AU604791B2 - Viral vector and recombinant DNA coding, in particular, for the p25 protein of the virus that is a causal agent of aids, infected cell culture, protein obtained, vaccine and antibodies obtained - Google Patents

Abstract

Viral vector comprising at least: - a part of the genome of a vector virus, - the complete gag gene or one of its subfragments, especially a gene coding for the p25 protein or a gene coding for the p18 protein of the HIV virus responsible for AIDS, - and the elements ensuring the expression of this protein in eukaryote cells in a culture.

Description

K

P/00/011 t 6U 4 7 PATENTS ACT 1952-1973 Form COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE jSE Class: Int. CI: Application Number: Lodged: **oComplete Specification-Lodged: o :*.Accepted: 0 Published: Priority: 0 00 0 Related Art: This documcnt contains thel umendments made under Section 49 and is correct for 040001 0 Name of Applicant: S t 'Address of Applicant: TO BE COMPLETED BY APPLICANT TRANSGENE S.A. INSTITUT PASTEUR, a French Body Corporate and Frdnch National Institute, of 16 rue, Henri Regnault, 92400 COURBEVOTE, FRANCE and 25 rue du Docteur-Roux, 75015 PARIS, FRANCE.

Actual Inventor: Address for Service: Marc Girard, Luc Montagnier Guy Rautmann MOORE% TgITM01 A CARTEik PATLI.-IT TflADI-WARf( ATT'ORNEYS 7 1 QUEENS ROAD MCLUOURNE, 3004. AUSTRALIA Complete Specification for the invention entitled: VIRAL VECTOR AIfl, RECOMBINANT DNA CODING, IN PARTICULAR, FOR THE p25 PROT'EIN OF THE VIRUS THAT IS A CA~.USAL AGENT OF AIDS, INFECI'ED CELL CULTURE, PROTIEIN OBTAINTED, VACCINE AND ANTIT3DIES OBTAINED" The following statement is a full description of this invention, including the best mothod of performing it knowni to -1 'Note: The description is to be typed in double spacing, pica type face, in an area not exceeding 250 mm in depth and 160 mm in width, on tough white paper of good quality and it is to be inserted inside this form.

11710/76-L 117 tO/76-L C. J. TisomrtsoN.Coomonwealtlr Government Printer, Canberra S- 1A The present invention relates more especially to a vaccine intended for the prevention of AIDS.

The acquired immune deficiency syndrome (AIDS) is a viral condition which is now of major importance in North America, Europe and Central Africa.

Recent estimates suggest that approximately one million Americans may have been exposed to the AIDS virus.

The individuals affected show severe immunosuppression and the disease is generally fatal.

The transmission of the disease most frequently takes place by sexual contact, although people using narcotics intraveneously also represent a high-risk group; furthermore, a large number of individuals have been infected with this virus after receiving contaminated blood 15 or blood products.

The causal agent of this condition is a retrovirus.

S, Many animal conditions have been attributed to retroviruses, but only recently has it been possible to describe human retroviruses.

20 Whereas types I and II human T-cell retroviruses (HTLV: human T leukemia virus) have been identified as the causal agent of certain T-cell leukemias in adults, the retrovirus associated with lymphadenopathy (LAV), also known as HTLV-III virus or AIDS-related virus (ARV) and recently S 25 dubbed HIV-1 (standing for Human Immunodeficiency Virus), is generally recognized as the agent responsible for AIDS.

The genome of several isolates of HIV has been characterized very completely (Wain-Hobson et al., 1985; Ratner et al., 1985; Muesing et al., 1985; Sanchez-Pescador et al., 1985) and the similarities in the genetic organization to visna virus, the prototype of the Lenti- S~ virinae, suggest a relationship with this group of retroviruses.

The Lentivirinae are agents of slowly progressing persistent diseases which possess a prolonged incubation period. Furthermore like visna virus, HIV shows some tropism for the nervous tissue of the brain.

As well as the three genes commonly found in all retroviruses, an designated gag, pol and env, the HIV ~~lll I-lll"~ _Ci a? 1- 2 virus genone possesses at Least 4 additional genes: Q or sor, TAT, ART, and F or 3'orf (Rosen et al., 1986; Fisher et aL., 1986).

The gag gene codes for the structural proteins of the virion. The messenger derived from its transcription is translated in the form of a pro'tein of 55,000 daltons and this precursor is then matured through the action of a specific protease encoded by the poL gene.

The first endoprotolytic cleavage generates a protein of 40,000 daltons (p 4 0) and one of 15,000 daltons and the second cleevage then generates a protein of 18,000 daltons (p1 8 and one of 25,000 daLtons (p25); the order of the reading-frames being H 2 N-p18-p25-p15-COOH. The

NH

2 -termina.L end of the p55, coinciding with that S 15 of the p18, is acyLated by myristic acid, and this corroborates the theory according to which the p18 would act as a link between the genomic ribonucleic acid and the membrane in the viral particle. The p25 is the major constitutent of the capsid of the virion. The strongly S 20 basic nature of the p15 suggests its intimate linkage S, with the viral ribonucleic acids (Wain-Hobson et al., 1985).

The subject of the present invention is, generally speaking, a viral vector which contains at least: a portion of the genome of a vector virus, the complete gag gene or one of its subfragments, in particular a gene coding for the p25 protein or a gene Scoding for the p18 protein of the HIV virus, responsible *for AIDS, as well as the elements which provide for the expression of this protein in eukaryotic cells in culture.

In a particular embodiment of the invention, the portion of the genome of a virus can be a portion of the pox virus genome. The pox virus can be vaccinia virus.

The DNA coding sequence can be under the control of a promoter of a pox virus gene.

The promoter can be a promoter of the vaccinia gene.

The DNA coding sequence can be under the control of a promoter of the gene for the 7.5 K protein of vaccinia.

The coding sequence can be cloned into the TK gene of vaccinia.

The subject of the present invention is hence also a culture of eukaryotic cells infected with a viraL vector according to the invention, as well as a method for the preparation of protein of the virus responsible for AIDS, wherein eukaryotic cells infected with a viral vector according to the invention are cultured and wherein the protein produced is recovered.

The subject of the present invention is hence also i V: the protein of the virus responsible for AIDS, obtained Sby carrying out the above method. The protein may be, 15 for example, the p25 protein, but also the p18 protein.

j It is easy to detect antibodies directed towards the proteins encoded by the gag gene in the sera of patients suffering from AIDS, with the exception of the protein. This immune response demonstrates the strongly immunogenic nature of these different proteins in subjects infected with the HIV virus. It is also noted that, when an individual is seropositive, most of his anti-bodies are directed towards the p25, and the acute S phase of the disease must be awaited in order to observe a S 25 drop in the titer of the anti-p25 antibodies. This observation, establishing a parallel between the immune S\ t response and the course of the disease, points to the as one of the targets against which an immune response must be raised in order to obtain protection against the fatal outcome of the disease.

Several publications emphasize the importance of the structural proteins of the virions in inducing cellular type mechanisms of immunity. Among these studies, there should be mentioned the case of influenza virus, for which a CTL (cytotoxic T-lymphocyte) reaction directed towards the nucleoprotein (the major component of the capsid of the virion) of one viral subtype is capable also of protecting against another subtype, whereas there is no immune cross-reaction between subtypes for the I i i~ -4antibodies induced by the surface gLycoproteins (hemagglutinin and neuramidase). The primary structure of these glycoproteins possesses variable regions in which the peptide sequences are specific to each subtype. This divergence is responsible for the Lack of immune cross-reaction.

There is also a divergence of this kind between the different isolates of HIV; it is maximal for the sequences of the ENV glycoprotein (up to but minimal (3 to for those of the gag gene (Starcick et al., 1986). Thus, the use of the p25 protein encoded by the gag gene for stimulating an immune response against infection with HIV is the strategy adopted in the present invention.

In its preferred aspect, the present invention relates, in effect, to the expression of the p25 protein us- 15 ing vaccinia virus as vector. Recent technical developments have made it possible to manipulate the vaccinia virus genome and to use it as a vector for the expression of cloned genes. Live recombinant viruses have enabled foreign antigens to be expressed, and have even enabled immunizations to be obtained against different viral diseases (herpes, influenza, hepatitis B, and the like) or parasitic diseases (malaria) Mackett et al., 1986; VB. Moss, 1985).

The expression of a sequence coding for a foreign protein by vaccinia virus (VV) necessarily involves two S stages: S, 1) The coding sequence must be aligned with a VV promoter, and be inserted in a nonessential segment of the VV genome, cloned into a suitable bacterial plasmid; 2) the VV DNA sequences situated on both sides of the coding sequence must permit homologous recombinations between the plasmid and the viral genome; a double reciprocal recombination event leads to a transfer of the DNA insert from the plasmid into the viral genome in which it is propagated and expressed (Panicali et Paoletti, 1982; Mackett et al., 1982; Smith et al., 1983; Panicali et al., 1983).

Naturally, the use of this type of vector frequently involves a partial deletion of the vector virus 5 genome.

The present invention relates more especiaLLy to a viral vector which contains at Least: a portion of the genome of a vector virus, a gene coding for the p25 protein of the HIV virus, responsible for AIDS, as well as the elements which provide for the expression of this protein in cells.

The invention also relates to the recombinant DNAs corresponding to the said viral vectors.

Virus responsible for AIDS is understood to designate, in particular, the HIV virus as well as possible Spoint mutants or partial deletions of these viruses, and also the related viruses.

I In the portion corresponding to the genome of the vector virus (in distinction to the virus responsible for AIDS), the viral vectors may be formed from the genome of a virus of any origin. However, it is preferable to use a portion of the genome of a pox virus, and more especially a portion of the vaccinia genome.

The conditions necessary for the expression of a i heterologous protein in vaccinia virus have been recalled above.

Generally speaking, the gene in question, for example the p25 gene, will, in order to be expressed, have to be under the control of a promoter of a vaccinia gene; this promoter will generally be the 7.5K protein promoter.

Furthermore, the coding sequence will have to be cloned into a nonessential gene of vaccinia which may optionally serve as a marker gene. In most cases, this will be the 4 30 TK gene.

The present invention relates, in the first place, to the use of viral vectors for obtaining the protein encoded by the p25 gene of HIV virus in cell cultures.

The cells are hence, initially, mammalian cells which have been infected with a viral vector according to the invention, or alternatively which can contain the corresponding recombinant DNA; among these cells, human diploid cells, Vero cells and also primary cultures should be 6 mentioned'more especially. Naturally, it is possible to envisage other types of cells.

Proteins thereby obtained may be used after purification for the production of vaccines.

It is also possible tc envisage the direct use of the viral vectors according to the invention in order to perform a vaccination, the p25 protein then being produced in situ and in vivo.

Finally, the present invention also relates to the antibodies raised against the p2 5 proteins, these antibodies being obtained by infecting a Living organism with a viral vector as described above and recovering the Santibodies induced after a specified time.

S'The techniques employed for obtaining the protein and the cell cultures and the vaccination techniques are identical to those which are currently practiced with the known vaccines, and will not be described in detail.

The examples below will enable further characteristics and advantages of the present invention to be revealed.

The attached figures are as follows: iFigure 1: construction of plasmid M13TG1124, Figure 2: construction of plasmid M13TG1125, S the abbreviations used are as follows: p15 reading frame of the p15 protein of HIV, H p18 reading frame of the p18 protein of HIV, c reading frame of the p25 protein of HIV, i nucleotide sequences originating from HIV, x x x 1 x' reading frame of the p25 protein of HIV, location lof the directed mutagenis, A fgt. RI deletion of the EcoRI restriction fragment, H3,R1,R5,B1,P2 Figure of p25 protein Figure Figure 7 demonstration of the restriction sites used, restriction enzymes, respectively: H'indIII, EcoRI, EcoRV, BamHI, PvuII, 3: autoradiograph showing the production by the recombinant viruses (3A and 38), 4: detection of antibodies to 5: immunoprecipitation of p2 5 r yT i if

C

-i 8 Example 1 Construction of plasmid M13TG1123.

The p25 protein is derived from a p55 muLti-protein precursor; its corresponding nucleic acid sequence is hence devoid of the translation initiation and termination signals at the ends of the reading frame. Two treatments for directed mutagenesis are performed, one for introducing an ATG upstream, the other for placing'a stop codon downstream from the coding sequence.

Cloning and mutagenesis of the sequence corresponding to the COOH-terminal portion of the The HindIII (coordinate 1258 in Wain-Hobson et al., 1985) EcoRV (coordinate 2523) restriction fragment of plasmid pJ19-13 (Wain-Hobson et al., 1985) was cloned into the corresponding sites of the vector M13TG131 (Kieny et al., 1983) to form plasmid M13TG1120.

The termination of translation at the end of the reading frame of the p25 wil:l be induced by the introduction of a stop codon in phase, at the nucleotide having the coordinate 1425. The synthetic oligonucleotide TG668, 5'-GGCTCATGAATTCCTACAAAACTC-3', was used for carrying out this mutagenesis on plasmid M13TG1120. The screening of S* the clones obtained after the directed mutagenesis. carried out with the oligonucleotide TG668 labelled with the isotope P, enabled plasmid M13TG1121 to be isolated S(Figure The small EcoRI restriction fragment of this plasmid was deleted so as to create plasmid M13TG1122.

Cloning and mutagenesis cf the sequence corresponding to the NH 2 -terminal portion of the The HindIII restriction fragment (coordinates 631- 1258 in Wain-Hobson et at., 1985) of plasmid pJ19-17 was cloned into the HindIII site of plasmid M13TG1122 to form plasmid M13TG1123. The initiation of translation at the NH2-terminal end of the reading frame of the p25 is achieved by adding an initiation codon upstream from the nucleotide having the coordinate 732. The synthetic oligonucleotide TG626, 5'-CACTATAGGGCCCATGGTGCTGACC-3', used for this mutagenesis was designed so as to produce mutation also of the sequences in the vicinity of the

I

9 iniLiation ATG, in conformity with Kozak's rules (Kozak M., 1986). The screening of the clones obtained after the direct mutagenesis, carried out with the oligonucLeotide 32 TG626 labelled with the isotope P, enabled plasmid M13TG1124 to be isolated. The latter contains a reading frame which codes for a-p25 protein elongated at its NH 2 terminal end by two additional amino acids: a methionine foLLowed by a gLycine. The remainder of the protein sequence is absolutely identical to that of the p25 of the HIV virus.

Example 2 Construction of plasmid M13TG1125.

This plasmid differs from plasmid M13TG1120 only in the substitution of the second glycine codon by the aLanine codon at the NH 2 -terminaL end of the protein.

S 15 The stages of cloning and mutagenesis of the COOH-terminaL portion, as well as the cloning of the NH2-terminal portion, are identical to those describe in Example 1.

Mutagenesis of the NH2-terminal portion.

The synthetic oligonucLeotide TG694, GGTGCCATGGTGCTGACCTGGATCCTGTGTCC-3', was used for introducing a methionine codon followed by an alanine codon upstream from the first proline codon of the p25 of the i HIV virus. In addition, a BamHI restriction enzyme site Swas placed 17 nucleotides upstream from the ATG during the mutagenesis of plasmid M13TG1123. Screening of the clones with the oligonucleotide TG694 labelled with the radioactive isotope P enabled plasmid M13TG1125 to be isolated (see Figure 2).

Example 3 Construction of plasmid pTG2101 To obtain the expression of the p25 by the vaccinia vector, the coding sequence for this protein was placed under the control of the 7.5K gene promoter of vaccinia virus.

Plasmid M13TG1124 was cut with the restriction enzymes PvuII and EcoRI, and the DNA fragment coding for the p25 was introduced into plasmid pTG186POLY (French Patent 86/05,043), treated successively with the enzyme BamHI, DNA polymerase and the enzyme EcoRI. The clones obtained, after the action of T4 DNA ligase and transfection into bacteria, are analyzed by re'striction mapping;plasmid pTG2101 was selected.

Example 4 Construction of pLasmid pTG2102.

The p25 sequence of plasmid M13TG1125 was also placed under the control of the 7.5K gene promoter of vaccinia virus.

Plasmids M13TG1125 and pTG186POLY were cut with the restriction enzymes BamHI and EcoRI. The products of these reactions were mixed, subjected to the action of T4 DNA ligase and transfected into bacteria. The clones obtained were analyzed by restriction mapping and plasmid pTG2102 was selected.

SExample 5 Introduction of the p25 gene into the vaccinia S* virus genome and isolation of recombinant 15 viruses.

I The strategy described by Smith et al. (1983) rests on the genetic recombination which takes place in vivo between the homologous sequences of the vaccinia virus genomes in the infected cell. This phenomenon enables a DNA fragment cloned into a plasmid to be transferred into the viral genome. The use of the thymidine kinase (TK) gene of vaccinia virus not only enables foreign DNA to be integrated in the locus of this gene, but also makes available phenotypic marker for the selection of the recombinant viruses which have become TK The TK viruses may be selected by plating on Sa TK cell line, in the presence of (Mackett et al., 1982). A TK virus is capable of replicating its DNA normally thereon, and forming visible plaques.

Vaccinia virus reproduces in the cytoplasm of infected cells rather than in their nucleus. For this reason, it is not possible to make use of the machinery for replication and transcription of the host's DNA, and it is necessary for the virion to possess the components for the expression of its genome. Purified VV DNA is non-infectious.

In order to generate the recombinants, it is necessary to perform the cellular infection with VV .4J 11 virio.n simultaneously with a transfection with the cloned foreign DNA st ment which carries a region of homology with the vaccinia DNA. However, the generation of the recombinants is limited to the small proportion of celLs which are competent for transfection with DNA.

The use, as a live infectious virus, of a temperature-sensitive (ts) mutant of vaccinia which is incapable of reproduction at a non-permissive temperature of 39.5 0 C (Drillien et Spehner, 1983) decreases the background formed by the non-recombinant viruses. When the ceLLs are infected with a ts mutant under non-permissive conditions and transfected with the DNA of a wiLdtype virus, viral multiplication will take place only in the cells which are competent for transfection and in 15 which a recombination between the wild-type viral DNA and the genome of the ts virus has taken place; no virus will multiply in the other cells, in spite of the fact that they have been infected. If a recombinant plasmid containing a vaccinia DNA fragment, such as pTG2101 or pTG2102, is included in the transfection mixture, at the appropriate concentration, with the wild-type DNA, it is also possible to procure its participation in the homologous recombination with the vaccinia DNA, in the competent cells.

Monolayers of chick embryo fibroblasts (CEF) primary cells are infected at 33 0 C with VV-Copenhagen ts7 (0.1 pfu/cell) and transfected with a calcium phos- S phate coprecipitate of the DNA of the VV-Copenhagen wildtype virus (50 ng/10 6 cells) and the recombinant plasmid ng/106 cells).

After incubation for 2 hours at a temperature of 33°C, the cells are incubated for 48 hours at 39.5 0

C,

which temperature does not permit the growth of the ts virus. Dilutions of ts virus are used for infecting monolayers of 143B-TK- human cells at 37°C in the presence of 5BUDR (150 jg/ml). Various plaques of TK virus are obtained from these cells which have received the recombinant plasmid, while the control cultures without plasmid do not show visible plaques. The TK viruses are then subcloned by means of a second selection in the presence 12of A double reciprocal recombination event between the pTG2101 or pTG2102 plasmid and the VV genome Leads to the exchange of the TK gene carrying the insert with the TK gene of the virus, the recombinants thereby becoming TK The DNAs purified from the different TK recombinant viruses are digested with HindIII and subjected to agarose geL eLectrophoresis. The DNA fragments are transferred to a nitroceLLulose filter according to the techniques by Southern (1975). The filter is then incubated with plasmid pTG2101 or pTG21202, previously labelled with 32 the isotope P. The filter is washed and autoradiographed. After development, the presence of fragments whose size testifies to the transfer of the gene coding for the p25 into the vaccinia genome is observed on the film.

For each of plasmids pTG2101 and pTG2102, a recombinant virus was selected and designated, respectively, and Example 6 Synthesis of the p25 protein in cells infected with the recombinant vaccinia viruses.

The procedure described below is applied in identical fashion to the recombinant viruses VV.TG.25MA or A semi-confluent monolayer of BHK21 cells is infected at a multiplicity of 0.2 pfu/cell. The infection is carried out with the recombinant vaccinia virus in a G-MEM culture medium supplemented with 5% of fetal calf serum. After incubation at 37 0 C for 18 hours, the culture medium is substituted with a medium containing methionine labelled with the radioactive isotope "i As a general rule, 10 4l of 3 5 Slmethionine mCi/300 Il) are added to 1 ml of culture medium to obtain efficient labelling of the proteins. After further incubation at 37°C, the culture incubation at 37°C, the culture supernatant is separated from the cells by centrifugation. The two fractions are incubated separately with the serum of a patient suffering from AIDS, and then with protein A-Sepharose. The immunoglobulin acc~ 13 fraction adsorbed on the protein A-Sepharose is then separated by centrifugation and this material is subjected to SDS-polyacrylamide gel electrophoresis followed by autoradiography.

Figure 3 shows the results obtained with the recombinant viruses VV.TG.25MG and VV.TG.25MA. In Figure 3A, which shows the results obtained with the cell pellets, Lanes 1, 2, 3, 4, 5 correspond to the recombinant virus 4, 6, 10 and 24 hours after labelling with 35 S-Met); lanes 6, 7, 8, 9, 10 correspond to the recombinant virvs VV.TG.25MG 4, 6, 10 and 24 hours after labelling); and lane M corresponds to a molecular weight marker (in daltons). Figure 38 shows the results obtained with corresponding culture supernatants.

These res'lts clearLy demonstrate that a protein having a molecular weight of approximately 25,000 daltons has been immunoprecipitated by the serum of a patient suffering .rom AIDS. This protein is present in the BHK cells infected with the recombinant vaccinia viruses, but absent on infection with the wild-type vaccinia virus (result not shown in the figure). The recombinant vaccinia viruses VV.TG.25MG and VV.TG.25MA hence provide for the synthesis of a protein having a structure similar to that of the p25 HIV, since it is fully recognized by the patient's serum.

Example 7 Production of anti-p25 antibodies in mice vaccinated with the recombinant vaccinia viruses i VV.TG.25MA and 4 seven-week-old male C57/B1 mice were inoculated by scarifying the base of the tail with 4 x 10 pfu for each of the recombinant vaccinia viruses VV.TG.25MG and After 4 weeks, a blood sample was drawn from the mice and a second inoculation was carried out as described above. 3 weeks later, the animals were sacrificed and their blood was removed in toto by cardiac puncture.

The capacity of these sera to recognize the HIV proteins was assessed by the immunoblotting technique.

This technique rests on the incubation of sera with a nitrocellulose sheet onto which the HIV proteins have 14 14 previously been adsorbed after SDS-polyacrylamide gel electrophoresis.

The results obtained with the first samples of sera, drawn one month after the vaccinations, show that the sera of mice inoculated with the recombinant vaccinia viruses contain antibodies capable of specifically recognizing the p25 protein of HIV. In addition, some sera are also capable of recognizing the common epitopes between the p25 and its p40 precursor, as attested by the presence of a band at the p40 level. These antibodies are not present in the sera of mice vaccinated with a wildtype vaccinia virus (results not shown in the figure).

1 rFigure 4 shows the results obtained with the sera drawn 25 days after the booster injection. Lanes 1, 2, 3, 4 correspond to the serum of 4 mice vaccinated with the recombinant virus VV.TG.25MA, and lanes 5 and 6 correspond to the serum of 2 mice vaccinated with the recombinant virus VV.TG.25MG. The sera of the 2 mice vaccinated with the recombinant vaccinia virus VV.TG.25MG which are not shown in Figure 4 give similar results. Lane T cor- 'responds to the serum of a patient suffering from AIDS.

,K The presence in Figure 4 of bands corresponding to the p25, to the p40 precursor and to the doublet characteristic of the p55 precursor demonstrates a strong immunological response in the mice vaccinated with the recombinant vaccinia viruses VV.TG.25MA and The results shown in Figure 4 clearly demonstrate that the vaccination of mice with the recombinants VV.TG.

and VV.TG.25MG which produce the p25 protein of HIV induces a specific immune response in these animals. In addition, the antibodies synthesized also recognize the and p40 precursors, thereby confirming the high specificity of the immune response in the vaccinated mice.

The recombinant protein thereby obtained may be used in diagnostic kits for detecting potential antibodies present in blood of patients who have been in contact with the virus. These tests may be carried out according to processes known to those versed in the art, for example ELISA, RIPA and Western blotting (immunoblotting).

151 15 This protein may also be used for the production of hybridomas and monoclonal antibodies intended for detecting the presence of virus in samples.

The following plasmids were deposited on 28th November 1986 at the Collection Nationale de Cultures de Microorganismes (National Collection of Microorganism Cultures) of the INSTITUT PASTEUR, 28 rue du Docteur-Roux, 75724 PARIS CEDEX E. coli 1106 pTG2101 under no. 1-631 E. coli 1106 pTG2102 under no. 1-632.

Example 8 Immunoprecipitation of the p25 protein of HIV-1 j I by antibodies of l inoculated with the recom- S, binant vaccinia virus A preparation of HIV-1 virus in which proteins had been radiolabelled by incorporation of 3 5 Scysteine was I used as a source of antigens. The latter were immunoprecipitated with serum of C57/bl mice inoculated with the recombinant vaccinia virus according to the protocol described in Example 7.

'50 Il of viral preparation were incubated with pl of serum in RIPA buffer overnight at 40C, and then brought into contact with protein A-Sepharose. The immunoglobulin fraction adsorbed onto this matrix was then sepa- 25 rated from the reaction medium by centrifugation. The antigen-antibody complexes were subjected to SDS-polyi acrylamide gel electrophoresis followed by fluorography.

Figure 5 shows the results obtained. Lanes a, b and c correspond, respectively, to immunoprecipitations carried out with the mouse serum and two different human sera. Autoradiography reveals that the p25 protein is the only protein of the HIV-1 that is immunoprecipitated by the serum of a mouse inoculated with the recombinant vaccinia virus VV.TG.25MA (its position is indicated by an arrow).

These mouse antibodies are hence capable of recognizing not only the p25 bound to nitrocellulose (on analysis by the Western blotting technique see Example but also the native p25 as found in the viral I 1 -1 S- 16particle.

This property demonstrates the value of this recombinant virus VV.TG.25MA, since it induces, in mice, the appearance of antibodies capable of recognizing the sequential an conformational epitopes of the p25 protein of HIV-1.

Example 9 Reconstruction of the complete gag gene and insertion into vaccinia virus.

Phage M13TG1120 contains the HindIII-EcoRV fragment of plasmid pJ19-13 (nucleotides 1258 to 2523) and codes for the C-terminal end of the p25 and the whole of p13. This phage has the following structure: PstI HindIII EcoRV SailI HIV Stra BglII SstI BarfHI EcoRI The remainder of the gag gene is contained in the 2 mids pJ19-1 and pJ19-17 (HindIII fragments).

The first stage consists in adding the portion coding for the p18 upstream from the HindIII-EcoRV fragment of M13TG1120.

Plasmid pJ19-1 contains the sequences of the genome of the HIV-1 virus between nucleotides 77 and

BRU

631 in plasmid pUC18: SstI (224) AccI H I V Hind III (77) HindIII (631) t I The direction of transcription of the DNA runs from SstI to AccI, and the codon corresponding to the gag initiation ATG is situated at position 335 (Wain-Hpbson, 1985).

The SstI-HindIII fragment is hence excised from plasmid pJ19-1 and inserted between BamHI and HindIII sites of bacteriophage M13TG1120 by means of the adaptor 5'-GATCAGCT-3', to generate M13TG191, whose structure is as follows: r.

-17- PstI HindIII EcoRV SalI Hl V HIV Smal BglII SstI BamHIo/SstIo EcoRI The following stage consists in inserting the HindIII central fragment (nucleotides 631-1258) of plasmid pJ19-17 into the HindIII site of M13TG191. This generates the bacteriophage M13TG192: HindIII HindIII RV PstI Smal

HIV

Z L /1 Sphi

I

BglII SstI BglII SalI p18 p 25 p13 Ec The SalI-EcoRI fragment of M13TG192 is then inserted between the SalI and EcoRI sites of plasmid pTG186POLY to generate plasmid pTG1144, in which the coding sequence of gag gene is placed under the control of the K promoter of vaccinia virus. This plasmid enables the gag expression block to be integrated, by recombination, into vaccinia virus.

Example 10 Introduction of a stop codon at the end of.

the reading frame of the p18.

The p18 terminates with the amino acids Glu Asn Tyr. A termination codon has hence to be introduced at the level of this sequence. For this purpose, a localized mutagenesis is performed on M13TG192 with the oligonucleotide: 5'-GCACTATAGACTAGTTTTGGCTG-3' to generate the following sequence (M13TG1154): Val Ser Gln Asn Tyr Pro Ile Val Gln GTC AGC CAA AAT TAC CCT ATA GTG CAG C AGC CAA AAC TAG TCT ATA GTG C Spe I Glu Asn Stop Pro r-i 18 The SalI-Sphl fragment of bacteriophage M13TG1154 is then cloned between the SalI and SphI sites of plasmid pTG186POLY to generate plasmid pTG1197. In this plasmid, the reading frame of the p18 is placed under the control of the 7.5 K promoter of vaccinia virus. This plasmid enables the expression block for the p18 to be integrated, by recombination, in vaccinia virus.

Example 11 Construction of a plasmid permitting the expression of a p18 fused at its N-terminal portion with the N-terminal hydrophobic peptide of the HA gene of measles virus.

The p18 protein is myristylated in vivo. It may hence be anchored in the cytoplasmic membrane via this fatty acid. In order to improve the presentation of this antigen, this anchoring can be replaced by a hydrophobic peptide, such as, for example, that of the hemagglutin (HA) gene of measles virus.

Plasmid pTG2155 (French patent 87/12,396) contains the coding sequence for the hydrophobic N-terminal peptide of the HA gen, downstream from the 7.5 K promoter of vaccinia virus. A BamHI site is situated after the I II sequence for the hydrophobic region and enables a foreign rI 'DNA to be integrated.

.A BglII site is introduced upstream from the initiation ATG of the p18 in order to enable the gene to be integrated in the BamHI site of plasmid pTG2155. This is accomplished by means of the oligonucleotide CACCCATAGATCTCCTTCTAG-3', mutating phage M13TG1154: t t 19 p18 GGCTAGAAGGAGAGAGTGGTGCGAGA... 3' 3' GATCTTCCTCTAGATACCCACGCTC BgLII The phage obtained is referred to as M13TG2161.

The DNA of the Latter is then excised with BglII and inserted into the BamHI site of plasmid pTG2155 to generate plasmid pTG2160, which enables the transfer into the vaccinia virus genome to take place. The corresponding recombinant virus will be referred to as VV.TG.2161.

Example 12 Expression of the p55 and p18 gag proteins in vaccinia virus.

The recombinant vaccinia viruses carrying the inserts described in the above examples, VV.TG.1144 and 1197, code, respectively, for the p55 and p18 proteins of the HIV-IBRU virus. These viruses were used for infecting cell lawns, as described in Example 6.

By immunoprecipitation, it is possible to demonstrate the production of the 2 proteins which migrate with the expected mobility after SDS-PAGE (polyacrylamide gel electrophoresis), and whose N-terminal ends are myristylated. The virus VV.TG.2161 enables an 23 unmyristylated 23-kD protein to be synthesized, as expected. Another form of the p18 migrating with an apparent mobility of 30 kD may be observed on the autoradiograph. This band does not correspond to a glycosylated form of the p18.

An immunofluorescence experiment on unfixed LTK mouse cells enables the p18 produced by the viruses VV.TG.1197 and 2161 to be visualized at the surface of the infected cells.

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Claims (15)

1. A viral vector, which contains at least: a portion of the pox virus genome, the complete gag gene or one of its subfragments hereinafter referred to as the DNA coding sequence, in particular a gene coding for the p25 protein or a gene coding for the p18 protein, of the HIV virus responsible for AIDS, as well as the elements which provide for the expression of this protein in eukaryotic cells in culture.

2. The viral vector as claimed in claim 1, in which the pox virus is vaccinia virus.

3. The viral vector as claimed in claim 1 or claim 2, in which the DNA coding sequence is under the control of a promoter of a pox virus gene.

4. The viral vector as claimed in claim 3, in which the promoter is a promoter of the vaccinia gene. The viral vector as claimed in claim 4, in which the DNA coding sequence is under the control of the promoter of the gene for the 7.5 K protein of vaccinia.

6. The viral vector as claimed in any one of the claims 3 to 5, in which the DNA coding sequence is cloned into the TK gene of vaccinia.

7. The viral vector as claimed in any one of the preceding claims, which contains at least: a portion of the pox virus genome, a gene coding for the p25 protein of the HIP virus responsible for AIDS, as well as the elements which provide for the expression of this protein in eukaryotic cells in culture.

8. The viral vector as claimed in claim 7, which is chosen from and VV.TG.25MA, each as hereinbefore defined.

9. The viral vector as claimed in any one of claims 1 to 6, which contains at least: a portion of the pox virus genome, a gene coding for the p18 protein of the HIV virus responsible for AIDS, as well as the elements which provide for the expression of this protein in eukaryotic cells in culture. The viral vector as claimed in claim 9, which is chosen from VV.TG. 1197 and 4VV.TG.2161, each as hereinbefore defined. 1 11. Recombinant DNA corresponding to the vectors as claimed in any one of J A% 23 claims 1 to

12. A culture of eukaryotic cells infected with a viral vector as claimed in any one of claims 1 to

13. A method for the preparation of protein of the virus responsible for AIDS, wherein cells are cultured as claimed in claim 12 and wherein the protein produced is recovered. 14 Protein of the virus responsible for AIDS, obtained by carrying out the method as claimed in claim 13. Ct 15. The protein as claimed in claim 14, which is the p25 protein. t tr

16. The protein as claimed in claim 14, which is the p18 protein. 0 t V i 17. The protein as claimed in claim 14, in which the p18 protein is fused at its N- terminal portion with the N-terminal hydrophobic peptide of the HA gene of measles I t I virus.

18. An immunogenic agent comprising the protein as claimed in claim 14.

19. A vaccine, which consists of a viral vector as claimed in any one of claims 1 to 10 and/or the protein as claimed in claim 14. An antibody corresponding to a protein of the virus responsible for AIDS, wherein a living organism is infected with a viral vector as claimed in any one of Sclaims 1 to 10 or wherein the former is inoculated with the protein as claimed in claim 14, and wherein the antibodies formed are recovered after a specified time.

21. A diagnostic agent comprising the protein as claimed in claim 14 or an F antibody as claimed in claim 20, labelled or otherwise.

22. A viral vector, recombinant DNA corresponding to said vector, cultures of eukaryotic cells infected with said vector, methods of preparation and protein produced therefrom, immunogenic agents, containing said protein, vaccines consisting of said protein and/or viral vector, antibodies and diagnostic agents substantially as hereinbefore described. DATED this 14th day of September, 1990. TRANSGENE S.A. INSTITUT PASTEUR.