SK82593A3 - Derivatives opf 160 and vaccines based on gp 160 or a derivative thereof, containing an adjuvant - Google Patents

Abstract

This invention provides novel, substantially uncleavable forms of gp160, and vaccine formulations containing gp160 or a derivative thereof, adjuvanted with 3-D Mpl. The compositions are useful for the immunotherapeutic and immunoprophylactic treatment of HIV infections.

Description

Technical field

The invention relates to a gp10 derivative and a vaccine based thereon. These materials are intended for use in the treatment of AIDS. In particular, the invention relates to novel types of said vaccine which contain 3-D monophosphoryl lipid A as adjuvant.

BACKGROUND OF THE INVENTION

Retroviruses, i.e. viruses of the family Retroviridae, are a large group of icosohedral viruses with a developed envelope, approximately 150 nm in size, which have a spiral nucleocapsid in the core structure and contain RNA as genetic material. This family contains oncoviruses, such as sarcoma virus and leukemia virus, some immunodeficiency viruses and lentiviruses.

Human immunodeficiency virus type 1 (HIV-1) is the etiological agent of acquired immunodeficiency syndrome, commonly referred to as AIDS. This retrovirus has a complex genetic organization, including long terminal repeats (LTRs), gag, pol and env genes and other genes. This retrovirus carries a variety of viral antigens that may be capable of eliciting, either individually or in a group, a protective immunological response upon vaccination.

The HIV-1 envelope protein is synthesized in the form of a polyprotein precursor, which is then glycosylated in the infected cell to produce a glycoprotein with a molecular weight of 160,000 (gp110), which is then processed by proteolysis into gp120 glycoprotein and gp41 transmembrane protein.

The HIV DNA coding region can be prepared from any immunodeficiency virus genome clone as reported in the literature, for example, in Shaw et al., Science, 226: 1165 (1984) and Kramer et al.

Science, 231: 1580 (1986). The genomic clone of the immunodeficiency virus can also be prepared from virus samples by conventional DNA cloning, U.S. Pat. No. 4,520,113 (Gallo et

U.S. Pat. No. 4,708,818 (Montagnier et al.).

The DNA proviral sequence for HIV-BH10-2 has been described by Ratner et al., Human Retroviruses and AIDS,

1989, HIV Sequence Database ed. Dr. Gerald Meyers, Los Alamos National Laboratory. The chain length is 8932 base pairs, the env gene is located between bases 5580-8150 and the cleavage site in this gene at positions 7088 and 7112 correspond to amino acids 503 and 511 (cleavage occurs after these residues), numbering according to the Los Alamos database.

SUMMARY OF THE INVENTION

The present invention provides gp160 HIV, which has been modified to contain, independently, amino acids different from lysine or arginine at positions 502 and 510.

Suitable lysine replacements are residues that are lysine-like in terms of hydrophilicity and size, for example, histinine, threonine, serine, asparagine, aspartic acid, glutamine, and glutamic acid. Of these amino acids, glutamic acid is particularly preferred. The preferred protein is non-cleavable due to the introduced mutations and, moreover, is capable of inducing the generation of cross-linked neutralizing antibodies that are dependent on the correct protein configuration.

In a preferred embodiment, the modified protein according to the invention can be obtained in oligomeric form, in particular with a relative molecular weight of 640 000. Depending on the molecular weight of the gp160 monomer, this is apparently a tetameric form. This is highly advantageous since the surface proteins of the virus naturally occur as oligomers, which in vivo form spikes protruding from the surface of the virus. Since it is necessary to preserve neutralizing epitopes, the natural form of the antigen should be preserved as much as possible in order to achieve a corresponding immunological response. In a preferred embodiment, the modified protein of the invention can be obtained in oligomeric and substantially pure form.

By substantially pure form is meant a purity of at least 75%, preferably at least 90% and especially 99%.

The present invention also provides a method for producing a modified HIV gp110 by expressing the DNA encoding the modified protein in a recombinant eukaryotic host cell and isolating the modified protein.

The present invention also provides a polymeric DNA comprising a nucleotide sequence which encodes a modified protein.

The recombinant DNA molecule of the invention can be prepared by the method of the invention by condensation of the appropriate mono-, di or oligomeric nucleotide units.

This procedure may be performed chemically, enzymatically, or a combination of the two, in vivo or in vitro as appropriate. The DNA molecule may be prepared by enzymatically binding the appropriate DNA fragments in a conventional manner, for example, as described by D. M. Roberts et al., Biochemistry 1985, 24, 5090-5098.

DNA fragments can be obtained by digesting the DNA containing the desired nucleotide sequences with restriction enzymes, chemical synthesis, enzymatic polymerization, or a combination of these.

The restriction enzyme digestion can be carried out in an appropriate buffer at a temperature of 20 to 70 ° C, usually in a volume of 50 microliters or less using 0.1 to 10 micrograms of DNA.

Enzymatic polymerization of DNA can be performed in vitro using a DNA polymerase, for example DNA polymerase I (Klenow fragment) in a suitable buffer containing nucleoside triphosphates dATP, dCTP, dGTP and dTTP at a temperature of 10 to 37 ° C, usually in a volume of 60 microliters or lower. Fragments can be polymerized and amplified by PCR reaction using Tag polymerase (e.g., according to PCR Protocols 1989 and Guide to Methods and Applications, Ed. M.A. Innis et al., Academic Press).

Enzymatic binding of DNA fragments can be performed using a DNA ligase, for example T4 DNA ligase, in an appropriate buffer at 4 ° C to room temperature, usually in a volume of 50 microliters or less.

Chemical synthesis of the DNA molecule or fragments thereof is carried out in a conventional manner using solid phase phosphotriesters, phosphides or phosphoramidites, for example according to the Chemical and Enzymatic Synthesis of Gene Fragment s - A Laboratory Manual, ed. HG Gassen and A. Lang, Verlag Chemie, Veinheim, 1982 or other scientific publications such as MJ Gait, HV D-Matthes, M. Singh, BS Sproat and RC Titmas, Nucleic Acids Research, 1982, 10, 6243, BS Sproat and V. Bannwarth, Tetrahedron Letters, 1983, 24, 5771, MD Matteucci and MH Caruthers, Tetrahedron Letters, 1980, 21, 719, MD Matteucci and MH Caruthers, Journal of the American Chemical Society, 1981, 103, 3185, SP Adams et al., Journal of the American Chemical Society, 1983, 105, 661, ND Sinha, J. Biernat, J. McMannus and H. Koester, Nucleic Acids Research, 1984, 12, 4539, and HVD Matthes et al., EMBO Journal, 1984 3, 801. Preferably, an automatic DNA synthesis device is used.

A DNA polymer coding for a modified protein can be prepared by site-directed mutagenesis of a cDNA coding for an unmodified protein using conventional procedures, for example, by G. Vinter et al., Nature 1982, 299, 756-758 or Zoller and Smith 1982, Nucl. Acids Res., 10, 6487-6500.

The invention also relates to a vector comprising a recombinant DNA molecule of the invention and a recombinant vaccine virus comprising the vector.

The vector may be prepared by digesting a linear DNA segment containing an intact replicon and linking the linear segment to one or more DNA molecules that form a complete or recombinant DNA molecule of the invention with the linear segment.

A recombinant host cell of the invention can be prepared by transforming a vaccine virus using a vector of the invention.

The modified protein product can be isolated from the environment by conventional protein isolation and purification procedures. Wetting agents such as Decyl PEG-300, DO Decyl PEG Triton X100 may be added. Deoxycholate can also be added to cleave cells and release the modified protein from the cell membrane material. Of these, particularly preferred are Decyl PEG-300 and Thesit Deoxycholate, as well as DO Decyl PEG Triton X100. The modified protein can then be purified by multi-stage ultrafiltration, centrifugation in an ultra-centrifuge, selective precipitation, for example using ammonium sulfate or polyethylene glycol PEG, centrifugation using a density gradient in CsCl or sucrose, or using a gradient of metrizamide and / or chromatography, e.g. preferably immunoaffinity chromatography, HPLC, reverse phase HPLC, cation exchange or anion exchange chromatography, size limiting chromatography and isoelectric point chromatography. Purification using immunoaffinity chromatography is preferred. During or after purification, the modified protein may be treated, for example, with formaldehyde, glutaraldehyde, or NAE to enhance stability or enhance the immunological response.

It is convenient to use mild conditions during purification to prepare oligomeric forms. In particular, the use of a reducing agent should be avoided, preferably using nonionic surfactants such as Decyl PEG-300 (polyethylene glycol 300 monodecyl ether) to disrupt cells and solubilize during chromatography. A preferred medium for affinity chromatography is Sepharose with a lentil lectin, and a preferred medium for performing immunoaffinity chromatography is a monoclonal anti-gpl10 antibody, e.g., antibody 178.1 (WO 90/06358) bound to a suitable carrier, e.g. Trisacryl (IBF), activated glutaraldehyde.

The modified protein can be adsorbed from the monoclonal antibody in the presence of octylglucopyranoside as a wetting agent, which can then be removed by dialysis. The wetting agents are preferably used at a concentration higher than its theoretical critical micelle concentration.

The modified protein obtained by the method of the invention can be used as a diagnostic agent to detect exposure to HIV. However, the modified protein can also be used to produce vaccines to prevent infection or to inhibit or prevent disease progression.

The invention also relates to a vaccine and pharmaceutical compositions comprising a modified protein of the invention. These compositions contain an immunoprotective amount of a modified protein of the invention and can be prepared by conventional methods.

An aqueous solution of said protein may be used directly in the vaccine of the invention. Alternatively, the protein, optionally after prior lyophilization, is mixed or absorbed onto any known composition. Such agents are, for example, aluminum hydroxide, muramyl dipeptide and some saponins, such as Quil A, 3D-MPL (3-acylated monophosphoryl lipid A) or TDM. It is also possible to encapsulate the protein in microparticles such as a liposome. Yet another possibility is to conjugate the protein to an immunostimulatory molecule, for example, killed Bordetella bacteria or tetanus toxoid.

Vaccine preparation is generally described in New Trends and Developments in Vaccines, ed. Aviary et al., University Park Press, Baltimore, Maryland, USA,

Encapsulation into liposomes has been described, for example, in U.S. Pat. 4,235,877 (Fullerton). Conjugation of proteins to macromolecules has been described, for example, in U.S. Pat. No. 4,474,757 (Armor et al.).

The amount of the modified protein of the invention in each vaccine dose is selected as an amount producing an immunoprotective response without significant adverse side effects. This amount will vary depending upon the use of the specific immunogen and also whether or not the vaccine contains an adjuvant. Typically, each dose will contain from about 1 to about 1000 micrograms of modified protein, preferably from about 10 to about 200 micrograms. The optimal amount for a given vaccine can be verified by routine experiments, including observation of antibody titer and other responses. After the initial vaccination, the next dose is likely to be given within 4 weeks to achieve an increase in antibody production and then further doses every 6 months as long as the risk of infection persists.

Thus, the modified protein of the invention can be used to vaccinate a possible host and to prepare vaccines.

In addition to the vaccination of persons who could be infected with HIV, the pharmaceutical compositions of the invention may be used to treat patients suffering from HIV infection by immunotherapy.

Accordingly, the pharmaceutical compositions of the invention are also intended for the treatment of persons suffering from HIV infection by administering to said persons an effective amount of a modified gp110 protein as described above.

In addition to the idea of utilizing certain components of the material, for example by recombinant technology, it may also be necessary to employ adjuvants and / or carriers for immunogenic agents in order to achieve both the usual immunological response, i.e. the generation of neutralizing antibodies and effector cell-mediated immunity. (DTH). It has been shown that by using the materials of the invention for the prophylaxis or treatment of HIV infection, both types of immunological responses can be achieved using 3D MPL as an adjuvant.

Accordingly, in a preferred embodiment, the pharmaceutical composition of the invention comprises gpl10 or an immunology derivative thereof and 3D monophosphoryl lipid A (3D-MPL) together with a suitable carrier.

In another preferred embodiment of the invention, the pharmaceutical composition comprises gp160 or an immunological derivative thereof and 3D-MPL in an oil-in-water emulsion. This system has increased efficiency.

Thus, according to a preferred embodiment of the invention, the pharmaceutical composition or vaccine comprises gp-160 or an immunological derivative thereof and 3D-MPL in an oil-in-water carrier, the carrier comprising an emulsion of tetrapolyol and a non-toxic mineral oil in saline containing buffer.

The carrier preferably comprises a Pluronic-type polyol, for example Pluronic L121, squalane or squalene or any other metabolized oil. An emulsifier such as Tween 80 or Tween 28 is preferably used to stabilize the emulsion.

Preferably, the carrier comprises only particles smaller than 1 micron, for example particles in the range of 100 to 400 nm.

The antigen concentration in the final pharmaceutical composition is preferably in the range of 10 to 150 and especially 20 to 100 micrograms / ml.

The 3D-MPL adjuvant is preferably present in the vaccine at a concentration of 10 to 100 and especially at a concentration of 25 to 50 micrograms / ml.

The present invention provides a vaccine for use in medicine, in particular for use in immunotherapy and prophylaxis of HIV-1 infection, for example AIDS syndrome or ARC-associated complex.

The present invention also provides a method of producing a vaccine comprising gp160 or an immunological derivative thereof, 3D-monophosphoryl lipid A and a suitable carrier, comprising mixing gp160 or an immunological derivative thereof with said carrier and 3D monophosphoryl lipid A.

This can be accomplished, for example, by using an oil-in-water carrier, wherein the emulsion is processed until a particle size of less than 1 micro meter is achieved, and then the emulsion is mixed with gp100 or its immunological derivative and 3D MPL.

In a typical procedure, the 3D-MPL is first mixed with the emulsion and then the antigen is added to the resulting mixture.

3D-MPL can be obtained according to the method of British Patent Specification No. 5,940,549. 2,211,502.

Suitable carriers for use herein are oil-in-water emulsions. The compositions of the invention may provide a more advantageous neutralization titer compared to conventional vaccines that contain only alum as an adjuvant (the only adjuvant authorized for use in human medicine).

The term immunological derivatives refers to immunogenic fragments of the gp10 protein that are capable of eliciting neutralizing antibodies to HIV-1 in the presence of the 3D-MPL adjuvant. Because these derivatives also include, for example, the HIV-1 outer membrane gp120 glycoprotein, immunological derivatives also include a naturally occurring gp110 isolate. Particularly preferred are those derivatives which can also elicit a DTH response.

Thus, in a very preferred embodiment, it is possible according to the invention to obtain a modified form of the protein in oligomeric form which, when purified under mild, non-reducing conditions, has an apparent molecular weight in the range of 600-700,000, preferably 640,000, apparently tetramer. chromatography on an SDS-gel under non-reducing conditions to give a dimer with a molecular weight of 330,000 and a monomer. The dimer can be further reduced under conventional reducing conditions to form a monomer.

All of the above forms of gp100 can be used in the compositions of the invention.

The gp10 protein or derivatives thereof can be obtained by known methods. Typically, this will involve the cloning and expression of the gp110 gene in a suitable host. Recombinant gp110 (rgp 160) can be obtained according to Maniatis et al., Molecular Cloning - A laboratory Manual, Cold Spring Harbor 1982.

A variety of eukaryotic cells and expression systems can be used to express recombinant DNA molecules. The most commonly used systems are yeast and insect and mammalian cells, although the invention is not intended to be limited thereto. In these systems, the recombinant DNA molecule of the invention is used, optionally together with a label for selection and optionally, for example, with an origin for replication.

Useful insect cells are, for example, Drosophila and Lepidoptera cells. Useful Drosophila cells are, for example, S1, S2, S3, KC-0 and D. hydei cells, as described, for example, in Schneider et al., J. Embryol. Exp. Morph., 27: 353, (1972), Schulz et al., Proc. Natl. Acad. Sci., USA, 83: 9428 (1986); Sinclair et al., Mol. Cell. Biol., 5: 3208 (1985).

Transfection of Drosophila cells can be accomplished by conventional procedures, such as calcium phosphate precipitation, cell fusion, pore opening, and transfection using virus. Cells are grown according to known techniques in a variety of nutrient media, including M3 media, which contain a balanced mixture of salts and basic amino acids, as described in Lindquist, DIS, 58: 163 (1982).

Promoters that can be used in Drosophila are, for example, promoters from mammalian cells, such as SV40, and also promoters from Drosophila cells, which are more preferred. Examples of useful promoters from Drosophila cells include the metallothionein promoter, the HSP70 promoter for protein formation in the case of a thermal shock of 70,000 molecular weight, and the COPIA LTR promoter described, for example, in DiNocera et al., Proc. Natl. Acad. Sci., USA, 80: 7095 (1983); McGarry et al. Cell 42: 903 (1985).

Useful Lepidoptera cells include Trichoplusia ni, Spodoptera frugiperda, Helithis zea, Autographica californica, Rachiplusis ou, Galleria melonella, Manduca sexta, or other cells that can be infected with baculian Russia, including nuclear polyhedrosis virus (NPV) virus, single nucleocapsid containing virus (SNPV) and multiple nucleocapsid (MNPV) viruses. Preferred baculoviruses are NPV or MNPV because they contain a polyhedrin gene promoter that is highly expressed in infected cells. MNPV from Autographica californica (AcMNPV) is an example. However, other MNPV and NPV viruses, such as the silkworm virus, Bombyx mori, may also be used. Lepitoptera cells may be co-transfected with the recombinant DNA molecule of the invention and infectious baculovirus DNA in a conventional manner. The cells are then grown in a conventional manner using a variety of media, for example, in the TC100 medium (Gibco Europe) described by Gardiner et al., J. Inverteb. Pathol., 25: 363 (1975) and supplemented with 10% FCS fetal calf serum according to Miller et al., In Setlow et al., Eds., Genetis Engineering: Principles and Methods, Vol. 8, New York, Plenum, 1986, p. 277 to 298.

Promoters for use in Lepidoptera cells include promoters from the baculovirus genome. The polyhedrin gene promoter is particularly preferred, since this protein usually exhibits a higher level of expression than other baculovirus proteins. Particularly preferred is the polyhedrin gene promoter from AcMNPV described in Summers et al., TAES Bull. NR 1555, May 1987, Smith et al., EP. 127,839, Smith et al., Proc. Natl. Acad. Sci. USA 82: 8404 (1985); and Cochran, EP-A-228 036.

For expression in mammalian cells, a recombinant DNA molecule can be cloned in a cloning vector and then used to transfect mammalian cells. The vector usually contains additional gene amplification functions, for example a cassette for DHFR expression, and may contain additional selection and / or amplification functions, for example, a neomycin resistance cassette for G418 selection. Still other functions can be used - for example, to facilitate transcription. In addition, functions to maintain episome stability, such as those from bovine papilloma virus, can be used. Preferably, a recombinant mammalian virus, such as a vaccinia virus, vaccinia virus, is used as the cloning vector.

This virus is a particularly suitable vector because recombination can be readily accomplished by integrating the foreign gene into non-essential regions of its DNA while maintaining the infectious virus. In a carefully selected procedure, proteins can be synthesized, processed and transferred to the membrane of infected cells. Although infection with this virus leads to cell death, there is little cell disruption and most cells remain intact, allowing easy isolation of the desired protein from infected cells. Said expression system has been described by Barett et al., Aids Research and Human Retroviruses 1989, 5: 159-171.

Useful mammalian cells include Chinese hamster ovary (CHO) cells, NIH3T3, COS-7, CV-I, BHK-21 cells, mouse or rat myeloma cells, HAK, Vero, HeLa cells, human diploid cells such as MRC-5, and VI38 or chicken lymphoma cell lines CV-I and BHK-21 are particularly preferred.

Growing of cells during transfection and subsequent cultivation is carried out in a conventional manner. Protein production in mammalian cells can also be accomplished by expression in transgenic animals.

Control chains suitable for achieving gene expression in mammalian cell lines or in primary mammalian cells include SV40 promoters, a metallothionein promoter, LTR viruses such as LTR of Rous sarcoma, LTR of Moloney sarcoma virus (MSV), and LTR of the mammary tumor tumor MMV or mouse MMTV tumor virus adenovirus as well as hybrid promoters, for example the hybrid virus promoter BK and the adenovirus promoter. The control element region may also include functions such as polyadenylation regions or other functions, for example, chains to facilitate transcription.

Among the yeasts which can be used in the practice of the invention are, in particular, the genera Hanensula, Pichia, Kluveromyces, Schizosaccharomyces, Candida and Saccharomyces. A preferred host from this group is Saccharomises cerevisiae.

Useful promoters include the copper inducible (CUP1) promoter, glycolytic gene promoters such as TDH3, PGK and ADH, and PHO5 and ARG3 promoters, which have been described in Miyanohara et al., Proc. Natl. Acad. Sci., USA, 80: 1 (1983), Mellor et al., Gene, 24: 1 (1983), Hitzeman et al., Science 219: 620 (1983), Cabezon et al., Proc. Natl. Acad. Sci. USA 81: 6594 (1984).

The practice of the invention will be explained by the following examples.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

i) Expression of modified gp160 in CV-1 cells

The HIV env gene of the molecular clone BH10 was mutagenized to avoid cleavage of the precursor.

To this end, the lys-ala-lys-arg and arg-glu-lys-arg cleavage sites present in these chains were modified by targeted mutagenesis to produce lys / arg and glu / arg.

cleavage site 1 cleavage site 2 position 502 position 510

kloň BH10: AAG GCA ΑΑθ AGA AGA GTG GTG CAG AGA GAA AAA AGA LYS ALA LYS ARG ARG VAL VAL GLN ARG GLU LYS ARG mutated 1 1 1 string: AAG GCA GAG AGA AGA GTC GTG CAG AGA GAA GAA AGA LYS ALA GLU ARG ARG VAL VAL GLN ARG GLU GLU ARG

The complete mutagenized env gene (nucleotides 5902-8478) was cloned in a pGS20 transfer vector (Mackett MG, Smith L. and Moss B., (1984), J. Virology, 49: 875-864) for use in vaccinia virus and was transferred to this infectious virus by recombination. The plaque of the recombinant virus was then screened for expression of the env gene by EIA and the positive plaques were recloned twice in CV-1 cells. The production of uncleaved gp160 in these cell lines was further verified by RIPA following metabolic labeling of cells infected with recombinant virus. Surface antigen expression was confirmed by fluorescence labeling of intact cell surface using anti-gp120 antibodies.

ii) Purification of the modified gp160 from the cells

The HIV gp160 envelope protein of step i) was purified in three steps. The host cells were disrupted and the antigen was extracted with a wetting agent and two affinity chromatography steps were used. The entire purification was carried out at 4 ° C.

Step 1: Disruption of CV-1 cells and extraction of gp160 antigen

After thawing, the suspension of cells and carrier microspheres, corresponding to 20 liters of culture, are centrifuged at 2200 g for 15 minutes and the supernatant discarded. The precipitate is resuspended in 1 liter of 30 nM tris / HCl pH 8, supplemented with 150 mM NaCl, 1% polyethylene glycol 300 monodecyl ether (Decyl PEG) and 20 micrograms / ml aprotinin. Cells were disrupted for 1 hour on ice with occasional shaking by hand and then centrifuged at 11300 g for 20 minutes. After separating the supernatant, the pellet was washed again with 400 ml of the cell disruption buffer and then centrifuged at 11300 g for 20 minutes. Cell debris and carrier microspheres were discarded and the supernatants were pooled and used for further purification.

Step 2: Sepharose 4B Affinity Chromatography with Lentil Lectin

The lysate was chromatographed on a 2.5 x 20 cm column containing Sepharose 4B with Lentil Lectin (Pharmacia-LKB), equilibrated in 30 mM Tris / HCl pH 8 buffer supplemented with 150 mM NaCl and 0.1% Decyl PEG. After loading the lysate at a flow rate of 1 ml / min, the column was washed with 30 mM Tris / HCl pH 8 buffer supplemented with 1 M NaCl and 0.1% Decyl PEG. Then, the antigen from the column was eluted using 0.5 M methyl-alpha-D-mannopyranoside in the same buffer and fractions containing gp10 were pooled. The column was washed and eluted at a flow rate of 5.5 ml / min.

Step 3: Immunoaffinity Chromatography on 178.1-Trisacryl

The monoclonal antibody 178.1 against gplo0 described in published patent application WO 90/06358 was purified from ascites fluid on a protein-G-Sepharose column (Pharmacia-LKB) and then the material bound to Trisacryl activated by glutaraldehyde (IBF) according to the manufacturer's instructions . Thereafter, the anti-epitope antibody on the gp120 antigen group (loop Vj) was bound at 1.5 mg / ml gel.

The resin was placed in a 2.5 x 10 cm column and equilibrated in 30 mM tris / HCl pH 8 buffer supplemented with 150 mM NaCl and 0.1% Decyl PEG.

The eluate of Sepharose 4B with lentil lectin was applied to the column and repeatedly passed through the column overnight at a rate of 1 ml / min. The column was then washed at a flow rate of 3.3 ml / min with 20 column volumes of 30 mM tris / HCl pH 8 buffer supplemented with 1 M NaCl and 1% n-octyl-beta-D-glucopyranoside OGP. Finally, the antigen was eluted at the same flow rate and using 0.1 M citric acid buffer pH 3.3 supplemented with 1% OGP. The fractions were immediately neutralized by addition of 1 M tris / HCl pH buffer

8.8 and antigen-containing fractions were pooled.

(iii) Protein determination

Protein concentrations were determined according to the Bradford method (Bradford, 1976), using bovine sulfur albumin as standard.

iv) Determination of antigen

The amount of gp110 antigen was measured by self-sandwich ELISA using a sheep anti-gp41 antibody to bind the monoclonal antibody and a mouse anti-gp120 antibody as an indicator of the monoclonal antibody. Further detection was performed using classical biotinylated anti-mouse tissue antibody, streptavidin and peroxidase system.

(v) Polyacrylamide gel electrophoresis and method

Vestern blott

SDS-gel plate electrophoresis was performed using a 10% polyacrylamide gel according to the method of Laemmli (Laemmli, 1970). After migration, proteins were stained with silver after oxidation with periodic acid (Pas staining, Dubray et al., 1982).

Electrophoresis was performed in the presence and absence of a reducing agent. Protein bands were further identified by Western blot on nitrocellulose according to the method of Towbin (Towbin et al., 1979) as probes using antibodies against the gpl10 antigen or the host cell CV-1 or against vaccinia virus proteins.

(vi) Antibody detection 178.1

The presence of monoclonal antibody 178.1 in purified gp110 antigen was measured by ELISA. Antibodies were captured to goat antibody against mouse tissues, detection was performed with a biotinylated antibody against mouse tissues. Further detection was performed with the streptavidin-peroxidase system.

(vii) Chromatography of purified env gp110 using size sizing material

Purified gp110 antigen was chromatographed on a 7.5 x 300 mm TSK 4000 SV HPLC column at steady state in 0.2 M phosphate buffer pH 7 supplemented with 1% OGP. The flow rate was 0.75 ml / min, fractions were analyzed for antigen by ELISA.

(viii) Determination of purity

Purity was determined after each step of SDS-polyacrylamide gel electrophoresis purification under reducing conditions. Protein bands were visualized by PAS staining and the protein was further identified by Western blotting.

The results clearly showed that the antigen was free of detectable protein impurities after chromatography on an immunoaffinity column. However, the resulting product could be contaminated with a trace amount of monoclonal antibody released from the immunoaffinity column. For this reason, the content of monoclonal antibody 178.1 in pure gp110 was measured by ELISA, and the content of 0.004 to 0.02% of the total amount of protein in the obtained samples was demonstrated.

The purified antigen was analyzed for the presence of oligomeric forms of gp110. The formation of oligomers could reflect the structure of gp10 in a virus in which the envelope protein is arranged in the form of spikes on the surface of the virion. The presence of cysteine residues in the primary chain of the antigen allows the formation of (homo) -oligomers linked by disulfide bridges. This could be demonstrated by SDS-polyacrylamide gel electrophoresis in the presence and absence of beta-mercaptoethanol. In the absence of a reducing agent, the molecular weight of the antigen in the protein bands was greater than 160,000 even in the presence of a wetting agent SDS. However, when the antigen was boiled in the presence of a reducing agent, only a single band of protein of 160,000 molecular weight could be observed. In order to determine the size of the oligomeric structures, the antigen was analyzed by size exclusion HPLC. Calibration of the column with known molecular weight standards indicated that most gplso molecules were based on a column having a retention time corresponding to a molecular weight of 640,000. Thus, it is evident that in the presence of a wetting agent, but without the presence of a reducing agent, most gplso protein was grouped into tetrameric structures. .

ix) Analysis

The resulting product has an antigen / protein ratio of about 2, which corresponds to the antigen content as determined by ELISA in the cell lysate, i. 0.8 to 1.4 mg of antigen per liter of culture.

Example 2

Expression of modified gp110 in BHK-21 and purification of gp110 envelope antigen from cells grown in suspension

The HIV gp110 envelope protein expressed in BHK-21 cells in an analogous manner to Example 1 (i) using recombinant vaccinia virus was purified by the method of Example 1 (ii) except for some minor modifications in cell disruption.

Step 1: BHK-21 cell disruption and gp110 antigen extraction

After thawing, the 10 µl cell suspension was centrifuged at 2200 g for 15 minutes and the supernatant discarded. The cell pellet was resuspended in 50 ml of 30 nM tris / HCl pH 8 supplemented with 150 mM NaCl, 1% polyethylene glycol 300 monodecyl ether (Decyl PEG) and 20 micrograms / ml aprotinin. Cells were disrupted for 1 hour on ice with occasional manual shaking and then centrifuged for 15 minutes at 11300 g. After separating the supernatant, the pellet was washed with 20 ml of the cell disruption buffer and then centrifuged again at 11300 g for 15 minutes. The pellet was discarded and the supernatants were collected and used for subsequent purification.

Stages 2 and 3

Steps 2 and 3 and subsequent analysis were carried out in the same manner as in Example 1 (ii) - (ix).

Example 3

Production of vaccine gpl60 - 3D-MPL

3 (a) rgp10 O-aluminum hydroxide with 3D-MPL

Purified rgp10 (100 or 20 micrograms per dose) from vaccinia virus of Example 1 was adsorbed overnight at 4 ° C onto aluminum hydroxide (alum) in an amount corresponding to 0.5 micrograms of Al ^{2+ +} in 1 ml of buffer containing 150 mM NaCl and 10 mM phosphate pH 6.8. After overnight incubation, the mixture was centrifuged and the supernatant discarded. An equal volume of adsorption buffer containing 100 micrograms of 3D-MPL was then added to the alum-bound rgp10. It has been shown that more than 95% of rgp10 was adsorbed to aluminum hydroxide.

3 (b) RP-100 3D-MPL in an oil-in-water emulsion

The vehicle was prepared as follows: Pluronic L121 (BASF Vyandotte, New Jersey) at a concentration of 5% v / v and 10% aqualan (Aldrich) was added to saline with phosphate buffered saline PBS at 0.4% v / v of Tween 80. This mixture was then passed through a microfluidization apparatus (Model M110 Microfluidics Corp., Newton, Mass.) Ten times. The resulting emulsion contained only particles smaller than 1 micrometer. One volume of this emulsion is mixed with an equal volume of twice concentrated rgp10 (either 20 or 100 micrograms) and mixed briefly to ensure thorough mixing of the ingredients. 100 micrograms / ml 3D-MPL is then added to the oil-in-water emulsion rgp10. The resulting formulation contained 0.2% Tween 80, 2.5% Pluronic L121, 5% squalane, 100 micrograms of 3D-MPL and 100 or 20 micrograms of rgp10 in 1 ml of injectable vaccine material.

Example 4

Immunogenicity in guinea pigs

ELISA and neutralization titers

Five guinea pigs were immunized with three injections of 50 micrograms of modified gp160 of Example 1 in SAF-1 (Syntex adjuvant) described by Byars NE and Allison AC (1987) Vaccine 5, 223-227. The injections were administered at monthly intervals . Sera were tested two weeks and one month after secondary immunization and two weeks after tertiary immunization in guinea pigs. The results are summarized in Table 2.

An EIA enzymatic immunoassay using disrupted HIV-1 (IIIB) infected cells was used to determine the ELISA titer in antisera after the first and second booster injections. The assay used is very similar to that described in Moore J. P. et al., 1989, AIDS 3, 155, (63).

Neutralization assays on microplates are based on the detection of HIV gag antigen in indicator cells. SupT1 cells, described in Hecht et al., 1984, Science 226: 1445, are used as indicator cells. The viral inoculum is composed of cell-free HIV-1 (IIIB) supernatant from a lymphoid cell line. This supernatant is centrifuged at high speed to remove cells and cell debris, dispensed into 1 ml vials, and stored at -80 ° C until use. The serum samples to be examined are inactivated by heating to 56 ° C for 30 minutes before testing. The negative control consisted of a mixture of sera from unimmunized animals or animals given only the adjuvant, animals of the same species as the sera studied. For neutralization, 750 TCID 50 are incubated with a serum dilution series for one hour at 37 ° C. SupT1 cells were then added at 4 x 10 ⁴ cells for wells and incubated for 4 days at 37 ° C. The cytopathic effect is monitored microscopically, Triton X-100 at a final concentration of 1% is added to each well and the plate is frozen. The sandwich ELISA method was used to monitor the relative amount of viral antigen produced in culture. The plates are then coated with an anti-p55 monoclonal antibody. Samples with Triton X-100 are incubated in the plate and the presence of gag antigen visualized with a mixture of human biotinylated HIV-1 and IgG, followed by treatment with streptavidin peroxidase. The percent reduction in HIV-1 antigen production compared to the control is then evaluated for all serum dilutions. Using a curve corresponding to the data in non-linear least-squares analysis, it is possible to achieve extrapolation of that serum dilution, which leads to a 50% reduction in antigen production compared to the control if such reduction occurs.

Table 2 summarizes the neutralizing antibody titer after tertiary immunization.

The neutralization titers observed after the second booster injection, i.e. after tertiary immunization, are higher than those that can be detected in infected humans. The neutralization titer of these antisera against HIV IIIB isolate is on average four times higher than that observed for a group of five seropositive VHO reference sera according to McKeating et al., 1989, J. Gen. Virol. 70: 3326-3333. Neutralization of a series of HIV-1 isolates (cross-neutralization) was tested in the laboratory of Dr. Veissa (Chester Beatty Laboratories, U.K.) using a more limited condition neutralization assay that can be shown to be less sensitive and titer. The results, which are summarized in Table 3, were reproduced in three cases at two different blood donations and showed good cross-neutralization for different strains of HIV-1, including the African strain CBL-4, as shown in Table 3.

Guinea pig sera immunized with gp10 produced by vaccinia virus according to Example 1 (01-05) show good titers for cross-neutralization after tertiary immunization.

Due to the strong cross-neutralization response observed after three doses, vaccinia gplo10 may be considered suitable for potential use in HIV-1 vaccination.

Example 5

Investigation of the effect of various vaccines on immunogenicity of purified vaccinia HIV gplo0 (isolate IIB) in Rhesus monkeys

In these assays, the ability of various vaccines to enhance the immunogenicity of purified recombinant gp110, rp110 in Rhesus monkeys (Macaca mulatta) was evaluated. The adjuvants to be tested were aluminum hydroxide (Alhydrogel, Superfos, Denmark) in combination with 3D-MPL of Example 2b) (3D Monophosphoryl Lipid A, Ribi) or 3D-MPL in an oil-in-water emulsion of Example 2a).

Rhesus monkeys (Macaca mulatta) weighing 3.5 to 5 kg were randomly divided into seven groups of three or four animals per group.

Individual groups were immunized with different doses of rgp10 in three adjuvant formulations as follows:

Group 1 (4 monkeys):

100 micrograms of rgp10, adsorbed on a mixture of aluminum hydroxide and 3D-MPL.

Group 2 (4 monkeys):

micrograms of rgp10, adsorbed on a mixture of aluminum hydroxide and 3D-MPL.

Group 3 (4 monkeys):

100 micrograms of rgp10 and 3D-MPL in an oil-in-water emulsion.

Group 4 (4 monkeys):

micrograms of rgp10 and 3D-MPL in an oil-in-water emulsion.

1.5 Antigen and adjuvant compositions

All formulations containing rgp10 were prepared just prior to use, each injection being given in a volume of 1 ml.

Groups of Rhesus monkeys were injected intramuscularly into the trigeminal shoulder muscle with a dose of 1 ml of various gpplO preparations on days 0 to 35. Two weeks after the second immunization, animals were bled for antibody determination.

2.5 Method of evaluation

Sera collected from these animals two weeks after the second injection were subjected to ELISA and neutralization tests.

3.5 ELISA

A modification of the immunoassay described by Thiriart et al., 1989, J. Immunol. 148, 6:

832-836. A commercially available gp41 monospecific reagent (Biochrom) is used in this assay. The crude lysate of vaccinia gp110 infected BHK 21 cells was used as the antigen.

4.5 Neutralization test

The microplate neutralization assay is based on the detection of HIV gag antigen in indicator cells. SupT1 cells according to Hecht et al., 1984, Science 226: 1455 were used as indicator cells. The viral inoculum was composed of cell-free supernatant of a lymphoid cell line producing HIV-1 (IIIB). The supernatant was centrifuged at high speed to remove cells and cell debris, dispensed into 1 ml vials and stored at -80 ° C until subsequent use. The sera to be examined were inactivated at 56 ° C for 30 minutes prior to the assay. For neutralization, 750 TCID4 g are incubated with a serum dilution series for 1 hour at 37 ° C. The cytopathic effect is monitored microscopically, Triton X-100 is added to each well to a final concentration of 1% and the plate is frozen. A Sanchwich ELISA was used to demonstrate the relative amount of viral antigen produced in culture. Plates are coated with monoclonal anti-p 55 antibody. Troton X-100 treated samples are incubated on the plate and the presence of gag antigen visualized by the addition of human biotinylated HIV-1 and IgG, followed by a step using streptavidin peroxidase. Thereafter, the percent reduction in HIV-1 antigen production compared to controls is evaluated for all serum dilutions. Using a curve corresponding to the data in a linear regression analysis, the serum dilution is extrapolated to achieve a 50% reduction in antigen production compared to controls if such a concentration exists.

5.5 The results

Table 4 summarizes the ELISA results and neutralizing antibody titer after the second immunization. In general, the immunogenicity of HIV rgpl10 in 3D-MPL in an oil-in-water emulsion is very good. When animals were dosed with 20 micrograms of gp160, very good neutralizing antibody (NT) titers and ELISA titers were achieved in the group of animals given rgp10 in 3D-MPL in an oil-in-water suspension (group 4). These data confirm the very good adjuvant effect of 3D-MPL in an oil-in-water emulsion. The immunological response of animals immunized with gp160 adsorbed to Alum in the presence of 3D-MPL is very low, although some neutralizing antibodies are produced. HIV gp160 contains a hydrophobic group, which is believed to cause high affinity of the molecule for lipids. For this reason, a composition containing 3D-MPL in an oil-in-water emulsion seems to be a suitable environment for HIV rgp10 when it is delivered to the immune system.

Example 6

6.1. Perform an experiment

Two chimpanzees no. 1 and no. 2 were immunized with purified recombinant vaccinia gplo0 at a dose of 100 micrograms using the material of Example 1.

Two other chimpanzees no. 3 and no. 4 were immunized with 100 micrograms of purified recombinant gp110 expressed in Drosophila Schneider cells according to Culp et al., Biotechnology, 9: 173-177, 1991.

The recombinant proteins were processed into the vaccine together with 3D MPL at 100 micrograms per dose in an oil-in-water emulsion. The formulation contained 0.2% Tween 80, 2.5% Pluronic L121, 5% squalane, 100 micrograms for rgp10 or rgp120 in a 1 ml injection volume.

Animals were immunized intramuscularly in the lower limb at month 0, 1 and 2.

Animals were bled every two weeks to determine the antibody titer by ELISA and to determine the titer of neutralizing antibodies (the method of performing the ELISA is shown in Example 4.3), the HIV neutralizing antibody assay was little modified as described below. Delayed hypertensive response (DTH) was also evaluated as described below.

2.6 Induction of humoral immunity

As shown in Table 5, chimpanzees vaccinated with rgpl20 or rgpl20 were allowed together with adjuvant

3D MPL in oil-in-water emulsion observed high antibody titer in ELISA as well as high titer neutralizing antibodies after three immunizations. Neutralizing antibodies could be detected in three serum samples of the four samples examined after two injections and in all four samples after the next injection. An increase in neutralizing antibody titer was observed after the third immunization. Both ELISA and neutralizing antibody titers after the third immunization are very similar to those obtained in Rhesus monkeys immunized three times with the same rgp10-containing formulation.

3.6 Call DTH

DTH assays were performed two weeks after the third immunization. All injections were given into the abdomen in a volume of 100 microlitres per injection.

Two chimpanzees immunized with rgp10 and both chimpanzees immunized with rgp120 were skin tested using three different antigens (rgp10, rgD2t and tetanus toxoid) and two control buffers (PBS control buffer for rgD2t and control buffer for rgp10). Different doses of recombinant antigen were tested, namely 40, 20 or 10 micrograms for rgp10 and 20, 10 or 5 micrograms for rgD2t.

The DTH response was followed after an additional 24 hours by measuring skin thickness. The results are shown in FIG. 1 to 3.

A specific DTH response against tetanus toxoid and rgp10 was observed in animals immunized with rgp10 or rgp1020.

The results show that resources that contain 3D

MPLs in an oil-in-water emulsion are capable of eliciting a specific T-cell response, as shown in Table 6.

Thus, it can be concluded that the results obtained in chimpanzees clearly demonstrate that formulations that contain 3D MPL in an oil-in-water emulsion as an adjuvant significantly improve the humoral response, i.e. titer of neutralized antibodies, as well as cell-mediated immunological response (DTH). ) in primates.

Assay for neutralizing antibodies

The assay for neutralizing antibodies performed on microplates is based on a visual evaluation of CPE after induction of HIV-1 infection in indicator cells. SupT1 cells according to Hecht et al., 1984, Science 226: 1445 were used as indicator cells. The viral vaccine material consisted of a cell-free lymphoid cell line producing HIV-1 (IIIB). The supernatant was centrifuged at high speed to remove cells and cell debris, dispensed into 1 ml vials and stored at -80 ° C until use. The sera to be examined were inactivated at 56 ° C for 30 minutes prior to testing.

As a negative control, a mixture of sera collected from the same animals and prior to immunization was used. To perform the neutralization assay, 750 TCID 50 are incubated with the dilution of the serum samples to be examined for 1 hour at 37 ° C. SupT1 cells were then added at 2 x 10 6 / well and incubated for 4 days at 37 ° C. The cytopathic effect is assessed microscopically on day 4 and the neutralizing antibody titer is determined visually. The neutralizing antibody titer corresponds to the reciprocal of that dilution of the serum of interest which, when used, can achieve an 80% reduction in syncytia formation compared to control samples taken prior to immunization. The visually determined liters are further evaluated objectively on day 7 by measuring cell viability in each well using the MTT assay described by Pauwels et al., J. Virol. Methods 20: 309-321, 1988. The titers determined in this assay are the reciprocal of that serum dilution that provides 80% protection against CPE compared to uninfected cells. Titers determined visually or determined by MTT are very reproducible in the course of further tests, and, not shown, titers determined by MTT are two to four times lower than those determined visually.

Table 1

Purification of HIV gp10 after his Expression v CV-1 cells and BHK-21 Method of cleaning volume (Ml) total protein (Mg) protein extract (%) antigen yield (%) CV-1 cell disruption 1400 2000 100 100 Lentin lectin 350 100 5 80 immunoaffinity chromatography 60 8 0.4 65 BHK-21 cell disruption 1480 2688 100 100 Lentin lectin 495 ¹¹³ 4 79 immunoaffinity chromatography 60 7 0.3 58

(1) 20 liters of culture as starting material (2) 14 liters of culture (2.1 x ΙΟ ^ θ cells) as starting material.

Table 2

Immunogenicity of gp110 in guinea pigs

H1V EIA titer (1) neutralization titer (111B) after II to III to II to III antigen guinea pig dose day 15 1 month day 15 day 15 1 month prostr. no.

gp10 vaccine

Ex. 1 I 50pg SAF-1 01 21653 20327 158589 183 <100 2207 02 22348 16694 179971 165 117 1930 03 25360 20327 151254 445 394 6630 04 35905 26175 161124 353 195 2034 05 ND 8658 77884 133 <100 677 GMT 25764 17330 140203 229 117 2079

(1) Inverted serum dilution value that provides an O.D of 0.5.

Table 3

Illb homology assay heterology assay SF2 MN RF CBL-4

01 02/05/90 80 10 / <10 - - - 02 02/05/90 <40 80 10 - 10 03 02/05/90 80 20 - - 10 04 02/05/90 40 10 - - 10 05 02/05/90 80 40 10 - 10 01 14/05/90 40 20 - - 10 02 14/05/90 80 20 - - 10 03 14/05/90 160 20 10 - 10 / <10 04 14/05/90 20 10 - - 40 05 14/05/90 40 40 0 / <10 - 10 positive u man ^ 160 320 80 160 80 negative u Makeover - - - - -

A positive neutralizing effect means a 90% inhibition of syncitia formation in visual assessment.

Table 3 compares the neutralizing effect of guinea pig serum against gp160 to the homologous (IIIb) and heterologous isolators.

Table 4

Immunogenicity of recombinant vaccinia gp160 in Rhesus monkeys ELISA titer and neutralization titer

group adjuvant / dose den 15 identification 11 L 50% NT ^X ELISA ^XX middle titer 1 ALUM 3D IP 111 <100 576 MPL / 100 ug IP 123 <100 301 IP 151 <100 411 IP 167 <100 630 GMT 460 2 ALUM 3D IP 101 <100 1871 MPL / 20pg IP 103 <100 1641 IP 105 <100 1437 IP 196 <100 94 GMT 802 3 3D MPLOV IP 120 486 9043 ΙΟΟμβ IP 125 282 5537 IP 150 285 3981 IP 152 918 10629 GMT 435 6789 4 3D MPLOV IP 114 462 5413 ² θμδ IP 117 576 5220 IP 118 <100 2529 IP 197 804 5268 GMT 322 4404

It corresponds to the inverted serum dilution value that can provide a 50% reduction in antigen production compared to control.

x ^xx Corresponds to the reciprocal of the serum dilution at which an absorbance of 50% of the maximum value can be achieved.

GMT = geometric mean titer.

3D-MPLOV = 3D-MPL in an oil-in-water emulsion.

Table 5

Antibodies to HIV in chimpanzees after vaccination with rgp10 and rgp120

vaccine chimpanzee po I 28 days titer po II po III 14 days 28 days titer 14 days 28 days neutral titer ELISA titer neutral. titer ELISA titer neutral. titer rgpl0 (100 gg) 1 <50 2592 <50 / 50 / 16509 200 400 3D MPL I / O 2 <50 9884 100 100 15665 200 / 400 rgpl10 (100 pg) 3 <50 3059 100 100 22679 800 800 3D MPL I / O 4 <50 506 <50 / 50 3917 200 200 /

Titer ELISA: corresponds to the reciprocal of the serum dilution that achieves 50% absorbance compared to maximum absorbance (mean titer).

Neutralization titer: corresponds to the reciprocal of the serum dilution value, which gives 80% protection against the cytopathic effect of HIV-1 IIIB isolate.

The slope means that the titer of neutralizing antibodies lies between two successive serum dilutions. For example, 200 means that the titer is between 200 and 400.

o / w: oil-in-water emulsion.

Table 6

Antibody generation

HSV in vaccinated chimpanzees after II after 111

14 days 28 days 14 days 28 days chimpanzee vaccine ELISA neutral. ELISA neutr. ELISA neutral. ELISA neutral. titer titer titer titer titer titer titer titer

<100 µg (100 µg) 1

3D MPLs o / w <100 <50

Titer ELISA: corresponds to the reciprocal of the serum dilution that achieves 50% absorbance compared to maximum absorbance (mean titer).

Neutralization titer: corresponds to the inverted serum dilution value that provides 80% protection against the cytopathic effect of HSV2.

Claims (30)

PATENT CLAIMS 1. HIV gp110 derivatives modified to contain independently at positions 502 and 510 an amino acid different from nu or arginine lyses. Derivatives according to claim 1, wherein the amino acid residues at positions 502 and 510 are selected from the group consisting of histidine, threonine, serine, asparagine, aspartic acid, glutamine and glutamic acid. C Derivatives according to claim 1 or 2, wherein the amino acid residue at position 502 and 510 is a glutamic acid residue. The oligomer or dimer of HIV gp10 derivatives according to any one of claims 1 to 3. The oligomer of claim 4, wherein the relative molecular weight is about 640,000. The HIV gp100 derivative or the oligomer or dimer of claims 1 to 6, in substantially pure form. A vaccine comprising a protein derivative or oligomer according to claims 1 to 6 together with a pharmaceutically acceptable carrier. 8. A pharmaceutical composition comprising a protein derivative or oligomer according to claims 1-6 together with a pharmaceutical carrier. 9. A vaccine or pharmaceutical composition comprising gp110 or an immunological derivative thereof and 3D Monophosphoryl lipid A adjuvant together with a suitable carrier. The vaccine or pharmaceutical composition according to claim 9, wherein the immunological derivative of gp10 is a protein or oligomer according to any one of claims 1 to 8. Vaccine according to claim 9 or 10, characterized in that the carrier comprises an oil-in-water emulsion. The vaccine of claim 11, wherein the concentration of gp10 is in the range of 10 to 150 micrograms / ml. The HIV gp110 protein or oligomer thereof according to any one of claims 1 to 8, for use in medicine. A vaccine according to any one of claims 9 to 12 for use in medicine. Use of the HIV gp110 protein or oligomer thereof according to any one of claims 1 to 8 for the manufacture of a vaccine for the treatment of HIV-1 infection. Use according to claim 15 for the prophylaxis of HIV infection. A process for the production of a modified HIV gp110 protein according to any one of claims 1 to 3. characterized in that the DNA encoding the protein is expressed in a recombinant eukaryotic host cell and the modified protein is isolated. A DNA strand which is a coding strand for a modified protein according to any one of claims 1 to 3. 19. A method for producing a gp10-modified oligomer or diamond according to claim 4 or 5, wherein the modified protein is isolated from a th eukaryotic host and then purified according to purification conditions. the recombination is not reduced 20. A method for producing a vaccine comprising a gp110 immunologic derivative, 3D monophosphoryl lipid A tically acceptable by admixing 3D monophosphoryl lipid or a pharmaceutical carrier thereof, characterized by gp110 or an immunological derivative thereof, A and a carrier. 21. A method of providing an effective amount of a prophylaxis of a person at risk of infection by the treatment of such a person with a derivative according to any one of the claims Third A method indicating an effective amount of 12. prophylaxis of persons at risk of infection by administering to them a vaccine according to any one of the claims HIV, administered 1 to HIV, administered 9 to 23. A method of treating a person infected with HIV, which comprises administering to said person an effective amount of a derivative according to any one of claims 1 to 18. The vaccine or pharmaceutical composition of claim 9, wherein the immunological derivative is gp120. The vaccine of claim 24, wherein the carrier comprises an oil-in-water emulsion. The vaccine of claim 24 or 25, comprising gp120 at a concentration of 10 to 150 micrograms / ml. A vaccine according to any one of claims 24 to 26 for use in medicine. 28. The method of claim 20 wherein gp120 is added as an immunological derivative. 29. A method for the prophylaxis of persons at risk of HIV infection, comprising administering to said persons an effective amount of the vaccine of claims 24 to 26. 30. A method of treating a person infected. HIV, characterized in that an effective amount of the vaccine according to any one of claims 24 to 26 is administered to said persons.