DK175461B1 - Expression of HIV proteins in Drosophila cells - Google Patents

Description

DK 175461 B1

The present invention relates generally to the expression of HIV proteins in Drosophila cells.

More particularly, the present invention relates to the preparation of novel mutant gp160 and gp120 gene products by this expression system.

Human immunodeficiency virus type 1 (HIV-1) is the etiologic agent of acquired immunodeficiency syndrome, also known as AIDS. This retrovirus has a complex genetic organization, including the long terminal repeats (LTRs), gag, pol and 10 env genes, as well as 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 env gene encoding the 160 kilodalton (kd) precursor glycoprotein of the viral envelope. gp160 is cleaved post-translationally to a 120 kd glycoprotein (gp120) and a 41 kd glycoprotein (gp41) present on the virus surface.

gp120, a 511 amino acid glycoprotein, is located on the two-thirds amino terminal 20 of the gp160 glycoprotein and is laid open on the outside of the virus. gp120 is essential for interaction or interaction between the virus and its cellular receptor, the CD4 protein, which is present on the surface of helper T4 lymphocytes, macrophages and other cells of the immune system. Thus, gp120 determines the tissue selectivity of the viral infection and contributes to the cytopathogenicity of HIV through its involvement in syncytia.

gp41, a 345 amino acid protein derived from the 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 30 gp41 is believed to project into the virus particle. gp41 or a portion thereof is believed to be responsible for association of the HIV glycoproteins expressed on the cell surface.

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Flat, with cells exhibiting surface T4 receptors. The part of gp41 which is believed to be

You are in charge of this association, located near the amino terminal. A so- I

In that association, it is believed to play a role in viral replication. See, e.g. M. Kowalski I

I et al., Science, 237; 1351-55 (1987); D.M. Knight et al., Science, 236: 837-36 (1987). IN

I 5 I

In these viral glycoproteins, a tertiary structure assumes like viral tips protruding I

In from the surface of the virus particle. Ca. 70 to 80 peaks are believed to be associated with I

In each new synthesized virus particle. As the virus particle ages, you disappear

In the forefront, apparently because the link between gp120 and gp41 is weak. For I

In 10 newly synthesized virus particles, this viral glycoprotein tip is believed to be the one

In the most immediate target available to the immune system after infection. IN

Virus neutralizing antibodies have been reported targeting gp120 and gp41 epitome I

I per. Specifically, it has been noted that a target site for type-specific neutralizing I

In 15 antibodies are located in the 3 'half of the gp120 glycoprotein molecule. IN

Thus, the env gene of HIV-1 has been the target of extensive, recent research. Ex- I

In the past, glycosylated gp160 has been previously obtained in mammalian cells and certain bacilli.

In lovirus insect cells in groups that have also reported the induction of both skin I

In 20 moral and cellular immune responses to these antigens, gp120 has been re-expressed

I combined using heterologous promoters in several systems. See, e.g. S. I

In Chakrabarti et al., Nature (London), 320: 535 (1986); S. I. Hu et al., Nature (London), I

I 320: 537 (1986); and Μ. P. Kieny et al., Biotechnology, 4: 790 (1986). IN

In 25 L. A. Lasky et al., Science, 233: 209-212 (1986) constructed a series of plasmids in

In containing mutant env genes for transfection in mammalian cells, especially Chinese ham- I

In steroidal (CHO) cells. These researchers secreted a gene product encoded into a plasmid

Containing the first 50 amino acids of glycoprotein D (gD) protein which were in phase I

In the to an amino acid sequence (Nos. 61-531) of the env protein, an HBsAg polyA signal; et i

In 30 DHFR gene and SV40 origin for replication. A recombined coat antigen was prepared

I readily containing 25 amino acids of gD at its amino terminus and missing 30 residues I

DK 175461 B1 3 from the fully developed form of gp120, and also comprising a deletion of the gp41 sequence (about 20 amino acids of carboxyl terminus to the current 160 kd precursor process site). The resulting gene had a length of 520 amino acids. By trans

fection into CHO cells, the cell-conditioned supernatants contained 130 kd of protein J

5 in, called gpl 30.

In a later report, L. A. Lasky et al., Cell, 50: 975-986 (1987) discuss interaction or interaction between the gp120 glycoprotein and its cellular receptor, CD4. Deleting 12 amino acids contained in amino acids # 410-421 of gp120 resulted in 10 complete binding loss. A single amino acid substitution at position 417 also resulted in diminished binding.

Kowalski et al., As cited above, introduced deletion and insertion mutations in a plasmid encoding envelope glycoprotein derived from the HTLV-niB strain of HIV-1.

The plasmid also contained the species gene. CD4 + and CD4 mammalian cell lines expressing the tat gene product were used as transfection recipients to study the connectivity of the transfected cells.

Knight et al., As cited above, describe expression of species / trs transactivator protein 20 of HIV in mammalian cells. The mammalian cell line used to express these H1V protions was COS-7 monkey cell line. These plasmids used HIV LTR as a promoter and RNA process signals from SV40 to express the inserted DNA as functional messenger RNA. To express gp120, a plasmid pENV160 was developed which contains the entire coding region of the env gene linked to HIV 25 LTR.

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

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DK 175461 B1 I

U.S. Patent No. 4,725,669 also discloses glycoproteins of 160 kd and 120 kd I

obtained from human H9 / HTLV-III cell line, each having an unglycosylated unit of I

ca. 90 kd. IN

Fox, Biotechnology, 6: 116 (1988) reports VAXSYN HIV-1 vaccine developed by I.

MicroGeneSys. The report does not describe any details regarding this vaccine. IN

D. L. Lynn et al. describes in "Mechanisms of Control of Gene Expression", published by I

Allan R. Liss Inc., pages 359-368 (1988) cloning the entire gp160 gene behind polyethylene

10 the drone promoter of the baculovirus Autographacalifomica. These insect cells which are infi- I

expressed with the recombinant virus, expresses a protein which is released from the cell by lyase I

see. This protein migrates with gp160, does not cleave to gp120 and gp41 and glycosylates in

and connects to the cell membrane. After deglycosylation with N-glycanase had pro- I

for example, a molecular weight of approx. 96 kd. The recombinant protein was immunoreactive with I

15 from HIV-infected H9 cells, with antisera to a recombinant fraction of I

gp120, with the gp120 itself, with a peptide fragment of gp41 and with human AIDS sera. IN

R. L. Willey et al., Proc. Nat'l Acad. Sci., USA, 83: 5038 (1986) discusses hypervasin

reliable regions of amino acids in gp120 protein. A later report by R. L. Willey et al., I

J. Virol., 62 (1): 139 (1988) investigated a region within the env gene that is required

for infectivity. This specification specifically describes amino acid substitutions by Asp-co-I

donate four N-linked glycosylation sites within the gp120 gene. IN

W. R. Gallagher, Cell, 50: 327 (1987) discusses a fusion peptide sequence of transmembrane

25 BR protein protein gp41 of HIV. IN

S. D. Putney et al., Science, 234: 1392 (1986) discloses the preparation of the neutralizer-I

the antibodies for an E. coli-produced fragment of the HIV env gene. I 1

B. J. Bond et al., Mol. Cell. Biol., 6 (6): 2080 (1986) describes the structure of Drosophila I

melanogaster actin 5C gen. This report discusses the two transcription start sites of I

5 The 1751 B1 actin 5C gene and associations 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 expression of HIV proteins in the yeast 5 Saccharomyces cerevisiae.

H. Johansen et al., 28th Annual Drosophila Conference, page 41 (1987) is a summary of the present inventors, which briefly indicates that E. coli gal K genes regulated by Drosophila metallothionein promoter were expressed in Drosophi-10 la-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", published by Y. Kuroda et al., Japan Scientific Societies Press, Tokyo, pp. 131-134 (1988) are also publications of the inventors of the present application in which a hygromycin B selection system for use in expression of foreign genes in D is discussed. melanogyster cells in culture. In summary, it is noted that the system was used to co-induce and overexpress the E. coli gal K gene and other mammalian origin genes.

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Thus, there is still a need in the field of high-level production of HIV proteins for use as vaccines and diagnostics.

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

In further related aspects, the present invention relates to an HIV env protein 30 or a derivative thereof prepared by the transfected insect cells of the present invention. The derivative encloses any HIV env protein, such as deletions,

DK 175461 B1 I

additions, substitutions or rearrangements of amino acids or chemical modifications

ones that retain the ability to be recognized by antibodies raised over I

for wild-type HIV env protein. IN

The present invention also relates to a method for producing an HIV I

env protein or an immunogenic derivative thereof. The method comprises cultivating I

Drosophila cells transfected with an HIV env gene expression unit in a medium such as I

is suitable for growth of the cells. The transfected cells grown in the suitable I

medium, is able to express the protein of interest. The protein can then be collected

10 ffa the cell or cell culture medium. IN

Other aspects and details of the present invention are described below. IN

The method and expression system of the present invention facilitates I

15 high-level production of HIV proteins comprising gp120, gp160 and derivatives I

thereof, in a Drosophila cell structure. The Drosophila cells are transfected using I

standard cloning techniques which allow the insertion of foreign DNA into a host cell I

without adversely affecting the foreign DNA or the host cell. They thus I

engineered recombinant Drosophila cells produce HIV proteins. IN

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In contrast to the known baculovirus system in which the HIV protein is only provided

after lysis of the infected insect cells, the method of the present invention provides

invention a continuous cell expression system for HIV proteins. After secretion I

For example, the protein is available by purification from the culture medium using conventional I

25 functional techniques. Alternatively, the protein can be produced intracellularly or membrane-I

bound. The protein can be extracted from the cells using conventional techniques

nods. Alternatively, membrane-bound protein can be used in a variety of cell-associated I

honored analyzes. I 1

A preferred Drosophila cell line for use in the practice of the present invention

The present invention is D. melanogaster S2 line. S2 cells [Schneider, J. Embryol. Exp. IN

7 DK 175461 B1

Morph. 27: 353 (1972)] are stable cell cultures of polyploid embryonic Drosophi la cells. Introduction of the cDNA coding sequence for gp160 or its subunits gp120 or gp41 or derivatives thereof into Drosophila S2 cells by DNA transfection techniques unexpectedly produces large amounts of the glycoprotein. Use of the S2-Droso-5 phila cell has many advantages, including but not limited to its ability to grow to a high density at room temperature. Stable integration of the selection system has produced up to 1000 copies of the transfected gene expression unit in the cell chromosome.

Other Drosophila cell culture systems may also be used in the practice of the present invention. Cells likely to be used are e.g. The KC-0 Drosophila Melanogaster cell line, which is a serum-free cell line [Schulz et al., Proc.

Nat'l Acad. Sci., USA, 83: 9428 (1986)]. Preliminary studies using the KC-0 line have suggested that transfection is more difficult than with S2 cells. Another cell line that may be useful is a cell line from Drosophila hydei. Protein expression can be obtained using the hyde cell line; however, transfection in this cell line may result in the transfected DNA being expressed at a very low efficiency [Sinclair et al., Mol. Cell. Biol., 5: 3208 (1985)]. Other available Drosophi-la cell lines which can be used in the practice of the present invention include Sj and S3.

Drosophila cells selected for use in the practice of the present invention can be grown in a variety of suitable culture media, including e.g. M3 medi-um. The M3 medium consists of a formulation of balanced salts and essential amino acids 25 at a pH of 6.6. Preparation of the medium is essentially as described by Lindquist in DIS, 58: 163 (1982). Other conventional media for growing Drosophila cells may also be used.

A recombinant DNA molecule or recombinant DNA vector containing an HIV protein gene expression unit may be used to transfect the selected Drosophila cells in accordance with the invention. The gene expression unit contains

DK 175461 B1 I

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a DNA coding sequence for a selected HIV protein or for a derivative thereof. Such you

derivatives can be obtained by manipulating the gene sequence using traditional I

le genetic engineering techniques, e.g. mutagenesis, restriction endonuclease treatment, li- I

other gene sequences, including synthetic sequences, and the like. See, e.g. T. I

Maniatis et al., Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory I

tory, Cold Spring Harbor, NY (1982). IN

The HIV DNA coding sequence has recently been published. See Ratner et al., Nature 313: I

277-284 (1985) or Wain-Hobson et al., Cell 40: 9-17 (1985). The nucleotide sequence is I

10 also available from GenBank (clone BH 10, Ratner et al., Supra). IN

DNA molecules comprising the coding sequence of the present invention can I

derived from HTLV-III infected cells using known techniques (see Hahn I

et al., Nature, 312: 166-169 (1984)) or alternatively may be synthesized by standard oligomer.

15 nucleotide techniques. Furthermore, there are numerous recombinant host cells containing I

the cloned DNA coding sequences that are widely available. IN

Derivatives can then be prepared by standard techniques, including DNA synthesis. So- I

forming derivatives can e.g. include gp120 or gpI60 molecules wherein one or more I

Twenty amino acids have been substituted, added or deleted without being significantly misused

you direction to influence the protein's binding ability or biological properties. Derivatives of I

these proteins may also be prepared by standard chemical modification techniques

ker, e.g. acylation and methylation. I 1 2 3 4 5 6

Also included in the gene expression assembly are regulatory regions or I

2

desirable for transcription of the HIV protein coding sequence and its subsequent I

3

translation and expression in the host cell. The regulatory region typically contains an I

4

promoter region that acts by binding RNA polymerase and by initiating RNA trans-I

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transcription. The promoter region is typically found upstream of the HIV protein coding sequence

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late. IN

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Preferred promoters are of Drosophila origin, e.g. 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. CuS04. Use of the Drosophila metallothionein promoter results in the expression system of the present invention which maintains full regulation even at very high copy capital. This is a direct contrast to the use of mammalian metal lothionein promoter in mammalian cells in which the regulatory effect of the metal diminishes as the copy number increases. In the Drosophila expression system, this retained inducibility effect increases the expression of the gene product in the Drosophila cell at 10 high copy capital.

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

Examples of other known Drosophila promoters include e.g. the inducible var mechoch (Hsp70) and COPIA LTR promoters. The SV40 early promoter provides lower levels of expression than the Drosophila metallothionein promoter. Promoters commonly used in cell expression vectors, including e.g. Rous fiiglesarcomvi virus LTR and simian virus (SV40 early promoter), show poor function and expression in the Drosophila system.

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

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DK 175461 B1 I

The HIV DNA coding sequence can also be followed by a polyadenylation (poly · I)

A) region, such as an SV40 early poly A region. The poly A region acting in polyad I

Nylation of RNA transcripts seems to play a role in stabilizing trans- I

transcription. A corresponding poly A region can be derived from a variety of genes in which it is I

5 naturally occurring. This region can also be modified to change its sequence

sequence, provided that polyadenylation and transcript stabilization functions are not significant

is adversely affected. IN

The recombinant DNA molecule may also carry a genetic selection marker, as well as I

10 as HIV protein gene functions. The selection cursor may be any or I

any genes that cause an easily detectable phenotypic change in a transfection

right host cell. Such a phenotypic change may e.g. be drug resistance, so-

as the gene for hygromycin B resistance. IN

Alternatively, a selection system using the drug may be methotrexate and

prokaryotic dihydrofolate reductase (DHFR) gene is used with Drosophi I

la-cells. The endogenous eukaryotic DHFR of the cells is inhibited by methotrexate. Do you know

transfection of the cells with a plasmid containing the prokaryotic DHFR, which is I

insensitive to methotrexate, and select with methotrexate, therefore, only cells will trans-

20 infected with and expressing the prokaryotic DHFR survive. Various methotrexate, selective I

tion of transformed mammalian and bacterial cells, methotrexate in Drosophila

the transfectants are used with initially high copy capital. Only cells that have in-

harboring the protective prokaryotic DHFR gene will survive. At the same time, these

cells the gene expression unit of interest. IN

25. IN

An illustrative plasmid produced according to the present invention is pgp160A32, I

which contains a gp160 derivative replacing the N-terminal 32 amino acid sequence of I

gp160 with the first amino acid of tPA, serine. This plasmid is further described in Eq

Sample 1. I

30 I

11 DK 175461 B1

Another such plasmid vector is pgp120FA32, which contains the gp160 sequence, the first 32 amino acids being replaced by serine, and which sequence contains a carboxyl deletion of 216 amino acids. This plasmid is also described in Example 1.

Another plasmid illustrating derivative proteins of the present invention is pgp120A32, which contains the entire coding sequence for gp120 minus the first 32 amino acids at the N-terminus replaced by serine. Furthermore, plasmid pgp120A274 contains a gp120 protein sequence in which the first 274 amino-terminal amino acids are replaced by the first amino acid of tPA, serine, and which sequence contains the remaining 10 amino acids of gp120 up to the process site of gp160. These vector constructs are described more fully in Example 1.

Once a recombinant DNA molecule or expression vector containing the HIV protein gene expression unit has been constructed, it or 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 e.g. calcium phosphate coprecipitation, cell fusion, electroporation, microinjection and virus transfection. 1 2 3 4 5 6 7 8 9 10 11

A two-weight system can be used in the practice of the present invention 2 to cotransfect into the Drosophila cell a gene expression unit for the desired 3 HIV protein or derivative and the coding region of the selection system to be used. A preferred illustrative embodiment of the present invention is the production of an HIV protein using a vector containing a 6 HJV protein expression unit, e.g. pgp120A32, and a vector containing a hygromycin B gene expression unit, e.g. pCOHYGRO. pgp120A32 contains an expression 8 expression unit comprising the Drosophila metaHothionein promoter, a derivative of the 9 gp120 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 11 by maximizing the benefit of hygromycin B resistance upon selection. With this system, the antibiotic hygromycin B can be used to select such cells as

I DK 175461 B1 I

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You contain the transfected vectors. A more complete description of this embodiment

The mode of embodiment is given in Example 2. I

I As another example, an expression system using DHFR gene / methotre I

In the 5 xate selection system consisting of the vectors pgp120A32 and pHGCO is used to I

In selecting methotrexate resistant cells expression of gp120 or a derivative thereof. Away

In the pgp120A32 tower, a gp120 gene expression unit comprising the promoter is Dro I

In the sophila-metallothionein promoter. The pHGCO vector comprises a DHFR gene expression I

In unit and cotransfected together with the pgp120A32 vector,

In 10, the DHFR gene is required for selection. These selectable markers are together I

In cotransfection of Drosophila cells, further described by Johansen et al., In US I

In patent application Serial No. 07 / 047,736, filed May 8, 1987 and incorporated herein by reference

In reference. IN

In accordance with the invention, two vectors are cotransfected into the S2 Drosophila cell using I

In the method described by Wigler et al. in Cell, 16: 777 (1979). The vectors co- I

You are transfused in varying ratios depending on the particular copy capital desired. The trans- I

In infected cells are then selected, such as in an M3 medium containing serum and the I

In the selection means, e.g. hygromycin B or methotrexate. IN

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Once an appropriate vector has been constructed and transfected into the selected Dro-I

In sophila cell line, expression of gp120 is induced by the addition of a suitable inducer

Inducing agent for the inducible promoter. Cadmium or copper is e.g. induction I

In agents for the metallothionein promoter. Heat is the inducer of Hsp70 promoto-I

In 25 clean. For constitutive promoters, such as the actin 5C promoter, no induction is required

In the agent of expression. IN

In Transcription and Expression of the HTV Protein Encoding Sequence in the I described above

In systems can be monitored. Eg. For example, Southem image analysis can be used to determine I

In the 30 me copy number of the gp120 gene. Northem Imprint Analysis provides information regarding I

I to the size of the transcribed gene sequence [see e.g. Maniatis et al

DK 175461 B1 I

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above]. The degree of transcription can also be quantitatively determined. Expression of it I

selected HIV protein in the recombinant cells can be further verified by means of I

Westem Imprint Analysis and Activity Studies on the Resulting Glycoprotein [see I

Example 5]. IN

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Drosophila S2 cells are particularly well suited for high protein production at I

the process of the present invention. The cells can be kept in suspension

oncultures at room temperature (24 ± 1 ° C). Culture medium is M3 supplemented with between I

5 and 10% (v / v) heat-inactivated fetal bovine serum (FBS). In a preferred I

In one embodiment of the invention, the culture medium contains 5% FBS. After induction I

the cells are grown in serum-free media. When the pCOHYGRO vector system is used, I provide

the media is also obtained with 300 µg / ml hygromycin B. In these media the S2 cells can be grown in I

in suspension cultures, e.g. in 250 ml to 2000 ml rotary flasks with stirring at I

50-60 p.m. Cell densities are typically maintained between 106 and 107 cells per ml. In one embodiment I

In accordance with the present invention, the cells are cultured prior to introduction into 1500 ml of I

rotary flasks in media containing 5% serum. IN

In Subsequent cell culture, the HIV protein can be isolated from the used media, e.g. do you know

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 media. Using the cell line S2 which secretes the gene product directly into the media is an important feature of the present invention. Direct secretion in the media allows the use of an effective one-step purification system. Using a monoclonal antibody column targeting the HIV protein, the spent culture media can be added directly to column I and the protein eluted using 1.5 M KSCN in phosphate buffered saline I (PBS).

In a preferred purification technique which enables efficient large-scale production of the HIV proteins of the invention, an immunoaffinity column containing a monoclonal antibody directed against an epitope present in gp160 and present in DK is used. 175461 B1 I 14 in fully developed secreted gp120 proteins. Such a monoclonal antibody is advantageous because of its ability to recognize the protein sequence in more than one configuration. An antibody with these properties that is useful in immunoaffinity columns for various HIV proteins, derivatives or fragments thereof is designated 178.1. This monoclonal antibody is described in more detail in Example 3. Such a column can be prepared by coupling an antibody having the properties of 178.1 to a conventional absorbent carrier, such as "Sephadex", under appropriate conventional pH conditions. value, temperature and the like. Such a purification column and procedure can be used 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 capable of binding HIV proteins, especially gp160 I or gp120, are described in the art and are available. Other novel monoclonal antibodies useful in the practice of the present invention can be developed by non-conventional techniques.

The glycoproteins produced by Drosophila cells in accordance with the present invention are completely free of contaminating material, e.g. teat- In 20 animal, yeast and bacterial material and more importantly other HIV viral materials. Droso-In-phila-produced HIV proteins have also been shown to exhibit different glycosylation patterns than have been reported for other systems, e.g. mammalian systems.

The HIV proteins and derivatives of the present invention may be useful in a variety of products. Eg. For example, these recombinant proteins can be used in pharmaceutical preparations to treat HIV infected individuals. Such a pharmaceutical composition comprises a therapeutically effective amount of the HIV protein or derivative of the invention in admixture with a pharmaceutically acceptable carrier. The preparation can be administered systemically either parenterally, intravenously or subcutaneously. When administered systemically, the therapeutic composition is for use in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such a parenterally acceptable protein solution having regard to pH, isotonicity, stability and the like is within the skill of those skilled in the art.

The dosage curve is determined by the physician, who takes various factors that modify the effect of 5 drugs, e.g. the patient's condition, body weight, gender and diet, the severity of the infection in question, the period of administration, and other clinical factors, are taken into account. The pharmaceutical carrier and other ingredients of a pharmaceutical formulation are selected by those skilled in the art.

Furthermore, recombinant proteins of the present invention can be used as constituents of vaccines to vaccinate mammalian subjects against HIV infection.

These proteins can be used alone or in combination with other recombinant proteins or therapeutic vaccine agents. Components of such a vaccine are determined by those skilled in the art.

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Finally, the proteins of the present invention may be useful as diagnostic agents for detecting the presence of HIV infection or antibodies to an HIV infectious agent in biological fluids such as blood, serum, saliva and the like. The proteins of the invention may also be used in methods to identify and / or isolate 20 HIV binding proteins or other HIV binding substances in biological fluids and tissues, e.g. sCD4 or derivatives thereof. Thus, the proteins may be constituents of kits for practicing such methods. For the identification of an HIV binding substance, a protein of the present invention is contacted with the substance or an impure mixture containing the substance in order to promote binding between the protein and the HIV binding substance. A conventional assay to detect the presence of binding, e.g. detection of radioactive labels or the like is also part of the method. Therefore, the presence of binding between the protein and the binding substance is indicative of HIV binding.

In a method for isolating an HIV binding substance from the mixture, the binding process could be followed similarly by a conventional procedure for purifying the bound I protein produced by the protein of the present invention and the HIV binding substance. from the mixture. Other components of such diagnostic systems and kits may | are conventional constituents of diagnostic kits and may be selected by those skilled in the art.

Ice' ·

The following examples illustrate the construction of representative vectors and transformants of the invention, the preferred purification system and assays for determining | Increase the production level of the glycoproteins gp120 and gp160. These examples are not to be construed as limiting the scope of the invention.

I 10

Restriction enzymes and other reagents were used substantially in accordance with the manufacturer's instructions. |

Examples I 15

Example 1. Vector Constructs ·

al pMTtPA

I As the base vector for gene expression in Drosophila, the tPA expression vector I pMTtPA (also called pDMtPA) was used. This vector is a derivative of vector pML1, a small pBR322 vector containing the β-lactamase gene, which has deleted the poison sequence [Mellon et al., Cell, 27: 297 (1982)]. These sequences inhibit amplification of the I vector. This vector was digested with SalI and Aat2, which removes a small portion of 25 pBR322, and the digested ends were filled. The missing piece of pBR322 is then replaced with a cassette containing the Drosophila metallothionein promoter on a

In end-filled EcoRl-Stul fragment followed by a pre-filled HindIII-Sacl fragment from pDSPI

I [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 I fragment is followed by an SV40 early polyadenylation site.

DK 175461 B1 I

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b) pgp! 60A32 I

A HindIII-XbaI fragment containing the entire env gene was isolated from an HIV isolate I

clone BH10 [L. Ratner et al, as cited above, GenBankJ. The entire gp160 sequence I

5 was then inserted into an NcoI-XbaI digested vector pDSP1. The resulting vector I

SU2 was digested with Ndel followed by treatment with mung bean nuclease,

it was digested with Sacl, thereby isolating the gp160 gene. The digestion of I

Ndel intersects the gp160 sequence at amino acid # 32. Sacl digest intersects 3 'of I

The gp160 gene including in the sequence a portion of the original pDSP1 vector contained within

10 holding a polylinker. This fragment was inserted into the expression described above

pMTtPA, which had been digested with BgHI, was filled and then cut in

with Sacl, which deletes the fully developed tPA sequence. The BglII site is located at I

In the first amino acid of tPA. Accordingly, the resulting vector pgp160A32 encodes an I-modified gp160 protein in which the N-terminal 32 amino acids of gp160 are replaced by serine.

I c) pepl 20FA32 In Another vector containing a modified gene sequence encoding HTV-1 surface glycoprotein gp160 was constructed by digesting pgp160A32 with HindIII and I Sac1, thereby ironing the carboxyl terminus of gp160. Ca. two-thirds of the sequence, which encodes gp41, is removed by this digestion. This gp160 sequence is thus missing the first 32 amino acids and the last 216 amino acids of the natural gp160 sequence. The deleted sequence was replaced with a short synthetic linker sequence which encodes a stop codon on a HindIII-Sac1 fragment. The 6-amino acid linker sequence is I as follows: I 5'AGCTTTGACTGACTGAGCT 3 '.

I 30 I DK 175461 B1 I 18 I d) pgp! 20A32

Yet another vector containing a mutant gp160 gene was constructed by digesting pgp160A32 with Styl and XbaI, whereby the entire sequence of protein gp41 and ca. Thirty amino acids at the carboxyl terminus of the gp120 glycoprotein sequence were deleted. This fragment was replaced with a synthetic Styl-Xbal linker coding for the correct carboxyl terminus from the Styl site to the process site of 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.

In e) pgpl20A274

Yet another representative 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 pgp120A32 with Bg111 followed by XbaI digestion. This fragment I was now inserted in place of the tPA gene in the BglIIDCbal cut pMTtPA expression vector.

In the resulting vector, p120Δ274 encodes a gp120 protein, which has replaced the first 274 amino-terminal amino acids with the first amino acid of tPA, serine.

I 20 f) pCOHYGRO

In A commercially available plasmid, pUC18 [BRL] containing a Bam HI and Sma I site was used. 5 'LTR from an integrated COPIA element (357 base pairs) was cloned into the I BamHI site of vector pUC18, resulting in the vector designated pUCO-I 25 PIA. COPIA is a representative member of the dispersal mid-repetition sequences found throughout the Drosophila genome [Rubin et al. in Cold Spring Harbor Symp.

I Quant. Biol., 45: 619 (1980)]. The vector pUCOPIA was cut at the Smal site, and E.

In the coli gene encoding hygromycin B phosphotransferase (hygromycin B cassette), I was cloned into pUCOPIA using standard cloning techniques. The hygromycin B cassette was isolated on a 1481 base pair HindIII-BamHI fragment from the vector I DSP hygro [Gertz et al. Gene, 25: 179 (1983)]. The hygromycin B cassette contains 19 DK 175461 B1

the sequence encoding the hygromycin B phosphotransferase gene and the SV40 early poly A region. The HindIII and Bam HI sites were filled using T4 DNA-I

polymerase, and the hygromycin B cassette was ligated into the Sma I site of the vector pU-I

COPIA-producing vector pCOHYGRO. IN

5

Example 2. Transfection in Drosophila S ^ cells. IN

pCOHYGRO was cotransfected into S2 Drosophila cells together with one of the vectors I

carrying a gp160 mutant gene under the control of Drosophila metallothionein promo-I

10 as described above. In this example, the vector used is pgp120A32. The transfected cells were unselected in M3 medium containing 5% serum and 300 pg / mT hygromycin B. After 2 to 3 days under identical conditions, the untransfected cells stopped dividing and began to die. The time of selection to obtain stable, growing hygromycin B resistant cells in the transfected cultures is approx.

15 2 to 3 weeks.

To obtain cultures that have integrated into their chromosomes different copy numbers of the gp120 mutant gene, the ratios of the two vectors were varied. The ratio of this In example was 20: 1. Similar ratios have been used for other gp 160 mutant vectors of the present invention. This ratio is the same for any of the gp 160 mutant vectors used.

Expression of the pgp120A32 gene product was verified after introduction of the metallothioin nein promoter together with 500 μΜ CuS04. Westem imprint analysis of the spent I supernatant from the induced cell cultures revealed a single band of ca. 100 kd.

The level of mutant gp160 gene product in the cell supernatants was measured using gp120 ELISA assay described in Example 4 and using purified viral gp120 as standards. 5 x 10 6 cells per ml were seeded in M3 medium without I serum and induced for 3 to 4 days. The level of gp120 measured in the supernatant is approx. 1-2 I mg / l.

I DK 175461 B1 I 20

Cells were kept as suspension cultures in 250 ml to 2000 ml rotary flasks. Culture medium was M3 supplemented with 300 µg / ml hygromycin B. Cultures were incubated H at 24 ± 1 ° C and stirred at 50-60 rpm. Cell densities were typically maintained between 106 and 107 cells per ml in M3 medium supplemented with hygromycin B. CuSO4 was added to a final concentration of 500 μΜ and cultures allowed to grow for 3 to 4 days in serum-free media prior to harvesting it. modified gpl 20 glycoprotein.

The proteins produced by this method had a molecular weight of approx.

100, and the degree of expression was higher than any other reported gpl 20 / - 10 gpl60 expression in any eukaryotic cell system. In standard biological activity assays, the purified, modified, expressed gp120 as described above is capable of inhibiting viral infection in tissue culture, binds T4 and reacts with antibodies to I gp120.

I 15 It is expected that professionals in the field could express the other gpl 60 and I

gpI20 proteins and fragments thereof described by the present invention, using essentially the same systems and procedures and exemplified above for the protein fragment encoded in pgp120A32.

Example 20 Monoclonal Antibody 178.1 In an affinity purification column using a novel monoclonal antibody, I was used in the purification scheme used for the mutant I gp160 / gp120 proteins described above. This monoclonal antibody can be characterized by being able to react with non-denatured HIV glycoprotein products present in cell lysate and with fully developed gp120 as secreted in the supernatant of a yeast culture. One such monoclonal antibody specific for the epitope contained both in the I non-processed recombinant gp160 molecule and in the full-sized gp120 protein is a mouse monoclonal antibody 178.1.

I 30

DK 175461 B1 I

21 I

An expression system using C. albicans glucoamylase promoter and sig- I

nal peptide, was used to produce partially purified yeast recombinant gp160 for pro-I

duction of 178.1. Production of this yeast-derived gp160 is described in U.S. Patent Application I

Application Serial No. 07 / 236,699, Bruck et al., filed August 25, 1988. This application I

5 is incorporated herein by reference. IN

Eight-week-old Balb / c mice received three times subcutaneously and intraperitoneally injected partially I

purified (1.5-3% purity) yeast recombinant gp160 in Freund's 4-week adjuvant

intervals. After a 3-month rest period, one mouse was killed and its spleen cells I

10 were joined to myeloma cells [see e.g. R. P. Siraganian et al., Meth. Etc., I

92: 17 (1983); EMBO Course on Hybridoma Production, Basel Inst, for Immunol. IN

(1980)]. The myeloma cells used are a subclone of the Sp2 / O-Agl4 lineage previously expressed.

selected for optimal growth in agar medium and high fusion efficiency [J. D. Franssen et al., I

Proc. XXIX Colloq. Protids Biol. Fluids, 29: 645-649 (1981)]. After approx. In time, 15 supernatants were taken for screening in a capture ELISA using a commercial monospecific anti-gp120 reagent [Biochorm. Seromed Ref. D7324] as a capture antibody.

Briefly, Nunc immunoplayer I (Nos. 4-39454) was coated overnight at 4 ° C with 50 I 20 μΐ of a solution of 5 µg / ml of sheep anti-gp120 IgGs in PBS. The plates were washed with 1 wash buffer (PBS, "Tween" 0.1%) and saturated with 100 µl! of a saturation buffer I [PBS, newborn calf serum 4%, bovine serum albumin (BSA) 1%, "Tween" 0.1%] for 1 hour at 37 ° C. Fifty µΐ / well of crude Molt3 / HTLV-inB or Molt3 cell lysate (107 I cells / ml in PBS, "Triton" X-100 1%) or of the supernatant fraction (S2-30) of recombinant yeast gp 160 (or similarly treated negative control) was used as antigen I and incubated in the plates for 3 to 5 hours at room temperature. The plates were thoroughly washed and 50 μΐ hybridoma supernatants were added to each well and incubated overnight at 4 ° C. After a washing step, 50 µl / well of a 1/500 dilution of biotinylated I anti-mouse immunoglobulins (Iger) (Amersham Ref. RPN 1021) in saturation buffer was cubed in the plates for 1 hour at 37 ° C. The plates were washed again and 50 μΐ / well of a I 22.

1/1000 dilution of streptavidin biotynylated horseradish peroxidase complex (Amer. Ref. RPN 1051) in saturation buffer was added to each well.

After a further washing step, 50 μΐ of a solution of 0.4 mg / ml of orthophenylene-I diamine dihydrochloride (OPD, Sigma PI526) and 1 μΐ / ml of H2O2 (30% in citric acid / sodium citrate 0.1 M pH · value 5) supplemented with 0.1% Tween 20 added to each well. The plates were then incubated for 20 min. at room temperature in the dark, and the reaction was stopped by the addition of 50 μΐ / well of 2M H2SO4. The optical density at X = 492 nm was monitored and 50 positive clones were selected for further subcloning in soft.

In 10 agarose according to P. Herion et al., Proo. XXIX Colloq. Protids Biol. Fluids. 29: 627 (1981). The cloned hybridoma cells were then cultured in vivo by injection of 2 to 5 x 10 6 hybridomas into the peritoneal cavity of Balb / c mice pretreated by intraperitoneal injection (2,6,10,14-tetramethylpentadecane).

The monoclonal antibodies selected by the above procedure were characterized by Westem Impression Assays (WB), Radio Immunoprecipitation Assay (RIPA), In Purification, Biotin Labeling and Competition Assays. Resulting monoclonal antibodies were further characterized by analyzing their responsiveness to various recombinant and naturally occurring antigens.

I 20

A high yield of hybridomas was obtained by this procedure. More than 200 wells I were positive in the screening analyzes. Of these, however, only 50 wells were selected, and after cloning, the cells were expanded in ascitic acid. All ascites fluids were examined by WB and RIPA. Among the 39 monoclonal antibodies studied, 37 showed a 25 gp160 band at RIPA. No one reacted with the gp120 form in the same assay. So * I monoclonal antibodies which showed only gp160 recognition by RIPA, being clearly responsive to gp120 by WB, were analyzed by subclass. Three I monoclonal antibodies, which were IgG2A, were purified on a protein A-Sepharose column I and biotin labeled. Competition assays using vaccine gp160 as the antigen were performed and the result obtained defined at least five different groups of epitope recognition with the gp160 protein. Monoclonal 178.1 was selected for an epitope present on fully developed gp120 and unprocessed intracellular gp160.

A Westemprint (WB) assay was performed according to conventional techniques to demonstrate that 178.1 is capable of binding HIV virus isolated from human cells infected with HTLV-ΠΙβ [Molt3 / HTLV-IIIB ].

Radioimmunoprecipitation assays (RIPA) were performed as described by P. J. Kanki et al. in Science, 228: 1199 (1985) to demonstrate that 178.1 could immunoprecipitate the 10 human cells infected with HTLV-III virus strains.

The responsiveness of monoclonal antibodies that recognize non-overlapping epitopes against a large panel of antigens was assessed using sandwich ELISA involving sheep antibody gp120 as capture reagent. Monoclonal antibody 178.1 was negative in ELISA on divergent HJY isoylates MoII3 / (FirLV-IIiB, H9 / HTLV-IIIRF) and Hut78 / Arv2, but clearly positive when tested on HTLV-IIIB in RIPA, WB or ELISA during use of recombinant antigens. This monoclonal antibody recognizes an epitope that is apparently conserved between gp160 and gp120, and thus, using the purification technique described in Example 4 below, provides an additional advantage for producing gp160 / gp120 glycoproteins in various constructs.

Example 4. Purification of ετ> 120Δ32 from Drosophila conditioned cell culture medium.

The recombinant gp120 protein of Example 2 was purified as follows: 30 l of Dro I-25 sophila conditioned media (CM) containing gp120A32 were prepared with 1 l mM phenylmethylsulfonyl fluoride (PMSF), 10 mM ethylenediaminetetraacetic acid (ED-I TA) and 70 Kallikrein inhibitor units. The CM was filtered through a 0.45 µm Durapo I re-membrane using a pellicon (millipore) device. Filtered CM was applied to S-Sepharose Rapid Flow (Pharmacia) (5 liters; 25.2 cm x 11 cm) at a linear

At 30 flow rate (LFR) of 37 ml / cm2.time equilibrated in Buffer A containing 20 mM

In 2- [N-morpholino] ethanesulfonic acid (MES) at a pH of 6.0. After applying everything in DK 24461 B1 to 24 H CM, the column was eluted in a buffer B step containing 20 mM MES, pH 6.0, in 0.4M NaCl.

H The S-sepharose-eluted gp120A32 was applied to an anti-gp120 mouse monoclonal cepharose 4B column (60 ml; 3.2 cm x 6.5 cm) at a LFR of 10 ml / cm2.h. This column H was equilibrated in buffer B. After application of a 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, 1.0M NaCl ( Buffer C) and 2 column volumes of Buffer A. gp120Å32 were eluted with H 0.1M acetic acid, pH 2.8, and fractions were immediately neutralized by the addition of 0.1 volume of 1M tris (hydroxymethyl) aminomethane ( Tris), pH 10.4.

In the Muse anti-gpl 20 monoclonal antibody hybridoma 178.1 was produced according to Example I 3 above. This hybridoma was seeded at 2 x 10 5 cells / ml and grown for four days in I Dulbecco's modified tender medium [Hazelton Research Products] supplemented with 4.5 15 g / l glucose, 2 μΜ 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 I x 10 cm) equilibrated in 0.1 M Tris, pH value 8.2. Antibody was eluted with 0.1 M sodium citrate, pH 3.5 and immediately neutralized with Tris.

In purified anti-gp120 monoclonal antibody was coupled to CNBr-activated sepharose 4B

I (Pharmacia), according to the manufacturer's instructions, at a density of 2 mg of antibody resin and with a coupling efficiency of 98%, resulting in an anti-gp120 Sepharose Dependent Resin. This affinity resin will specifically bind gp120 protein upon interaction of the antibody with a unique structural epitope of gp120.

I 25 I The purity of the finished gp120 protein product according to this purification technique is 80-90% with a calculated yield of 8.5 mg / 30 l of conditioned medium. Extraction is estimated at between 25-50%.

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

DK 175461 B1 I

25 I

Example 5 Analysis. IN

The assay described above is a non-isotope assay using an enzyme I

and a substrate for the detection of gpI20 or fragments thereof, which assay was used

I 5 turned to detect the gp120 proteins produced by the method and I

I by means of the compositions of the present invention. IN

In this assay, the criteria for the detection of gp120 are dependent on antibody specificity

In tet. An anti-gp120 monoclonal antibody [DuPont, cat. No. 9284], diluted in 0.1M sodium I

In 10 µm carbonate buffer (pH 9.5) to two µg / ml is used to capture the gp12O protein. IN

I 100 μΐ of this antibody dilution is added to each well twice on an assay plate, I

Except for such wells that are intended to be controls. The plates were incubated

In dish at 4 ° C overnight. The antibody was washed out the next day and the plate was blocked

In dish by adding 300 μΐ of a blocking buffer consisting of 1% BSA in PBS to each I

In 15 wells for 1 hour at room temperature. IN

The viral gp120 standards were diluted to 1 pg / ml, 0.5 pg / ml, 0.25 pg / ml, 0.1 pg / ml I

I and 0.2 µg / ml in a wash buffer consisting of PBS and 0.05% "Tween" 20. 100 µΐ aide I

In diluted standards, each well is added double. The plates were incubated on a plate shaker for 2 hours at room temperature, after which each plate was washed four times with wash buffer.

To each well was added 100 μΐ of rabbit anti-gp120 antibody (described by DeBouck et al. In U.S. Patent Application Serial No. 07 / 056,553, filed May 29, 1987) diluted I '25 1/1000 in wash buffer, and each platelets were incubated on a shaker for one hour. This second antibody brings gp! 20 into sandwich between the two antibodies. The plates were then washed four times with wash buffer. To detect this complex, a third anti-I substance, 100 μΐ of peroxidase (POD) labeled goat anti-rabbit antibody (mostly IgG and I IgM antibody) diluted in wash buffer without azide was added to each well. The plates were then incubated for 2 hours on a shaker at room temperature.

In DK 175461 B1 Η H After the plates were washed four times, 100 μΐ of a colorless substrate (1 mg / ml of OPD in citrate buffer together with 4 ml of 35% hydrogen peroxide per 10 ml of buffer) was added.

The hydrogen peroxide was added just prior to the addition of substrate to the wells. These plates were incubated for 8 min. on a shaker and the reaction was stopped by adding 5 100 μΐ 0.1M sodium fluoride to each well. In the presence of peroxidase-conjugated antibodies, the substrate turns dark yellow. Optical density or color intensity, proportional to the amount of gp1 captured, was read on a plate reader at 450 nm and a standard curve constructed of unknown material concentrations was constructed. 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 to the described methods, e.g.

Using other truncated gp 160 / gp 120 sequences which are obvious to those skilled in the art of molecular genetics and related sciences, it is intended to fall within the scope of the following claims.

/

Claims (10)

27 IN PATENT CLAIM I A method of producing an HIV protein in Drosophila cells, characterized in that it comprises culturing in a suitable medium of Drosophila cells transfected with an HIV protein gene expression unit capable of for expressing the II protein and wherein the HIV protein gene expression unit comprises the gp120 or gp160 II DNA coding sequence for the protein and a regulatory element necessary II for transcription of the coding sequence and translation into a Drosophila cell. I I 10 I Process according to claim 1, characterized in that the regulatory element comprises an actin 5C promoter or a Drosophila-metallothionein promoter. IN Method according to claim 1, characterized in that the DNA coding sequence comprises the HIV-DNA coding sequence present in pgp160A32, pgp120FA32, i1 pgp120A32 or pgp120A274. IN HIV-gp120 or HIV-gp160 protein, characterized in that it is produced by the method of claim 1. A method for producing an HIV protein in cultured Drosophila cells, characterized in that it comprises transfecting Drosophila cells with an HIV protein I, gene expression unit comprising the gp120 or gp160 DNA coding sequence and a selection marker, and culturing the cells in a suitable medium and under conditions such that the protein is expressed. Method according to claim 5, characterized in that the cells are transfected with a first vector containing the coding sequence for hygromycin B phosphotransferase I and a second vector containing the coding sequence for an HIV protein gene expression unit. I DK 175461 B1 I 28 Method according to claim 6, characterized in that the first vector is pCOHY-I GRO. Method according to claim 6, characterized in that the second vector comprises an HIV gene expression unit present in pgp160A32, pgp120FA32, pgp120A32 I or pgp120A274. Method according to claim 5, characterized in that the Drosophila cells are D. melanogaster S2 cells. 10 Method according to claim 9, characterized in that the S2 cells are cotransfected with the H vector pCOHYGRO and a vector comprising a gene expression unit present in pgp160A32, pgp120FA32, pgp120A32 or pgp120A274. I I 15