CA2003794C - Expression of hiv proteins in drosophila cells - Google Patents

Abstract

The present invention provides a novel method for expression of high levels of HIV glycoproteins in Drosophila cells.

Description

_1_ Title EXPRESSION OF HIV PROTEINS IN DROSOPHIL~A CELLS
Fi~ld of Tnventian The present invention relates generally ~o expression of HIV proteins in Drosophila cells and purification of the expressed gene products. More specifically, this invention relates to the production of novel mutant gp160 and gp120 gene products by this expression system.
Background of the Invention Human immunodeficiency virus type d (HIV--1) is the etiological agent of acquired immune deficiency syndrome, also known as AIDS. This retrovirus has a complex genetic organization, including the long germinal repeats (LTRs); the gag, ~, and env genes, and other genes. This retrovirus carries a number of viral antigens which are potential candidates either alone or in concert as vaccinal agents capable of inducing a protective immune response.
Among the more p~omisin.g of the HIV-l antigens is the viral envelope glycoprotein (gp160) or specific fragments thereof. The env gene encodes the 160 kilodalton (kd) precursor glycoprotein of the viral 1 envelope. gp160 is cleaved posttranslationally into a 120 kd glycoprotein (gp120) and a 41 kd glycoprotein (gp41), which are present at the virus surface.
gpI20, a 511 amino acid glycoprotein, is located on the amino terminal two-thirds of the gp160 glycoprotein and is exposed on the outside of the virus. gp120 is crucial to the interaction of the virus with its cellular receptor, the CD4 protein present on the surface of helper T4 lymphocytes, macrophages, and other cells of the immune system. gpI20 thus determines the tissue selectivity of viral infection and contributes to the cytopathogenicity of HIV through its involvement in syncytium formation.
gp4l, a 345 amino acid protein derived from the ~-5 carboxyl terminus of gp160, is an integral membrane protein of HIV-1. gp41 contains a series of hydrophobic amino acids which anchor the protein in the lipid bilayer of the cellular plasma membrane. The carboxyl end of gp41 is believed to protrude into the viral particle. gp41 or a portion thereof is believed to be responsible for fusion between the HIV glycoproteins expressed at the surface of the cell with cells displaying surf ace T4 receptors.
The portion of gp41 which is believed to be responsible for this fusion is located at the amino terminal. Such fusion is believed to play a role in viral replication.
See, e.g., M. Kowalski et al, Science, 237: 1351-55 (1987); D.M. Knight et al, Science, 236: 837-36 (1987).
These viral glycoproteins assume a tertiary structure as viral spikes protruding outwards from the surface of the viral particle. About 70 to 80 spikes are believed to be associated with each newly synthesized viral particle. As the viral particle ages, the spikes disappear, apparently because the association between the gp120 and gp41 is weak. Thus, for newly synthesized viral particles, this viral glycoprotein spike is believed to be the most immediate target accessible to the immune system following infection.

ia~~~ i':~~='~
1 Virus neutralizing antibodies have been reported directed against gp120 and gp41 epitopes. It has been specifically noted that a 'target site for type specific neutralizing antibodies is located in the 3' half of the gp120 glycoprotein molecule.
The env gene of HIV-1 has thus been the target a~ numeraus recent investigations. Expressian of glycosylated gp160 has previously been obtained in mammalian cells and pertain baculavirus insect cells by groups which have also reported the induction of both humoral and cellular immune responses to these antigens.
gp120 has been expressed recombinantly with the use of heterologous promoters in several systems. See, e.g., S. Chakrabarti et al, Nature (London), 320: 535 (1986);
S.I. Hu et al, Nature (London), 320: 537 (1986); and M.P. Kieny et al, Biotechnology, _4: 790 (1986).
L.A. Lasky et al, Science, 233: 209-212 (1986) constructed a number of plasmids containing mutant env genes for tranfectian into mammalian cells, specifically Chinese hamster ovary (CHO) cells. These researchers secreted a gene product encoded in a plasmid containing the first 50 amino acids of the glycoprotein D (gD) protein joined in phase to an amino acid sequence (#61-#531) of the env protein, an HBsAg polyA signal, a DHFR gene and the SV40 origin of replication . A
recombinant envelope antigen was produced containing 25 amino acids of gD at its amino terminus and lacking 30 residues from the mature processed from of gp120, and also having a deletion of the gp41 sequence (about 20 amino acids of the carboxyl terminus to the actual 160 kd precursor processing site). The resulting gene was 520 amino acids in length. When transfected into CHO cells, the cell-conditioned supernatants contained a 130 kd protein, Balled gp130.
A later report, L.A. Lasky et al, Cell, _50:
975-986 (1987), discussed the interaation between the _4_ 1 gp120 glycoprotein and its cellular receptor, CD4. By deleting 12 amino acids contained within amino acids #410-421 of gp120, a complete loss of binding resulted.
Similarly, a single amino acid substitution at position 417 resulted in decreased binding, Kawalski et al, cited above, introduced deletion and insertion mutations into a plasmid that encodes the envelope glycoprotein derived from the HTLV-IIIB strain of HIV-1. The plasmid also contained the art gene.
Mammalian CD4+ and CD4- cell lines expressing the tat gene product were used as transfection recipients to study ability of the transfected cells tn 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 HTV proteins was the COS-~-7 monkey cell line. These plasmids utilized the HIV LTR as a promoter and RNA
processing signals from SV40 to express the inserted DNA
as a functional messenger RNA. To express gp120, a plasmid pENV160 was developed which contains the entire coding region of the env gene fused to the HIV LTR.
S.W. Pyle, Aids Research and Human Retroviruses, 3(4): 387 (1987) disclose the purification of the gp120 glycoprotein from culture fluids of HIV infected H9 cells by immunoaffinity chromatdgraphy.
U.S. Patent 4,725,669 also discloses glycoproteins of approximately 160 kd and 120 kd obtained from the human H9/HTLV-III cell line, each having an approximately 90 kd unglycosylated moiety.
Fox, Hiotechnoloqy, _6: 116 (1988) reports the VAXSYN HIV-1 vaccine developed by MicroGeneSys. This report does not disclose any details of this vaccine.
D.L. Lynn, et al, in "Mechanisms of Control of Gene Expression", Eds. Allan R. Liss Inc., pp. 359-368 (1988) disclose the cloning of the entire gp160 gene behind the polyhedron promoter of the baculovirus , CA 02003794 1998-12-07 1 Autographa californica. These insect cells infected with the recombinant virus express a protein that is released from the cell upon lysis. This protein co-migrates with gpl6o, is not cleaved into gp120 and gp4l, and is glycosylated and associated with the cell membrane. When deglycosylated with N-glycanase, the protein had a molecular weight of approximately 96 kd. The recombinant protein was immunoreactive with protein from HIV-infected H9 cells, with antisera to a recombinant fraction of gp120, with gp120 itself, with a peptide fragment of gp4l, and with human AIDS sera.
R.L. Willey, et al, Proc. Nat'1 Acad. Sci., USA, 83: 5038 (1986) discusses hypervariable regions of amino acids in gp120 protein. A later report, R.L. Willey et al, J. Virol., 62(1): 139 (1988), studied a region within the env gene necessary for infectivity. Specifically disclosed herein are amino acid substitutions at the Asp codons of four N-linked glycosylation sites within the gp120 gene.
W.R. Gallagher, Cell, 50: 327 (1987) discusses a fusion peptide sequence of the transmembrane protein gp41 of HIV.
S.D. Putney et al, Science, 234: 1392 (1986) discloses the production of neutralizing antibodies to an E, coli-produced fragment'of the HIV env gene.
B.J. Bond et al, Mol. Cell. Biol., 6(6): 2080 (1986) disclose the structure of the Drosophila melanoqaster actin 5C gene. The report discusses the two transcription start sites of the actin 5C gene and fusions between the promoter sequences and bacterial chloramphenicol acetyltransferase gene inserted into D
melanoqaster host cells.
P.J. Barr et al, Vaccine, 5: 90 (1987) discloses the expression of HIV proteins in the yeast Saccharomyces cerevisiae.

-1 H. Johansen et al, 28th Annual Drosophila Conference, p. 41 (1987) is an abstract by the inventors of the present application which briefly states that _E.
call gal K genes regulated by a D_rosophila metallothionein promoter were expressed in DrOSOphila cell lines.
A. Vanderstraten et al, Proceedings of the 7th International Conference on Tnvertebrate and Fish Tissue Culture, Abstract, University of Tokyo Press, Japan, (1987) arid A. Vanderstraten et a1, in "invertebrate and Fish Tissue Culture", Eds. Y. Kuroda et al, Japan Scientific Societies Press, Tokyo, pp. 131-134, (1988) are also publications by the present inventors which discuss a hygromycin B selection system for use in expressing foreign genes in D. m_elanoaaster cells in culture. The abstract notes that the system was used to co-introduce and overexpress the E. coli gal K gene and other genes of mammalian origin, There thus remains need in the art for high-level production of HIV proteins for use as vaccinal and diagnostic agents.
Summary of Invention In one aspect, the present invention is an HIV
env gene expression unit which includes a DNA coding sequence for the desired protein and regulatory seguences necessary for transcription of the protein coding sequence and subsequent translation within a Drosophila cell.
In related aspects, this invention is a DNA
vector which comprises the gene expression unit of the present invention.
In yet another related aspect, this invention is a Drosophila cell transfected with the DNA vector of this invention.

_7_ 1 In further related aspects, this invention is an HIV env protein, or a derivative thereof produced by the transfected insect cells of this invention. The derivative encompasses any HIV env protein such as deletions, additions, substitutions or rearrangement of amino acids or chemical modifications thereof which retain the ability to be recognized by antibodies raised to the wild-type HIV env protein.
In another aspect, this invention is a vaccine far stimulating protection against HIV infection, which comprises an immunaprotective and non-toxic quantity of the HIV env protein produced by this invention.
Also provided by this invention is a diagnostic agent useful in detecting presence of HIV infection in a sample of biological fluid which contains a Drosophila cell-produced HIV protein of the invention. Additionally, the env protein of the present invention may be employed to identify or isolate HIV binding proteins or proteinaceous substances, such as CD4 or derivatives thereof.
This invention also relates to a method for production of an HIV env protein, or an immunogenic derivative thereof. The method ewtails culturing Drosophila cells transfected with an HIV env gene expression unit in a medium suitable for growth of the cells. The transfected cells, cultured in said suitable medium, are capable of expressing said protein of interest. The protein may thereafter be collected from the cell or cell culture medium.
The present invention also provides a method for purifying HIV proteins or fragments thereof which bind to a monoclonal antibody reactive with an epitope present on mature gp120 and also within the gp160 unprocessed intracellular protein. Among antibodies of this class is the mouse monoclonal antibody designated 178.1.

~(3~; j'°1~~~
1 Other aspects and details of the present invention are disclosed in the following description:
Detailed Description of the Invention The method and expression system of the present invention facilitate high-level production of HIV
proteins, particularly gp120, gp160 and derivatives thereof, in a Dros~ila cell structure. The Drosophila cells are transfected by using standard cloning techniques which permit introduction of foreign DNA into a host cell without adversely affecting the foreign DNA or the host cell. The recombinant Drosophila cells so constructed produce HIV proteins.
In contrast to the Baculovirus system of the prior art an which the HIV protein is provided only upon lysis of the infected insect cells, the method of this invention provides a continuous cell expression system for HIV prateins. Upon secretion, the protein is available by purification from the culture medium using conventional techniques. Alternatively, the protein may be produced intracellularly or membrane-bound. The protein may be extracted from the cells using conventional techniques.
Alternatively, membrane-bound protein may be employed in a variety of cell-associated assays.
A preferred Dr~hila cell line for use in the practice of the invention is the D_. melanoctaster S2 line. S2 cells [Schneider, ,3. Embryol. Exp. Morph. 27:
353 (1972)] are stable cell cultures of polyploid embryonic Drosophila cells. Introduction of the cDNA
coding sequence for gp160, or its subunits gp120 or gp41 or derivatives thereof into Drosophila S2 cells by DNA
trans~ection techniques produces unexpectedly large amounts of the glycoprotein. Use of the S2 Drosophila cell has many advantages, inoluding, but not limited to, _g_ 1 its ability to grow to a high density at room temperature. Stable integration of the selection system has produced up to 1000 copies of the transfected gene expression unit into the cell chromosomes.
Other Drosophila cell culture systems may also useful in the present invention. Some possibly useful cells are, for example, the KC-O Drosophi~ melanoaast- r cell line which is a serum-free cell Line [Schulz et al, Proc. Nat'1 Acad. Sci. USA, _83: 9428 (1986)]. Preliminary studies using the KC-O line have suggested that transfection is more difficult than with S2 cells.
Another cell Iine which may be useful is a cell line from Drosophila hydei. Protein expression can be obtained using the hydei cell line; however, transfection into this cell line can result in the transfected DNA being expressed with very low efficiency [Sinclair et al, Mol.
Cell. Biol., 5: 3208 (1985)]. Other available Drosophila cell lines which may be used in this invention include S1 and S3.
The Drosophila cells selected for use in the present invention can be cultured in a variety of suitable culture media, including, e.g., M3 medium. The M3 medium consists of a formulation of balanced salts and essential amino acids at a pH of 6.6. Preparation of the media is-substantially as'described by Lindquiat, DIS, 58:
163 (1982). Other conventional media for growth of Drosophila cells may also be used.
A recombinant DNA molecule or vector containing an HIV protein gene expression unit can be used to transfect the selected Drosophila cells, according to the invention. The gene expression unit contains a DNA coding sequence for a selected HIV protein or for a derivative thereof. Such derivatives may be obtained by manipulation of the gene sequence using traditional genetic engineering techniques, e.g., mutagenesis, restriction endonuclease treatment, ligation of other gene sequences including y ~ -lo-1 synthetic sequences and the like. See, e.g., T. Maniatis et al, Molecular Cloning, A Laboratory Manual., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).
The HIV DNA coding sequence has been recently published. See, Rather 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, .
Rather et al, su ra).
DNA molecules comprising the coding sequence of this invention can be derived from HTLV-III infected cells using known techniques (see, Hahn et al, Nature 312:166-169 (1984)), or, in the alternative, can be synthesized by standard oligonucleotide techniques.
Moreover, there are numerous recombinant host cells containing the cloned DNA coding sequences, which are widely available.
Derivatives can then be prepared by standard techniques, including DNA synthesis. Such derivatives may include, e.g., gp120 or gp160 molecules in which. one or ZO more amino acids have been substituted, added or deleted without significantly adversely affecting the binding capacity or biological characteristics of the protein.
Derivatives of these proteins may also be prepared by.
standard chemical modification techniques, e.g., acylation, methylation.
Also included in the gene expression unit are regulatory regions necessary or desirable for transcription of the HIV protein coding sequence and its subsequent translation and expression in the host cell.
The regulatory region typically contains a promoter region which functions in the binding of RNA polymerase and in the initiation of RNA transcription: The promoter region is typically found upstream from the HIV protein coding sequence.
Preferred promoters are of Drosophila origin, e.g., the Drosophila metallothionein promoter 1 CLastowski-Perry et al, J. Biol. Chem., _260: 1527 (1985)]. This inducible promoter directs high-level transcription of the gene in the presence of metals, e.g., CuSO~. Use of the Drasophila metallothionein promoter results in the expression system of the invention retaining full regulation even at very high copy number.
This is an direct contrast to the use of the mammalian metallothionein promoter in mammalian cells in which the regulatory effect of the metal is diminished as copy n~ber increases. In the Drosophila expression system, this retained inducibility effect increases expression of the gene product in the Drosophila cell at high copy number.
The Drosophila actin 5C gene promoter CB. J. Bond et al, Mol. Cell. Biol., 6: 2080 (1986)] is also a desirable promoter sequence. The actin 5C promoter is a constitutive promoter and does not require addition of metal. Therefore, it is better-suited for use in a large scale production system, like a perfusion system, than is the Drasophila metallothionein promoter. An additional advantage is that the absence of a high concentration of copper in the media maintains the cells in a healthier state for longer periods of time.
Examples of other known Drosophila promoters include, e.g., the inducible heatshock (Hsp70) and COPIA
LTR promoters. The SV40 early promoter gives lower levels of expression than the Drosophila metallothionein promoter. Promoters which are commonly employed in the cell expression vectors including, e.g., avian Rous sarcoma virus LTR and simian virus (SV40 early promoter) demonstrate poor function and expression in the Drosophila system.
A desirable gene expression unit or expression vector for the HIV protein may be constructed by fusing the HIV protein coding sequence to a desirable signal sequence. The signal sequence functions to direct , CA 02003794 1998-12-07 1 secretion of the protein from the host cell. Such a signal sequence may be derived from the sequence of tissue plasminogen activator (tPA). Other available signal sequences include, e.g., those derived from Herpes Simplex virus gene HSV-I gD [Lasky et al, Science, supra.].
The HIV DNA coding sequence may also be followed by a polyadenylation (poly A) region, such as an SV40 early poly A region. The poly A region which functions in the polyadenylation of RNA transcripts appears to play a role in stabilizing transcription. A similar poly A
region can be derived from a variety of genes in which it is naturally-present. This region can also be modified to alter its sequence provided that polyadenylation and transcript stabilization functions are not significantly adversely affected.
The recombinant DNA molecule may also carry a genetic selection marker, as well as the HIV protein gene functions. The selection marker can be any gene or genes which cause a readily detectable phenotypic change in a transfected host cell. Such phenotypic change can be, for example, drug resistance, such as the gene for hygromycin . B resistance.
Alternatively, a selection system using the drug methotrexate, and prokaryotic dihydrofolate reductase (DHFR) gene, can be used with Drosophila cells. The endogenous eukaryotic DHFR of the cells is inhibited by methotrexate. Therefore, by transfecting the cells with a plasmid containing the prokaryotic DHFR which is insensitive to methotrexate and selecting with methotrexate, only cells transfected with and expressing the prokaryotic DHFR will survive. Unlike methotrexate selection of transformed mammalian and bacterial cells, in the Drosophila system, methotrexate can be used to initially select high-copy number transfectants. Only cells which have incorporated the protective prokaryotic DHFR
gene will survive. Concomitantly, these cells have the gene expression unit of interest.

i~~~ ~~~~

1 An illustrative plasmid produced, according to 'the present invention, is pgp160032, which contains a gp160 derivative replacing 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 pgp120Fd32 which contains gp160 sequence having the first 32 amino acids replaced with serine and containing a carboxyl deletion of 216 amino acids. This plasmid is also l0 described in Example 1.
Still another plasmid which illustrates the derivative proteins of the present invention is pgp120~32, which contains the entire coding sequence for gp120 minus the first 32 amino acids at the N-terminal which are replaced with serine. Additionally, plasmid pgpl2onz74 contains a gp120 protein sequence which has replaced the first 274 amino terminal amino acids with the first amino acid of tPA, serine, and containing the remaining amino acids of gp120 up to the processing site of gp160. These vector constructions are described more completely in Example 1.
Once a recombinant DNA molecule or expression vector containing the HIV protein gene expression unit has been constructed, it can be transfected into the selected Drosophila cell using standard transfection techniques.
Such techniques are known to those of skill in the art and include, for example, calcium phosphate co-precipitation, cell fusion, 2lectroporation, microinjection and viral ~transfection.
A two-vector system can be used in the present invention to co-transfect into the Drosophila cell a gene expression unit for the desired HTV protein or derivative and the coding region for the selection system to be used. A preferred illustrative embodiment of this invention is the production of an HIV protein employing a vector containing an HIV protein expression unit, e.g.;

' CA 02003794 1998-12-07 -pgp120~32, and a vector containing a hygromycin B gene expression unit, e.g., pCOHYGRO. pgp120~32 contains an expression unit comprising the Drosophila metallothionein promoter, a derivative of the gpI20 gene, and the SV40 poly A site. This gp120 expression unit in combination with the pCOHYGRO vector system will produce a gp120 derivative in S2 Drosophila cells by maximizing the advantage of hygromycin B resistance for selection. With this system, the antibiotic hygromycin B can be used to select for those cells containing the transfected vectors. A more complete description of this embodiment is described in Example 2.
As another example, an expression system employing the DHFR gene/methotrexate selection system, consisting of the vectors pgp120032 and pHGCO, can be used to select methotrexate-resistant cells expression gp120 or a derivative thereof. The vector pgp120a32 comprises a gp120 gene expression unit in which the promoter is the Drosophila metallothionein promoter. The pHGCO vector comprises a DHFR gene expression unit and is co-transfected with the pgp120~32 vector, thereby providing the DHFR gene necessary for selection. These selectable markers along with cotransfection of Drosophila_ cells is -further described by Johansen et al, U.S. Patent 1 5,681 713 . issued October 28, 1997.
According to the invention, the two vectors are co-transfected into the S2 Drosophila cell using the method as described by Wigler et al, Cell, 16: 777 -(1979). The vectors are co-transfected in varying ratios depending upon the particular copy number desired. The transfected cells are then selected, such as in M3 medium containing serum and the appropriate selection agent, e.g., hygromycin B or methotrexate.
Once an appropriate vector has been constructed and transfected into the selected Drosophila cell line, ~~~ ~''~;~-~
-i5-1 the expression of gp120 is induced by the addition of an appropriate inducing agent for the inducible promoter.
For example, cadmium or copper are inducing agents for the metallothionein promoter. Heat is the inducing agent for the Hsp70 promoter. For constitutive promoters, such as 'the actin 5C promoter, no inducing agent i~, required for expression.
Transcription and expression of the HIV protein coding sequence in the above-described systems can be monitored. For example, Southern blot analysis can be used to determine copy number of the gp120 gene. Northern blot analysis provides information regarding the size of the transcribed gene sequence [see, e.g., Maniatis et a1, cited above]. The level of transcription can also be quantitated. Expression of the selected HIV protein in the recombinant cells can be further verified through Western blot analysis and activity tests on the resulting glycoprotein [see Example 5].
Drosophila S2 cells are especially suited to 20,high-yield production of protein in the method of the present invention. The cells can be maintained in suspension cultures at room temperature (24+i°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 media. When the pCOHYGRO vector system is used, the media is also supplemented with 3OO ug/ml hygromycin B. In this media, the S~ cells can be grown 30-in suspension cultures, for example, in 250 ml to 2000 ml spinner flasks, with stirring at 50-60 rpm. Cell densities are typically maintained between 105 and 10~
cells per ml. In one embodiment of this invention, the cells axe grown prior to induction in 1500 m1 spinner flasks in media containing 5% serum.

' CA 02003794 1998-12-07 1 Following cell culture, the HIV protein can be isolated from the spent media, e.g., by use of a monoclonal antibody affinity column. Other known protein purification steps, e.g., metal chelates, various affinity chromatography steps or absorption chromatography, can be used to purify the HIV protein from the culture media.
The use of the cell line S2 which secretes the gene product directly into the media is an important feature of the present invention. Direct secretion into the media allows utilization of an efficient one-step purification system. Using a monoclonal antibody column directed against the HIV protein, the spent culture media can be added directly to the column and the protein eluted using 1.5 _M KSCN in phosphate-buffered saline (PHS).
A preferred purification technique enabling large-scale efficient production of the HIV proteins of the invention employs an immunoaffinity column containing a monoclonal antibody directed against an epitope present in gp160 and present in mature secreted gp120 proteins.
Such a,monoclonal is advantageous because of its capacity to recognize the protein sequence in more than one configuration. An antibody having these characteristics and useful in immunoaffinity columns for various HIV
proteins, derivatives or fragments thereof is designated 178.1. This monoclonal aritibody is described in greater detail in Example 3. Such a column of the invention may be made by coupling an antibody with the characteristics of 178.1 to a conventional absorbant carrier, such as SEPHADEX, Wider appropriate conventional conditions of pH, temperature and the like. Such a purification column and procedure may be utilized to separate the HIV proteins and fragments of the present invention.
Other monoclonal antibodies may be used in this purification procedure. A variety of monoclonal antibodies which are capable of binding to HIV proteins, particularly gp160 or gp120, have been described in the 1 art and are available. Other new monoclonal antibodies useful in this invention may be developed by now-conventional techniques.
The glycoproteins produced by Drosotahila cells, according to this invention, are completely free of contaminating materials, e.g., mammalian, yeast, bacterial and more importantly, other HIV viral materials.
Drosophila-produced HTV proteins have also been demonstrated to possess different pattern of glycosylation than that reported by other systems, e.g., mammalian systems.
The HIV proteins and derivatives produced, according to the present invention, may be useful in a variety of products. For example, these recombinant proteins may be used in pharmaceutical compositions for the treatment of HIV-infected subjects. Such a pharmaceutical composition, according to the present invention, comprises a therapeutically effective amount of the HIV protein or derivative of the invention in admixture with a pharmaceutically acceptable carrier. The composition can be systemically administered either . parenterally, intravenously or subcutaneously. When systemically administered, the therapeutic composition for use in this invention is in the form of a pyrogen-free, z5 parenterally acceptable aqueous solution. The preparation of such a parenterally acceptable protein solution, having due regard to pH, isotonicity, stability and the like, is within the skill of the art.
The dosage regimen will be determined by-the attending physician, considering various factors which modify the action of drugs, e.g., the condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. The pharmaceutical carrier and other components of a pharmaceutical formulation would be selected by one of skill in the art.

Additionally, the recombinant proteins of the present invention may be used as components of vaccines to innoculate mammalian subjects against HIV infection.
These proteins may be used alone or with other recombinant proteins or therapeutic vaccinal agent s, Components of such a vaccine would be determined,by one of skill in the art.
Finally, the proteins of the present invention may be useful as diagnostic agents for the detection of the presence of HIV infection or antibodies to an HTV
infective agent in biological fluids, such as blood, sertun, saliva and the like. These proteins of the invention may also be employed in methods to identify and/or isolate HIV-binding proteins or other HIV-binding substances in biological fluids and tissues, e.g., sCD4 or derivatives thereof. The proteins may thus be components in kits to perform such methods. To identify an HIV-binding substance, a protein, according to the invention, is employed to contact the substance or an impure mixture containing 'the substance under conditions to promote binding between the protein and the I-IIV-binding . substance. A conventional assay to detect the occurrence of binding, e.g " detection of radioactive labels or the like, is also part of the method. The presence of binding between the protein and the binding substance is, therefore, indicative of HIV binding.
Similarly, in a method to isolate an HIV-binding substance from the mixture, the binding event could be followed by a conventional procedure to purify the bound entity formed by the protein of the present invention and the HIV-binding substance from the mixture. Other components of such diagnostic systems and kits may be conventional components of diagnostic kits and may be selected by those of skill in the art.
The following examples illustrate the construction of exemplary vectors and transformants of the ~(~~ ~'~~~

1 invention, the preferred purification system and assays for determination of the production level of the glycoproteins gp120 and gpl6o. These examples are not to be considered as limiting the scope of this invention.
5 Restriction enzymes and other reagents were used subst ant:ially in accordance with 'the vexxdars' instructions.
Examples example 1. Vector Constructions a) Mp ~'tPA
As the basic vector for gene expression in Drosophila, the tPA expression vector pMTtPA (also called pDMtPA) was used. This vector is a derivative o~ vector pMLI, a small pER322 vector containing the beta-lactamase gene which has deleted the poison sequences [Melton et al, Cell, 27: 297 (1982)]. These sequences are inhibitory to amplification of the vector. This vector was digested with Sall and Aat2 which removes a small piece of pBR322, and the digested ends were filled in. The missing piece of pBR322 is then replaced with a cassette containing the Drosophila metallothioneir~ promoter on an end-filled EcoRl-Stul fragment, followed by a filled-in _HindIII-Sacl fragment from pDSPI [D.S. Pfarr et al, DNA, _4(6): 461 (1985)] containing a tPA sequence containing the signal sequence, prepeptide and the entire coding region of tPA.
The tPA gene an this fragment is followed by an SV40 early polyadenylatian site.
b) p_p1~0~32 A HindIII-Xbal fragment containing the entire env gene was isolated from an HIV-isolate clone BH10 [L.

1 Ratner et al, cited above; GenBank]. The entire gp160 sequence was then inserted into a N_co_1-Xbal digested vector pDSPl. The resulting vector, _SU2, was digested with Ndel, followed by treatment with mung bean nuclease and subsequently digested with Sacl, thus isolating the gp160 gene. The digestion with Ndel cwt the gp160 sequence at amino acid #32. The Sacl digestion cuts 3' of the gp160 gene, including in the sequence part of the original pDSPl vector containing a polylinker. This fragment was inserted into the above-described expression vector pMTtPA which had been digested with Hc~lII, end-filled, and subsequently cut with Sacl, which deletes the mature tPA sequence, The Bg_lII site is positioned at the first amino acid of tPA. Consequently, the resulting vector pgp160~32 codes for a modified gp160 protein which has replaced the N-terminal 32 amino acids of gp160 with serine.
c) ~apl2oF~32 Another vector containing a modified gene sequence coding for HIV-1 surface glycoprotein gp160 was constructed by digesting pgp160d32 with H_indIII and Sacl, thereby removing the carboxyl terminal of gp160.
Approximately two-thirds of the sequence coding for gp41 is removed by this digestion. Thus, this gp160 sequence is missing 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 coding for a stop colon on an HindIII-_Sacl fragment. The 6-amino-acid linker sequence is as follows:
5'AGCTTTGACTGACTGAGCT 3'.
d) pap120A32 Yet another vector containing a mutant gp160 _21-gene was constructed by digesting pgp160~32 with S~1 and Xbal, thereby deleting all of the sequence of protein gp41 and about 30 amino acids at the carboxyl terminus of the gp120 glycoprotein sequence. This fragment was replaced by a synthetic S~1-Xbal linker sequence coding for the correct carboxyl terminus from the S~1 site to the processing site of gp120-gp4l. This sequence was followed by a stop colon. This sequence thereby contained all of 'the coding sequence for gp120 minus the first 32 amino acids and vane of the gp41 coding sequence.
e) pai~1200274 Still another exemplary vector containing a mutant gp120 gene was constructed as follows: a 720-base pair carboxyl terminal fragment of gp120 was isolated by partial digestion of pgp120~32 with Bc~lII followed by XbaI digestion. This fragment was now inserted in place of the tPA gene into the ~qlII-Xbal cut pMTtPA expression vector. The resulting vector p120~274 codes for a gp120 protein which has replaced the first 274 amino terminal amino acids with the first amino acid of tPA, serine.
f) pCOHxGRO
A commercially available plasmid, pUCl8 [BRL]
containing a BamHI arid SmaT site was used. The S' LTR
from an integrated COPIA element (357 base pairs) was cloned into the BamHI site of vector pUCl8, resulting in the vector designated pUCOPIA COPTA is a representative member of the disperse middle repetition sequences found scattered through the Drosophila genome (Rubin et al, in Cold Sprina Harbor Symp Quant ~ Biol , _45: 619 (1980)].
The vector pUCOPIA was cut at the SmaI site and the E.
coli gene coding for hygromycin B phosphotransferase~
(hygromycin B cassette) was cloned into pUCOPTA using ' CA 02003794 1998-12-07 1 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 coding for 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 Smal site of the vector pUCOPIA producing vector pCOHYGRO.
Example 2. Transfection into Drosophila S2 Cells pCOHYGRO was co-transfected into S2 Drosophila cells together with one of the vectors carrying a gp160 mutant gene under the control of the Drosophila metallothionein promoter as described above. For purposes of this example, the vector employed is pgp120~32. The transfected cells were selected in M3 medium containing 5% serum and 300 ug/ml of hygromycin B. After 2 to 3 days under identical conditions, the untransfected cells stop dividing and begin to die. The time of selection in order to obtain stable, growing hygromycin B-resistant cells in the transfected cultures is approximately two to three weeks.
To obtain cultures having integrated into their chromosomes different copy numbers of the gp120 mutant gene, the ratios of the two vectors were varied. The ratio in this example was 20:1. Similar ratios have been employed for other gpI60 mutant vectors of this invention. This ratio is the same when any of the gp160 mutant vectors are used.
Expression of the pgp120~32 gene product was verified after induction of the metallothionein promoter with 500 uM CuS04. Western blot analysis of the spent supernatant from the induced cell cultures revealed a single band at approximately 100 kd.

e~~%~li ~',~~
_23_ 1 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 viral gp120 as standards. 5 x 106 cells/m1 were seeded in S M3 medium without serum and induced for 3 to 4 days.
The level of gp120 measured in the supernatant is approximately lw2 mg/l.
Cells were maintained as suspension. cultures in 250 ml to 2000 ml spinner flasks. Culture medium was M3 supplemented with 300 ~g/ml hygromycin B. Cultures were incubated at 24+1°C and stirred at 50-~60 rpm. Cell densities were typically maintained between 106 and 10~ cells per ml in M3 medium supplemented with hygromycin B. CuSO~ was added to a final concentration 1S of 500 NM, and the cultures were allowed to grow for 3 to 4 days in serum-free media prior to harvesting the modified gp120 glycoprotein.
The proteins, according to this method produced, were approximately 100 MW, and the level of expression was higher than any other reported gg120/gp160 expression in any eukaryotic cell system. Tn standard biological activity assays, the purified modified gp120 expressed, as described above, is capable of inhibiting virus infection in tissue culture, binds T~ and reacts to antibodies to gp120.
It is expected that one of skill in the art could express the other gp160 and gp120 proteins and fragments thereof, described by the present invention, using substantially the same systems and procedures as exemplified above for the protein fragment encoded in pgpI20~32.
Example 3. Monoclonal Antibody 178.1 An affinity purification column employing a novel monoclonal antibody was used in the purification ' CA 02003794 1998-12-07 1 scheme applied to the above-described mutant gp160/gp120 proteins. This monoclonal antibody may be characterized as being capable of reacting with non-denatured HIV
glycoprotein products present in cell lysate and with mature gp120 as secreted into the supernatant of a yeast culture. One such-monoclonal antibody specific for the epitope which is contained both in the unprocessed gp160 recombinant molecule and in the full-size processed gp120 protein is a mouse monoclonal antibody 178.1.
An expression system employing the _C. _albicans glucoamylase promoter and signal peptide was employed to produce partially purified yeast-recombinant gp160 for production of 178.1. The production of this yeast-derived production of 178.1. The production of this yeast-derived 15-gp160 is described in European patent application No. 362,179, published April 4, 1990.
Eight-week-old Balb/c mice were injected three times subcutaneously and intraperitonally with the partially purified (1.5 - 3% purity) yeast-recombinant gp160 in Freund's adjuvant at 4-week intervals. After a resting period of 3 months-, one mouse was sacrificed, and its spleen cells were fused with myeloma cells [see, e.g., R.P. Siraganian et al, Meth. Enz., 92: 17 (1983); yMBp Course on:Hybridoma Production, Basel Inst. for Immunol.
(1980)]. The myeloma cells used are a subclone of the Sp2/O-Agl4 line previously selected for optimal growth in agar medium and high fusion efficiency [J. D. Franssen et al, Proc. XXIX Collog Protids Biol Fluids, _29:
645-649 (1981)]. After about ten days, supernatants were withdrawn for screening in a capture ELISA, using a commercial monospecific anti-gp120 reagent [Biochorm, Seromed Ref. D7324) as capture antibody.
Briefly, NUNC IMMUNOPLATE*'I (nr 4-39454) were coated overnight at 4°C with 50 ul of a solution of 5 ug/ml of sheep anti-gp120 IgGs in PBS. The plates *Trade-mark ' CA 02003794 1998-12-07 1 were washed with washing buffer (PBS, TWEEN* 20 0.1%) and saturated with 100 ul of saturation buffer [PBS, Newborn Calf Serum 4%, bovine serum albumen (BSA) 1%, TWEEN 20 0.1%] for 1 hour at 37°C. Fifty ul/well of crude-Molt3/HTLV-IIIB,or Molt3 cell lysate (107 cells/ml in PBS, TRITON x-100* 1$) or of the supernatant fraction (S2-30) of the recombinant-yeast gp160 (or similarly-treated negative control) were used as antigen and incubated in the plates for 3 to 5 hours at room temperature. The plates were washed extensively, and 50 ul of hybridoma supernatants were added to each well and incubated overnight at 4°C. After a washing step, 50 ul/well of a 1/500 dilution of biotinylated anti-mouse immunoglobulins (Igs) (Amersham Ref. RPN 1021) in saturation buffer were incubated in the plates for 1 hour at 37°C. The plates were washed again, and 50 ul/well of a 1/1000 dilution of streptavidin biotinylated horseradish peroxidase complex (Amersham Ref.
RPN 1051) in saturation buffer were added to each well.
After an additional washing step, 50 ul of a solution of 0.4 mg/ml of orthophenylene diamine dihydrochloride (OPD, Sigma P1526) and 1 ul/ml of H202 (30% in citric/Na citrate 0.1 M pH 5) supplemented with O.ls TWEEN 20 were added to each well.
The plates were then incubated for 20 minutes at room temperature in the dark, and the reaction was stopped by addition of 50 ul/well of 2M H2S04. The optical density at lambda = 492 nm was monitored, and 50 positive clones were selected for further subcloning in soft agarose, according to P. Herion et al, Proc. XXIX Colloq.
Protids Biol. Fluids, _29: 627 (1981). The cloned hybridomas were then grown in vivo by injecting 2 to 5 x 106 hybridoma cells in the peritoneal cavity of Halb/c mice pretreated by intraperitoneal injection of pristane (2, 6, 10, 14-tetramethyl pentadecane).
*Trade-mark ' CA 02003794 1998-12-07 1 The monoclonal antibodies selected from the above procedure were characterized by Western blot analysis (WB), radioimmuno precipitation assay (RIPA), purification, biotin-labeling and competition assays.
Resulting monoclonal antibodies were further characterized by analysis of. their reactivity toward various recombinant and native antigens.
A high yield of hybridomas was obtained by this procedure. More than 200 wells were positive in the screening assays. However, among them, only 50 wells were selected and after cloning, the cells were expanded in ascitic acid. All the ascitic fluids were tested in WB
and RIPA. Among the 39 monoclonals tested, 37 showed a gp160 band in RIPA. None reacted with the gp120 form in the same assay. Those monoclonal antibodies that displayed only gpl6o recognition in RLPA, while being clearly reactive to gp120 in WB, were analyzed by subclass. Three monoclonals that were IgG2A were purified on a protein A-SEPHAROSE* column and biotin-labelled.
Competition assays using vaccinia gp160 as antigen were performed, and the obtained result defined at least five different groups of epitope recognition with the gp160 protein. Monoclonal 178.1 was selected for an epitope present on mature gp120 and unprocessed intracellular gp160.
A Western blot (WB) analysis was performed according to conventional techniques to demonstrate that 178.1 is capable of binding HIV virus isolated from human cells infected with HTLV-IIIB [Molt3/HTLV-IIIB].
Radio immuno precipitation assays (RIPA) were performed, as described in P.J. Kanki et al, Science, 228:
1199 (1985) to demonstrate that 178.1 could immunoprecipitate the human cells infected with HTLV-III
virus strains.
The reactivity of the monoclonal antibodies recognizing non-overlapping epitopes towards a large panel *Trade-mark ' CA 02003794 1998-12-07 1 of antigens was assessed using a sandwich ELISA involving sheep anti-gp120 as capture reagant. Monoclonal antibody 178.1 was negative in ELISA on divergent HIV isolates Molt3/HTLV-IIIB, H9/HTLV-III~1 and Hut78/ARV2, while clearly positive when tested on HTLV-IIIB in RIPA, WB, or ELISA using recombinant antigens. This monoclonal 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 added advantage for the production of gp160/gp120 glycoproteins in various constructs.
Example 4. Purification of gp120032 from Drosophila conditioned Cell Culture Medium The recombinant gp120 protein from Example 2 was purified as follows: 30 liters of Drosophila-conditioned media (CM) containing gp120~32 was made with 1 mM
phenylmethylsulfonyl fluoride (PMSF), 10 mM
ethylenediamine tetraacetic acid (EDTA) and 70 Kallikrein inhibitor units. CM was filtered through a .45 um 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/cm2hr~equilibrated in Buffer A, containing 20 mM 2-[N-morpholino]ethanesulfonic acid (~S), pH 6Ø After application of all CM, the column was eluted .in one step with Buffer B, containing 20 mM
MES, pH 6.0, 0.4M NaCl.
The S-SEPHAROSE-eluted gp120~32 was applied to an anti-gp120 mouse monoclonal-SEPHAROSE 4B column (60 ml;
3.2 cm x 6.5 cm) at a LFR of 10 ml/cmZhr. This column was equilibrated in Buffer B. After application of one-half S-SEPHAROSE pool, the column was washed with 1 column volume of Buffer B, 2 column volumes of 20 mM MES, pH 6.0, 1. OM NaCl (Buffer C), and 2 column volumes of *Trade-mark ' CA 02003794 1998-12-07 1 Buffer A. gp120032 was eluted with O.1M acetic acid, pH 2.8, and fractions were immediately neutralized by addition of 0.1 volumes of 1M Tris (hydroxymethyl)aminomethane (Tris), pH 10.4.
Mouse anti-gp120 monoclonal antibody hybridoma 178.1 was produced according to Example 3 above. This hybridoma was seeded at 2 x 105 cells/ml and cultured for four days in Dulbecco's Modified Eagle Medium [Hazelton Research Productsl supplemented with 4.5 grams/liter glucose, 2 uM glutamine and loo serum.
CM containing 178.1 antibody was filtered (0.2 um membrane) and applied to a protein A-SEPHAROSE (Pharmacia) (I7 ml; 1.5 cm x 10 cm) equilibrated in O.1M Tris, pH 8.2.
Antibody was eluted with O.1M sodium citrate, pH 3.5 and immediately neutralized with Tris.
Purified anti-gp120 monoclorial antibody was coupled to CNBr-activated SEPHAROSE 4B* (Pharmacia), according to manufacturer's instructions at a density of 2 mg antibody/ml resin and with a coupling efficiency of 98~, resulting in an anti-gp120-SEPHAROSE-affinity resin.
This affinity resin will specifically bind gp120 protein through the interaction of the antibody with 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 conditioned media.
Recovery is estimated at between 25-500.
This purification technique and affinity resin is also believed to be effective with other HIV proteins or fragments thereof having this epitope.
Example 5. Assay The assay described below is a non-isotopic assay utilizing an enzyme and a substrate for the detection of gp120 or fragments thereof, which was *Trade-mark ' CA 02003794 1998-12-07 1 employed in detecting the gp120 proteins produced by the methods and compositions of the present invention.
In the assay, the criteria for detecting gp120 is dependent on antibody specificity. An anti-gp120 monoclonal antibody [DuPont, Cat. No. 9284 diluted in O.1M sodium carbonate buffer (pH 9.5) to two ugs/ml, is used to capture the gp120 protein. 100 ul of this antibody dilution is added to each well in duplicate in an assay plate, except for those wells designated 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 ul of blocking buffer consisting of to BSA in PBS to each well for 1 hour at room temperature.
The viral gp120 standards were diluted to 1 ~g/ml, 0.5 ug/ml, 0.25 ug/mI, 0.1 ug/ml, and 0.2 ug/ml in a washing buffer consisting of PBS and 0.05 TWEEN 20. 100 ~1 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 thereafter, each plate was washed four times with washing buffer.
To each well, 100 ul of rabbit anti-~p120 antibody (described by DeBouck et al, European Application No. 293,184 published November 30, 1988) diluted 1/1000 in washing buffer was added and each plated incubated on a shaker for 1 hour. This second antibody 'sandwiches the gp120 between the two antibodies. The plates were, thereafter, washed four times with washing buffer. To detect this complex, a third antibody, 100 ul of peroxidase (POD) labeled goat anti-rabbit antibody (mostly IgG and IgM antibody) diluted in washing buffer with no azide, is added to each well. The plates were then incubated for 2 hours on a sha'_~er at room temperature.
After the plates were washed four times, 100 ul of a colorless substrate (1 mg/ml of OPD in v~~~ ~ s ~:'~~

1 citrate buffer with 4 ul of 35% hydrogen peroxide per ZO ml of buffer) was added. The hydrogen peroxide was added just prior to adding substrate to the wells. These plates were incubated ~or 8 minutes on a shaker and the reaction stopped by adding 100 ~l of 0.1 M sodium flyoride to each well, rn the presence of peroxidase-conjugated antibodies, the substrate turns deep yellaw. Optical density, or intensity of the color, which is proportional to the amaunt of gp120 captured, was read on a plate reader at 450 manometers, and a standard curve was constructed with concentrations of unknowns aalcula~ted. The amount of gp120 in the supernatant culture was determined by comparison to this standard curve.
The above description and examples fully disclose the invention, including preferred embodiments thereof. Modifications of the methods described, e.g, employing other truncated gp160/gp120 sequences that are obvious to one of ordinary skill in the art of molecular genetics and related sciences, are intended to fall within the scope of the following claims:

Claims (24)

1. An HIV protein gene expression unit comprising a DNA sequence encoding an HIV gp120 or gp160 protein or a derivative thereof and a promoter of Drosophila origin which regulates the transcription of said DNA sequence and translation into protein within a Drosophila cell transfected with said expression unit.

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

3. The gene expression unit of claim 1 comprising the HIV DNA coding sequence as present in pgp160.DELTA.32, pgp120F.DELTA.32, pgp120.DELTA.32, or pgp120.DELTA.274.

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

5. A Drosophila cell transfected with the vector of claim 4.

6. A method for production of an HIV protein in Drosophila cells comprising culturing in a suitable medium Drosophila cells transfected with an HIV protein gene expression unit, which expression unit comprises a DNA
sequence encoding an HIV gp120 or gp160 protein or a derivative thereof and a promoter of Drosophila origin which regulates the transcription of said DNA sequence and translation into protein within said cells, said cells being capable of expression said protein.

7. The method of claim 6 wherein the Drosophila cells are D. melanogaster S2 cells.

8. The method of claim 7 wherein said S2 cells are co-transfected with the vector pCOHYGRO and a vector comprising a gene expression unit as present in pgp160.DELTA.32, pgp120F.DELTA.32, pgp120.DELTA.32 or pgp120.DELTA.274.

9. A method for identifying an HIV-binding substance comprising contacting said substance with an HIV
protein produced according to the method of claim 6, and assaying for the occurrence of binding between said substance and said protein, such binding being indicative of HIV binding.

10. A method for purifying the HIV protein produced by the method of claim 6 comprising employing an affinity resin containing a monoclonal antibody having the characteristics of monoclonal antibody 178.1 and capable of reacting with an epitope present in non-denatured gp160 protein products and in mature gp120 protein.

11. The gene transcription unit of claim 1 further comprising a polyadenylation region.

12. The gene transcription unit of claim 1 further comprising a selection marker.

13. The gene transcription unit of claim 1, wherein said Drosophila promoter is inducible.

14. The gene transcription unit of claim 1 wherein the Drosophila promoter is constitutive.

15. The gene transcription unit of claim 1, wherein said DNA sequence encoding said HIV gp120 or gp160 protein or a derivative comprises a signal sequence fused to said HIV encoding sequence.

16. The method of claim 6 further comprising transfecting said cells with a selection marker.

17. The method of claim 16, wherein the gene expression unit and the selection marker are located on different vectors, such that the Drosophila cells are co-transfected.

18. The method of claim 6 wherein said HIV
protein is expressed and secreted into the culture medium.

19. The method of claim 6 wherein said HIV
protein remains intracellular.

20. The method of claim 6 wherein said HIV
protein is bound to the outer cell membrane.

21. The method of claim 6 wherein said Drosophila cells are Drosophila malanogaster S2 cells.

22. The method of claim 6 wherein 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.

23. The method of claim 22 wherein said first vector is pCOHYGRO.

24. The method of claim 22 wherein said second vector comprises an HIV gene expression unit as present in pgp160.DELTA.32, pgp120F.DELTA.32, pgp120.DELTA.32 or pgp120.DELTA.274.