Symp. Quant. Biol., 45: 619 (1980)]. Vector pUCOPIA was excised at the SmaI site and the E.co// gene encoding hygromycin B phosphotransferase (hygromycin B cassette) was cloned into pUCOPIA using standard cloning techniques. The hygromycin B cassette was isolated on a HindIII-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 encoding the hygromycin B phosphotransferase gene and the SV40 early poly-A region. The HindIII and BamHI sites were filled in using T4 DNA polymerase and the hygromycin B cassette was ligated into the SmaI site in vector pUCOPIA producing vector pCOHYGRO.

Example 2. Transfection into Drosophila S2 cells

pCOHYGRO was co-transfected into S2 Drosop/w/a cells together with one of the vectors carrying a gp160 mutant gene under the control of the Drosophila metallothionein promoter as described above. For the purposes of this example, the vector used is pgpl 20A32. The transfected cells were selected in M3 medium containing 5% serum and 300 u/ml hygromycin B. After 2 to 3 days under

identical conditions, the non-transfected cells stop dividing and begin to die. The time for selection to obtain stable, adult hygromycin B-resistant cells in the transfected cultures is approximately 2 to 3 weeks.

In order to obtain cultures that have integrated different copy numbers of the gp120 mutant gene into their chromosomes, the ratio between the two vectors was varied. The ratio in this example was 20:1. Similar conditions have been used for other gp160 mutant vectors according to this invention. This relationship is the same when any of the gp160 mutant vectors are used.

Expression of the pgp120A32 gene product was verified after induction of the metallothionein promoter with 500 µM CuSO 4 . Western blot analysis of the supernatant used from the induced cell cultures revealed a single band of approximately 100 kd.

The level of the mutant gp160 gene product in the cell supernatants was measured using the gp120 ELISA assay, described in Example 4, and using purified virus gp120 as standards. 5 x 10<6> cells/ml were plated in serum-free M3 medium and induced for 3 to 4 days. The level of gp120 measured in the supernatant is approximately 1-2 mg/l.

Cells were maintained as suspension cultures in 250 ml to 2000 ml spinner flasks. Culture medium was M3 supplemented with 300 µg/ml hygromycin B. Cultures were incubated at 24+°C and stirred at 50-60 rpm. Cell densities were typically maintained between 10<6> and 10<7> cells per ml in M3 medium supplemented with hygromycin B. CuSO 4 was added to a final concentration of 500 M, and the cultures were allowed to grow for 3 to 4 days in serum-free medium, before harvesting the modified gp120 glycoprotein.

The proteins produced according to this method were approximately 100 MW, and the level of expression was higher than any other reported gp120/gp160 expression in any eukaryotic cell system. In standard biological activity assays, the purified, expressed modified gp120, as described above, is capable of inhibiting virus infection in tissue culture, binding T4 and reacting with antibodies against gp120.

It is expected that someone skilled in the field can express the other gp160 and gp120 proteins and fragments thereof, described in the present invention, by using essentially the same systems and methods as exemplified above for protein fragments encoded for in pgp120A32.

Example 3. Monoclonal antibody 178.1

An affinity purification column using a new monoclonal antibody was used in the purification scheme used for the mutant gp160/gp120 proteins described above. This monoclonal antibody can be characterized as being able to react with non-denatured HIV glycoprotein products present in cell lysate and with mature gp120 as secreted in the supernatant of a yeast cell culture. One such monoclonal antibody specific for the epitope contained both in the untreated gp160 recombinant molecule and in the missized gp120 protein is a mouse monoclonal antibody 178.1.

An expression system using the C. albicans glucoamylase promoter and signal peptide was used to produce partially purified yeast recombinant gp160 for production of 178.1. The production of this yeast cell-derived gp160 is described in US 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 intraperitoneally with the partially purified (1.5-3% purity) yeast cell recombinant gp160 in Freund's adjuvant at 4-week intervals. After a resting period of 3 months, a mouse was sacrificed and its spleen cells were added together with myeloma cells (see, e.g., R.P. Siaganian et al., Meth. Enz., 92:17 (1983);

EMBO Course on Hybridoma Production, Basel Inst. for Immunol. (1980)] The myeloma cells used are a subclone of the Sp2/0-Ag14 line previously selected for optimal growth in agar medium and high fusion efficiency [J.D. Franssen et al., Proe. XXIX Colloq. Protids Biol. Fluids, 29: 645-649 (1981)]. After approximately 10 days, supernatants were withdrawn for mapping in a capture ELISA, using a commercial monospecific anti-gp120 reagent [Biochorm, Seromed Ref. D7324] as capture antibody.

Briefly, Nunc Immunoplate I (No. 4-39454) was coated overnight at 4°C with 50 µl of a solution of 5 µg/ml sheep anti-gp120 IgG in PBS. Plates were washed with wash buffer (PBS, Tween 20 0.1%) and saturated with 100 µl saturation buffer [PBS, newborn calf serum 4%, bovine serum albumin (BSA) 1%, Tween 20 0.1%] for 1 h at 37° C. 50 µl/well of raw Molta/HTLV-lllB or Molt3 cell lysate (10<7> cells/ml in PBS, Triton X-100 1%) or of supernatant fraction (S2-30) of the recombinant yeast cell gp160 (or similarly treated negative control) was used as antigen and incubated in the plates for 3 to 5 hours at room temperature. The plates were washed thoroughly, and 50 µl hybridoma supernatant was added to each well and incubated overnight at 4°C. After a washing step, 50 µl/well of a V.500 dilution of biotinylated anti-mouse immunoglobulins (Igs) ( Amersham Ref. RPN 1021) in saturation buffer incubated in the plates for 1 hour at 37° C. The plates were washed again, and 50 µl/well of a 1:1,000 dilution of streptavidin biotinylated horseradish peroxidase complex (Amersham Ref. RPN 1051) in saturation buffer was added to each well.

After an additional washing step, 50 µl of a solution of 0.4 mg/ml orthophenylene diamine dihydrochloride (OPD, Sigma P1526) and 1 µl/ml H 2 O 2 (30% in citric acid/sodium citrate 0.1 M pH 5) was added to 0, 1% Tween 20 added to each well. The plates were then incubated for 20 minutes at room temperature in the dark, and the reaction was stopped by the addition of 50 µJ/well of 2M H 2 SO 4 . The optical density at lambda = 492 nm was measured, and 50 positive clones were selected for further subcloning in soft agarose, according to P. Herton et al., Proe. XXIX Colloq. Protids Biol. Fluids, 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-tetramethylpentadecane).

The monoclonal antibodies selected from the above procedure were characterized by Western blot analysis (WB), radtoimmunoprecipitation assay (RIPA), purification, biotin labeling and competition assays. Resulting monoclonal antibodies were further characterized by analysis of their reactivity against various recombinant and natural antigens.

A high yield of hybridomas was obtained by this method. More than 200 wells were positive in the mapping measurements. However, among them, only 50 wells were selected, and after cloning, the cells were expanded in peritoneal acid. All peritoneal fluids were tested in WB and RIPA. Among the 39 monoclonal antibodies tested, 37 showed a gp160 band in RIPA. No one reacted with the gp120 form in the same measurement. The monoclonal antibodies that showed only gp160 recognition in RIPA, while being clearly reactive against gp120 in WB, were analyzed by subclass. Three monoclonal antibodies that were IgG2A were purified on a protein A-Sepharose column and labeled with biotin. Competition measurements using vaccinia gp160 as antigen were performed and the obtained result defined at least five different groups of epitope recognition by gp160 proteins. Monoclonal 178.1 was selected for an epitope present on mature gp120 and untreated intracellular gp160.

A Western blot (WB) analysis was performed according to conventional techniques to show that 178.1 is capable of binding HIV virus isolated from human cells infected with HTLV-IIIB [MoIVHTLV-IIIb].

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

The reactivity of the monoclonal antibodies recognizing non-overlapping epitopes against a large panel of antigens was measured using a sandwich ELISA involving sheep anti-gp120 as the capture reagent. Monoclonal antibody 178.1 was negative in ELISA on divergent HIV isolates MoltVHTLV-lllu, H9/HTLV<RF1> and Hut78/ARV2, while clearly positive when

tested for HTLV-IIIb 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, provides an additional advantage for the production of gp160/gp120 glycoproteins in different constructs.

Example 4. Purification of gp120A32 from Drosop/i//a-conditioned cell culture medium

The recombinant gp120 protein from Example 2 was purified as follows: 30 liters of Drosop/irfa conditioned medium (CM) containing gp120A32 was prepared with 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM ethylenediaminetetraacetic acid (EDTA) and 70 Kallikrein inhibitor units. CM was filtered through a 0.45 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^ime 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, 0.4 M NaCl.

The S-Sepharose-eluted gp120A32 was applied to an anti-gp120 mouse monoclonal-Sepharose 4B column (60 ml; 3.2 cm x 6.5 cm) at an LFR of 10 ml/hr. This column was equilibrated in buffer B. After applying a half S-Sepharose load, the column was washed with 1 column volume of buffer B, 2 column volumes of 20 mM MES, pH 6.0, 1.0M NaCl (buffer C), and 2 column volumes -volume of buffer A. gp120A32 was eluted with 0.1 M acetic acid, pH 2.8, and fractions were immediately neutralized by addition of 0.1 volume of 1 M Tris(hydroxymethyl)aminomethane (Tris), pH 10.4.

Mouse anti-gp120 monoclonal antibody hybridoma 178.1 was produced according to Example 3 above. This hybridoma was seeded at 2 x 10<5> cells/ml and grown for four days in Dulbecco's Modified Eagle Medium [Hazelton Research Products] supplemented with 4.5 g/l glucose, 2 µM glutamine 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 0.1 M Tris, pH 8.2 . Antibody was eluted with 0.1 M sodium citrate, pH 3.5 and immediately neutralized with Tris.

Purified anti-gp120 monoclonal antibody was bound to CNBr-activated Sepharose 4B (Pharmacia), according to the manufacturer's instructions at a density of 2 mg antibody/ml resin and with a binding efficiency of 98%, resulting in an anti-gp120 Sepharose affinity resin. This affinity resin will specifically bind gp120 protein through interaction between the antibody and a unique structural epitope on gp120.

The purity of the final gp120 protein product according to this purification technique is 80-90% with an estimated yield of 8.5 mg/30 liters of conditioned medium. Recovery is estimated at between 25-50%.

This purification technique and affinity resin is also believed to be effective with other HIV proteins or fragments thereof that have this epitope.

Example 5. Measurement

The measurement described below is a non-isotopic measurement where an enzyme and a substrate are used for the detection of gp120 or fragments thereof, which were used in the detection of the gp120 proteins produced by the methods and mixtures of the present invention.

In the measurement, the criteria for detection of gp120 depend on antibody specificity. An anti-gp120 monoclonal antibody [DuPont, Cat. No. 9284], diluted in 0.1 M sodium carbonate buffer (pH 9.5) to 2 µg/ml is used to capture the gp120 protein. 100 µl of this antibody dilution is added to each well in duplicate in a measuring plate, except for such wells which are designed as 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.

The viral gp120 standards were diluted to 1 µg/ml, 0.5 µg, 0.25 µg/ml,

0.1 ug/ml and 0.2 ug/ml in a wash 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 incubated on a plate shaker for 2 hours at room temperature, and then each plate was washed four times with washing buffer.

To each well, 100 µl of rabbit anti-gp120 antibody (described by DeBouck et al, US Patent Application No. 07/056,553, filed May 29, 1987) diluted 1/1000 in wash buffer was added, and each plate incubated on a shaker for 1 hour. This second antibody "sandwiches" th gp120 between the two antibodies. The plates were then washed four times with wash buffer. To detect this complex, a third antibody, 100 µl peroxidase (POD) labeled goat anti-rabbit antibody (mainly IgG and IgM antibody) diluted in wash buffer without any azide is added to each well. The plate was then incubated for 2 hours on a shaker at room temperature.

After the plates were washed four times, 100 I of a colorless citrate (1 mg/ml OPD in citrate buffer with 4 µl of 35% hydrogen peroxide per 10 ml of buffer) was added. The hydrogen peroxide was added just before adding the substrate to the wells.

These plates were incubated for 8 minutes on a shaker, and the reaction was 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 strength of color, which is proportional to the amount of captured gp120, was read on a plate reader at 450 nm, and a standard curve was constructed with concentrations of unknowns calculated. The amount of gp120 in the supernatant culture was determined by comparison with this standard curve.

The above description and examples fully describe the invention, including preferred embodiments thereof. Modifications of the methods described, e.g. where using other truncated gp160/gp120 sequences that are obvious to one of ordinary skill in the art, molecular genetics and related sciences are intended to fall within the scope of the following claims.

Claims (17)

1. HIV protein gene expression unit for use in the production of active HIV proteins at a high level, characterized in that it comprises a DNA selected from the group that codes for gp120 or gp160, and a regulatory element necessary for transcription of the coding DNA sequence and translation in a Drosophila ceWe. 2. Gene expression unit according to claim 1, characterized in that said regulatory element comprises an actin 5C promoter or a DrøsopMa metallothionein promoter. 3. Gene expression unit according to claim 1, characterized in that it comprises the HIV DNA coding sequence present in pgp160A32, pgp120FA32, pgp120A32 or pgp120A274. 4. DNA vector, characterized in that it comprises the gene expression unit according to claim 1. 5. DrosopMa cell, characterized in that it is transfected with the vector according to claim 4. 6. HIV gp120 or gp160 protein which is, or gives rise to active proteins, produced in a culture of insect cells according to the preceding claim. 7. Vaccine to stimulate protection against HlV infection, characterized in that such a vaccine comprises an immunoprotective and non-toxic amount of the protein according to claim 6. 8. Diagnostic agent for detection of HlV infection in a human individual in vitro, characterized in that it comprises the protein according to claim 6. 9. Method for the production of an HIV protein in Drosoprw/a cells, characterized in that it comprises cultivation in a suitable medium of Drosop/)/7a cells transfected with an HIV protein gene expression unit according to claim 1. 10. The procedure according to claim 9, characterized in that said cells are transfected with a first vector containing the coding sequence for hygromycin B phosphotransferase and a second vector containing the coding sequence for an HIV protein gene expression unit. 11. The procedure according to claim 10, characterized in that said first vector is pCOHYGRO. 12. The procedure according to claim 10, characterized in that said second vector comprises an HIV gene expression unit which is present in pgp160A32, pgp120FA32, pgp120A32 or pgp120A274. 13. The procedure according to claim 9, characterized in that the Drøsopft/7a cells are D. methanogaster S2 cells. 14. The procedure according to claim 13, characterized in that said S2 cells are co-transfected with vector pCOHYGRO and a vector comprising a gene expression unit present in pgp160A32, pgp120FA32, pgp120A32 or pgp120A274. 15. Method for identifying an HIV-binding material, characterized in that said material is brought into contact with the protein according to claim 6, and measures the occurrence of binding between said substance and said protein, such binding being indicative of HIV binding. 16. Method for purifying the HIV protein according to claim 6, •■■ r* characterized in* that an affinity resin containing a monoclonal antibody capable of reacting with an epitope present in non-denatured gp160 protein products and in mature gp120 protein is used. 17. Process for purifying the HIV protein produced by the method according to claim 9, characterized in that the use of an affinity resin containing a monoclonal antibody capable of reacting with an epitope present in non-denatured gp160 protein products and in mature gp120 protein.