AU622129B2

AU622129B2 - Vector for the expression of proteins of the hiv-2 virus, one of the casual agents of aids, cell culture infected or transformed by this vector, proteins obtained, vaccine and antibodies obtained

Info

Publication number: AU622129B2
Application number: AU21902/88A
Authority: AU
Inventors: Marc Alizon; Marc Girard; Bruno Guy; Marie-Paule Kieny; Luc Montagnier; Guy Rautmann
Original assignee: Transgene SA
Current assignee: Transgene SA; Institut Pasteur
Priority date: 1987-09-07
Filing date: 1988-09-07
Publication date: 1992-04-02
Anticipated expiration: 2008-09-07
Also published as: FR2620030B1; PT88430B; DK495188D0; EP0314534A1; DK495188A; PT88430A; JPH02448A; KR890004727A; FR2620030A1; AU2190288A

Description

4A)AUSTRAIAAL 622129 Form PATENTS ACT 1952 COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE Short Title: Int. CI: Application Number: Lodged: .**Gomplete Specification-Lodged: Accepted:

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Prority: *0 o° o o Lapsed: Published: Related Art: *0 S A Name of Applicant: Address of Applicant: Actual Inventor: TO BE COMPLETED BY APPLICANT TRANSGENE S.A. INSTITUT PASTEUR, a French Body Corporate and French National Institute, of 16 rue Henri Regnault, 92400 COURBEVOIE, FRANCE and 27 rue du Docteur-Roux, 75015 PARIS, FRANCE.

MARIE-PAULE KIENY, GUY RAUTMANN, BRUNO GUY, LUC MONTAGNIER, MARK ALIZON and MARC GIRARD i ti 't i' 1 Care of: COWIE, CARTER HENDY, Address for Service: Patent Attorneys, 71 Queens Road, Melbourne, Vic., 3004, Australia.

Complete Specification for the invention entitled: "VECTOR FOR THE EXPRESSION OF PROTEINS OF THE HIV-2 VIRUS, ONE OF THE CASUAL AGENTS OF AIDS, CELL COLTURE INFEC

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ED OR TRANSFORMED BY THIS VECIOR, PROTEINS OBTAINED, VACCINE AND ANTIBODIS OBTAINED" The following statement is a full description of this invention, including the best method of performing it known to -1- SNote: 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 bu inserted inside this form.

14599/78-L Printed by C. J. THOMPSON, Commonwealth Government Printer, Canberra l- i;-r l -ca-IL;

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0 0 S. S 0 L: 1 A- The present invention relates 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 Africa.

Recent estimates suggest that one or two million Americans may have been exposed to the AIDS virus. The individuals effected exhibit severe immunosuppression, and the disease is generally fatal.

The transmission of the disease most frequently takes place by sexual contact, although intravenous drug users also represent a high risk group; moreover, a large number of individuals have been infected with this virus after receiving contaminated blood or blood products.

15 The causal agent of this condition is a retrovirus. Many animal conditions have been attributed to retroviruses, but only recently has it been possible to describe retroviruses affecting man.

Whereas type I and II human T cell retroviruses 20 have been implicated as a causal agent of certain T cell leukemias in adults (HTLV: human T leukemia virus), the HIV virus (human immunodeficiency virus) is recognized to be th- agent responsible for AIDS.

The genome of the HIV-1 retrovirus has been characterized very completely (Wain-Hobson et al., 1985; Ratner et al., 1985; Muesing et al., 1985; Sanchez-Pescador et al., 1985), and information regarding the genomic sequence indicates a CLose relationship with the lentivirus group. Lentiviruses, the prototype of which is ovine Visna virus, are the agents of very slowly progressing diseases which typically show a prolonged incubation period. HIV and Visna'virus share many similarities, especially in their tropism for neurat tissue.

A virus related to the HIV family, but different from HIV-1, has recently been isolated from AIDS patients originating, above all, from West Africa. This virus, referred to as HIV-2, has been cloned and sequenced (Guyader et al., 1987); this information on the genomic sequence confirms, despite certain divergences, that HIV-1 _bl~r 1 I I -2and HIV-2 viruses belong to the same group and have the same type of genomic map and organization.

The number of patients contaminated with this new virus still appears to be small. It is nevertheless essential, even now, to envisage a vaccination against AIDS comprising the vaccinating proteins of HIV-1 and HIV-2 viruses, since the epidemiological importance of HIV-2 may come to equal that of HIV-1.

HIV-1 and HIV-2 both exhibit a pathogenic character, in distinction to HTLV-4 described in Patent WO 87/02,892.

But HIV-2 possesses proteins whose sequence differs from that of the proteins of HIV-1, so that individuals who are seropositive for HIV-2 possess antibodies which cannot react with certain HIV-1 proteins.

15 The sequence of HIV-2 reveals a genetic structure very similar to that of HIV-1. In effect, apart from the GAG, POL and ENV proteins, both viruses code for Q, F, R, TAT and ART proteins which are unique among the other retroviruses. HIV-2 codes, in addition, for a putative X protein.

The sequence of the env gene product reveals the characteristics expected of a transmembrane envelope glycoprotein. The precursor possesses a molecular weight in the region of 160 kDa and, as is the case for HIV-1, 25 is cleaved to 2 proteins of 120 and 40 kDa.

Antibodies raised against the gp160 ENV protein, and .its gp120, gp40 and gp32 cleavage products under certain conditions (regarding gp32, see Montagnier et al.

1987), are commonly detected in the serum of patients infected with HIV-2, and the ENV glycoprotein represents Ithe major surface antigen of the AIDS viruses.

The ENV protein thus appears to be a promising candidate for developing a vaccination strategy, because it is exposed at the surface of the virus. Thus, attention has been concentrated on this protein and on its coding sequence.

The gag gene codes for the internal structural proteins of the virion. The messenger derived from its transcription is translated in the form of a 57-kDa protein i; ::NEW"1 rj goes Goes sees 0 t 0*SS :0.

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0@ 3 (P55-57) and this precursor is then matured through the action of a specific protease encoded by the poL gene.

The first endoproteolytic cleavage generates a and 12-kDa (P12) protein, and the second cleavage then generates a 16-kDa (P16) and 26-kDa (P26) protein; the order of the reading frames being H 2 N-P16-P26-P12-COOH.

The NH 2 -terminal end of the P55, coincident with that of the P16, can, by analogy with the P18 of HIV-1, be acylated by myristic acid. This corroborates the hypothesis according to which the P16 can act as a Link between the genomic ribonucleic acid and the membrane of the nucleocapsid in the viral particle. The P26 is the major constituent of the capsid of the virion. The strongly basic character of the P12 suggests its inti- 15 mate linkage with the viral ribonucleic acids, as has been suggested for the P13 of HIV-1 (Wain-Hobson et al., 1985).

It is easy to detect antibodies directed towards the proteins encoded by the gag gene in the sera of individuals infected with HIV-2. This immune response reveals the strongly immunogenic character of these different proteins. It is also noted that, when an individual is seropositive for HIV-2, a majority of the antibodies is directed towards the P26. This observation suggests, as in the case of HIV-1, that the proteins encoded by the gag gene are one of the targets against which an immune response must be raised in order to provide for better protection against the viral infection.

Several publications emphasize, in effect, the importance of the structural proteins of the virions in the induction of the mechanisms of cellular type immunity.

Among these studies, mention should be made of the case of influenza virus, for which a CTL (cytotoxic T lymphocyte) reaction directed against the nucleoprotein (major component of the capsid of the virion) can take place against different subtypes of the virus, whereas there is no immune cross-reaction between subtypes for the antibodies induced by the surface glycoproteins (hemagglutinin and neuramini-dase). The primary structure of these i;i i"j i I B::n -4 glycoproteins shows variable regions in which the peptide sequences are specific to each subtype. This divergence is the feature responsible for the absence of immune cross-reaction. Such a divergence also exists between the different isolates of HIV; it is maximal for the sequences of the ENV glycoprotein (up to 25 but minimal (5 to 10 for those of the gag gene (Starcick et al., 1986). Thus, the use of the proteins encoded by the gag gene for stimulating an immune response against infection by HIV is one of the strategies adopted in the present invention.

The F protein of HIV-1 is a myristilated protein, and in some isolates it is phosphorylated by protein kinase C. It was shown, in addition, that the F protein regu- 15 Lated the expression of the T4 antigen, and could thus play an important part in the establishment and maintenance of the infection caused by HIV. The induction of an immune response with respect to the F antigen hence seems important. The primary structure of the F protein, de- 20 duced from the genomic sequence of HIV-2, shows substantial similarities with the equivalent protein of HIV-1.

There is, in particular, a potential myristilation site, essential for anchorage in the membrane, and a potential site for phosphorylation by protein kinase C at the N- 25 terminal end. In addition, substantial sequence homologies exist in the C-terminal portion, probably the portion most exposed to the immune system, the N-terminal portion being anchored in the membrane via hydrophobic myristic acid. It hence appears advantageous also to induce an immune response against the F protein of the HIV-2 virus.

The present invention is thus directed towards providing useful means for developing a vaccine against the HIV-2 virus. Throughout this patent application, HIV-2 virus is understood to designate both the virus as described in the publication by Guyader et al. (1987) and possible point mutants or partial deletions of this virus, j as well as related viruses, in particular the hybrid Sviruses different from HIV-1 that are capable of inducing Sir 1 1 1 i u- 1 om 10 RoAoZ CIA• 1 /V'Z 0 an AIDS in man, and the viruses capable of hybridizing with HIV-2. The typical HIV-2 virus corresponds to the plasmids mentioned in the examples below.

The invention relates, generally speaking, to a viral or plasmid vector containing a nucleotide sequence coding for one of the proteins of the HIV-2 virus, and capable of directing the expression of this protein in a eukaryotic or prokaryotic cell.

In other words, the subject of the invention is a viral or plasmid vector for the expression of a protein of the HIV-2 virus in a cell infected or transformed by this vector, and which contains at least: a portion of the genome of a heterologous virus or of a plasmid a nucleotide sequence coding for one of the proteins of the HIV-2 virus selected from: env, gag and F proteins as well as the elements providing for the expression of the selected protein in eukaryotic or prokaryotic cells.

Among viral vectors which are usable, heterologous viruses, that is to say viruses different from HIV-2, are naturally employed; poxviruses and viruses such as adenoviruses, herpes viruses and baculoviruses should be mentioned more especially.

It will nevertheless be preferable to use a portion of the genome of a poxvirus, and more especially a portion of the vaccinia virus (VV) genome.

Vaccinia virus is a double-stranded DNA virus which has been used very widely throughout the world for controlling and eradicating smallpox. Recent technical developments have permitted the development of this virus as a cloning vector (Panicali and Paoletti, 1982), and live recombinant viruses have made it possible to express foreign antigens and even to obtain immunizations against different viral or parasitic diseases.

Thus, several groups have demonstrated the use of recombinants of this type for expressing the influenza and hepatitis B antigen and the rabies glycoprotein, in order to immunize against these diseases (Smith et al., 1983; Panicali et al., 1983; Kieny et al., 1984). The mwspe#7633 91 11 26 6 expression of the env gene of HIV-1 in vaccinia virus has also been described by Chakrabarti et al., 1986; Hu et at., 1986 and Kieny et at., 1986.

The expression of a sequence coding for a foreign protein by the vaccinia virus (VV) necessarily involves three stages: 1) the coding sequence must be aligned with a VV promoter, and be inserted in a non-essential segment of the VV DNA, cloned into a suitable bacterial plasmid; 2) the VV DNA sequences situated on each side of the coding sequence must permit homologous recombinations between the plasmid and the viral genome in a receptor cell; a double reciprocal recombination event leads to a transfer of the DNA insert from the plasmid into the viral genome 15 in which it is propagated and expressed (Panicali et Paoletti, 1982; Mackett et al., 1982; Smith et al, 1983; oo*o Panicali et al., 1983); 3) the expression of the DNA sequence integrated in the 9':066 recombinant VV genome in a suitable cell.

20 Naturally, the use of this type of vector often involves a partial alteration of the genome of the vector virus.

The present invention relates more especially to a vector for the expression of a protein of the HIV-2 25 virus in a cell infected or transformed by this vector, S' in which the nucleotide sequence coding for one of the proteins of the HIV-2 virus is a nucleotide sequence coding for one of the envelope glycoproteins (gp) of the a HIV-2 virus.

It is appropriate to note that the envelope glycoproteins (gp) of the HIV-2 virus are 3 in number, designated by their mass in kDa, namely the gpl60, the and the gp40 (or gp32); the first, gp160, is, in fact, the precursor of the latter two proteins.

It is desirable to express these three proteins.

The first tests performed with a viral vector in which the gene coding for the whole ENV prote.in was cloned led to the proposal for modification of this gene in order to improve the immunogenicity of thev expression S- 7 products.

As in the case of HIV-1, a substantial release of the ENV protein into the culture supernatants (a release which takes place, probably in vivo, in the circu- Lating fluids), was observed. This may be due to a poor attachment of the protein in the cell membranes; it is known, in addition, that the presentation of the antigens at the surface of the cells is very important for the induction of an immune response with the vaccinia system.

It is hence proposed to modify the env gene so as to improve the anchorage of the glycoprotein in the cell membrane.

This is accomplished by modifying the env gene between the coding sequences for the gpl20 and for the 15 gp40 (or gp32), in order to abolish the sites of cleavage by proteases situated between the gp120 and the gp40, in *o ge"o particular by abolishing the KEKR site (amino acids 501-504).

The present invention is also directed towards i 20 the expression of the GAG proteins, and especially the p57, p26 and p16 proteins.

Its subject is hence also a viral or plasmid vector for the expression of a protein of the HIV-2 virus in a cell infected or transformed by this vector, in I 5 25 which the nucleotide sequence coding for one of the proteins of the HIV-2 virus is a nucleotide sequence coding for .at least one of the GAG proteins of the HIV-2 virus.

Finally, the present invention relates to the expression of the F gene. It thus relates more especially to a viral or plasmid vector for the expression of a protein of the HIV-2 virus in a cell infected or transformed by this vector, in which the nucleotide sequence coding for one of the proteins of the HIV-2 virus is a nucleotide sequence coding for the F protein of the HIV-2 virus.

To increase the immunogenicity of the F protein, it can be advantageous to anchor the latter in the cell membranes. According to a characteristic of the invention, E the sequence coding for the F protein is fused in phase

J

8 with an N-terminal anchorage sequence, such as that of the hemagglutinating (HA) protein of the measles virus.

Generally speaking, when the vaccinia virus is used for expressing the gene in quPstion, it is preferable that the gene is under the control of a vaccinia virus promoter, and the 7.5 K protein promoter is preferably chosen. In addition, the coding sequence is cloned into a non-essential gene of the vaccinia virus. In most cases, this will be the TK gene, which may optionally be used as a marker The present invention hence relates mainly to the use of viral or plasmid vectors for obtaining the proteins encoded by the env, gag and F genes of the HIV-2 virus, as well as their cleavage products, in cell cul- 15 tures. It also relates to cells which have been infected by a viral vector or transformed by a plasmid according to the invention, or alternatively which can contain the corresponding recombinant DNA; among these cells, mammalian cells such as human diploid cells, Vero cells or primary 20 cultures, should be mentioned more especially. It is naturally possible to envisage other types of cells, such as insect cells, yeasts or bacteria.

The proteins thereby obtained can be used after purification for the production of vaccines.

25 In particular, in the case of the ENV glycoprotein, it is possible to express a soluble gp160, that is to say bereft of a membrane anchorage region, and which may be collected in the supernatants of infected cells in order to make a "subunit vaccine".

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

It is advantageous to envisage the combined use of several vaccinating agents administered jointly or separately, in particular the vaccinating agents corresponding to the vectors expressing the ENV protein and one of the GAG proteins or the F protein.

It is also advantageous to use a vaccine jointly 9 employing protection against HIV-1 and HIV-2.

These vaccines may be obtained either by the use of the corresponding recombinant viruses described above, Live or inactivated, or alternatively by using the products of cultures of infected cells or alternatively certain components of these cultures.

The vaccines in question are usable according to known techniques, in particular with adjuvants that improve their immunogenicity for the products of the cell cultures.

The administration routes clearly depend on the type of vaccine chosen.

Finally, the present invention also relates to the antibodies raised against the proteins of HIV-2, ob- 15 tained by the infection of a living organism with a viral vector as described above and the recovery of the antibodies induced after the specified time.

The antigens obtained, like the corresponding antibodies, may be used in diagnostic kits enabling the 20 corresponding viral infection to be detected.

The recombinant proteins thereby obtained may be used in diagnostic kits for detecting the potential antibodies present in the blood of patients who have been in contact with the virus. These tests can be carried out 25 according to processes known to those versed in the art, for example by ELISA, RIPA or "Western blotting" (immunoblotting).

These proteins can also be used for the production of hybridomas and of monoclonal antibodies designed to detect the presence of virus in samples.

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

METHODS

Cloning Maniatis et al., 1982.

Enzymes Used according to the supplier's directions.

1 :;sl :I LL i Localized mutagenesis Method derived from Zoller and Smith, 1983.

Transfer into vaccinia Kieny et al., 1984.

Only difference: 143B human cells replace the LMTK cells.

Preparation of the stock virus "Germ free" chick primary cells are infected at 0.1 pfu/ cell for 4 days at a temperature of 37 0 C (medium MEM

NCS).

Purification of the virus A centrifugation of the above stock virus is performed for minutes at 2,500 r.p.m. (Sorvall rotor GSA). The supernatant is set aside. The pellet is taken up in an RBS buffer (10 mM Tris-HCL pH 7.4, 10 mM KCL, 1 mM MgCL 2 for 15 15 minutes at 40C. Grinding is performed in a potter, j followed by a centrifugation for 15 minutes at 2,500 r.p.m.

The supernatant is added to the previous supernatant and a second grinding is then performed in the same S manner.

20 All the supernatants are deposited on 10 ml of a 36 sucrose cushion (10 mM Tris pH A centrifugation is performed for 2 hours at 14,000 r.p.m. (Beckman s rotor SW28). The pellet is taken up, dispersed and replaced on a second identical cushion. The 2nd pellet is 25 taken up in 5 Am of PBS and Loaded onto a 20-40 sucrose gradient (10 mM Tris pH 8) (same rotor). A centrifugation is performed for 45 minutes at 12,000 r.p.m..

The band of virus is recovered; it is pelleted by centrifugation for 1 hour at 20,000 r.p.m. The pellet is taken up in 10 mM Tris pH 8.

Immunoprecipitations An infection of BHK21 cells (dishes 3 cm in diameter, 106 cells per dish, cultures in G-MEM 10 FCS) is performed at 0.2 pfu/cell for 18 hours. The medium is decanted and replaced by 1 mL of medium without methionine and 10 pL of C35S methionine (Amersham) per dish.

An excess of non-radioactive methionine is added after 2 hours.

S When the Labeling is complete, the following are 11 performed; scraping of the infected cells, centrifugation for 1 minute in an Eppendorf centrifuge, separation of the supernatant fractions and pellet, washing of the pellet once in PBS buffer, and then immunoprecipitation and gel electrophoresis (according to Lathe et at., 1980).

Western blotting This technique for detecting antibodies directed towards the proteins of the HIV-2 virus is derived from that described in the procedure for LAV-BLOTR Western blotting sold by Diagnostics Pasteur.

The examples below will enable other characteristics and advantages of the present invention to be demonstrated.

Example 1 Construction of the bacteriophage M13 carrying 15 the env sequence.

Plasmid pROD35, deposited at the Collection Nationale de Cultures de Microorganismes (National Collection a of Microorganism Cultures) of the Institut Pasteur, 28 *eas Rue du Docteur Roux 75015 PARIS, contains the 4.3 kb of 20 the right-hand end of the HIV-2 genome, and hence contains all the coding sequence of the env gene. (This plasmid bears the deposition number 1-633).

.1 The KpnI-KpnI restriction fragment (position 5304- 0 00 00 o 9243) is inserted in the KpnI site of the bacteriophage 25 M13TG131, in the orientation such that the direction of transcription is in the direction of transcription of the S-galactosidase gene carried by M13 (M13TG1162).

Example 2 Construction of the plasmid for transfer into vaccinia virus bearing the env gene.

The Kpnl site situated upstream from the coding sequence of env, which was used for the construction of the phage M13TG1162, is at a distance of 843 bp from the initiation ATG of env. It is hence necessary to create a restriction site in the vicinity of the ATG. This was carried out by means of the oligonucleotide: CATCATACTCACAGATCTGGTGTAGG 3' BgllII A BglII site is thus introduced into the bacteriophage M13TG1162, generating the phage M13TG1163. The 1 I i ,b ,V -12- BglII-BglII restriction fragment of M13TG1163 is then inserted into the BamHI site of plasmid pTG186POLY, which permits the transfer of the env gene into vaccinia virus (pTG2151).

Example 3 Elimination of the stop signal present in the coding sequence for the The coding sequence of the env gene of plasmid contains a translation stop codon at position 8304 (this is a feature of pLasmid pROD35, but other isolates also contain the stop signal). In effect, a nucleotide T at this position generates the codon TAG.

The sequence corresponding to this region has been accomplished on other clones, and this established that the nucleotide T must be replaced by a C. It is hence appro- 15 priate to mutate the stop codon in order to obtain the sequence coding for the whole env gene.

This is carried out by means of a localized mutagenesis using the oligonucleotide: ATGGATCTGCTGGATAT 3'

CTATT

S*

GAT

stop I non-homologous positions The bacteriophage obtained is referred to as M13TG1164.

S The BgLII-BglII restriction fragment of M13TG1164 is then introduced into the BamHI site of the plasmid pTG186POLY, to generate plasmid pTG2152.

Example 4 Construction of the plasmid carrying the uncleaved env gene.

The gpl20 protein and the gp40 protein are generated by the proteolytic cleavage of the gp160. The gp120 is rapidly released into the culture medium. It would hence be advantageous to obtain an uncleaved ENV protein.

In effect, soluble proteins are poorly i7iur nogenic in the vaccinia expression system.

To this end, the nucLeotide sequence of the env gene is modified in M13TG1164 in the portion corresponding

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13 to the cleavage site (position 7635-7648) by means of the following oligonucleotide: GAGCAGAGGAGTAGTTGATATCTTGTGTAGGTGCGAA 3'.

Mutagenesis permits the introduction of an EcoRV restriction site, which permits the identification of the mutant clones, and the new sequence is as follows: T K E K R original clone: CCT ACA AAA GAA AAA AGA TAC mutated: CCT ACA CAA GAT ATC AAC TAC T Q D Q N EcoRV The bacteriophage generated is referred to as M13TG1165.

The BglII restriction fragment is then cloned into the BamHI site of the transfer plasmid pTG186POLY, to generate the plasmid pTG2158.

Example 5 Construction of a bacteriophage M13 containing the coding sequence of the gag gene.

Plasmid pROD27-5', deposited at the Collection Nationale de Cultures Microorganismes (National Collection of Microorganism Cultures) of the Institut Pasteur, 28 Rue du Docteur Roux 75015 PARIS, contains an EcoRI restriction fragment which contains the left-hand end of 25 the HIV-2 virus genome. (This plasmid bears the deposition number 1-626).

The BglI-EcoRI restriction fragment (position 502-2658) is inserted between the BamHI and EcoRI sites of the bacteriophage M13TG131, after filling in the Bgll and BamHI ends with Klenow polymerase.

The phage M13T.1155, which contains all the sequence of the gag gene, is generated in this manner.

Example 6 Construction of a plasmid permitting the expression of the proteins encoded by the gag gene.

The BglII-EcoRI restriction fragment of the phage M13 TG1155 is cloned between the BamHI and EcoRI sites of the transfer plasmid pTG186POLY, generating pTG2112. The BglII site in M13TG1155 originates from the polylinker of the vector bacteriophage.

1 4 I 1 14 Example 7 Construction of a plasmid permitting the expression of the P26.

The PstI-EcoRI restriction fragment of the bacteriophage M13TG1155 is cloned between the PstI and EcoRI sites of the bacteriophage M13TG130. A Localized mutagenesis carried out with 2 oligonucleotides permits the simultaneous introduction of a BglII restriction site and an ATG codon at the 5' end (position 951) of the sequence coding for the P26, and an SstI restriction site and a STOP codon at the 3' end (position 1641). The oligonucleotides are the following: BglII M G P 5' GGAGGAAATTACAGATCTATAATGGGTCCAGTGCAACAT 3' ease -3 15 M Stop SstI 5' GCTAGATTAATGTGAGAGCTCCTGAAAGAGGTC 3' positions not homologous with the parent sequence.

20 The bacteriophage M13TG1158 is thus generated from the phage M13TG1157.

The BglII-SstI restriction fragment of M13TG1158 is then cloned between the BamHI and SstI sites of the transfer plasmid pTG186POLY (pTG2111).

Example 8 Construction of a plasmid permitting the expression of the P16.

In the bacteriophage M13TG1155, the BglII site is positioned downstream from the initiation ATG of the gag gene (and hence of the sequence coding for the P16).

It is hence necessary to carry out a localized mutagenesis in order to introduce a STOP codon and a restriction site at the 3' end of the sequence coding for the P16. This is achieved by means of the following Soligonucleotide:

(AGT)

CCGCCTACTGTTGAATTCAGTAATTTCCTCCC 3' EcoRI The bacteriophage M13TG1156 is thereby generated.

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15 The BglII-EcoRI restriction fragment of M13TG1156 is then inserted between the BamHI and EcoRI restriction sites of the transfer plasmid pTG186POLY, generating plasmid pTG2110.

Example 9 Construction of plasmid pTG1198 containing the F gene.

The sequence coding for the F protein of HIV-2 originates from a cDNA cloned into plasmid PSPE2 (Institut Pasteur, Paris). The 950-bp fragment obtained by cleavage using the enzyme PvuII and containing the F gene (nucLeotides 8429 to 9378) is cloned into the phage M13MP8, open at the Smal site. The phage obtained is M13TG1152. In order to create a BglII site upstream from *Soo the translation initiation ATG of the F gene, a mutagenesis was performed by means of the oligonucleotide I having the following sequence: 5' ACTCGCACCCATATTAGATCTAGGCTGTTCTAAGTC 3' g L BglII The resulting phage is M13TG1153. The BgLII-SalI fragment of M13TG1153, containing the coding sequence of the F gene, is cloned into a transfer plasmid, pTG186POLY, in order to accomplish the transfer into vaccinia virus as described above. The resulting plasmid is pTG1198.

Example 10 Construction of plasmid pTG2157, containing the F gene preceded by the sequence coding for the anchorage region of the HA protein of the measles virus.

Plasmid pTG1169 was deposited at the Collection Nationale de Cultures de Microorganismes (National Collection of Microorganism Cultures) of the Institut Pasteur on 3rd April 1987 under No. 1-657.

The PstI-BamHI fragment of plasmid pTG1169 (see French Patent 87/09,629) containing sequence coding for the hemagglutinin (HA) of the measles virus is cloned into the phage M13TG131 (same patent), opened at the same sites. The resulting phage is M13TG1159.

A BamHI site is created just downstream from the sequence coding for the hydrophobic anchorage region of the HA of measles in M13TG1159 using the oligonucLeotide: L 1 i.

-16- CTCTGCGGTGTAGATGGATCCCCGATGAAGTCTAAT 3' BamHI The resulting phage is M13TG1160.

The 200-base pair PstI-BamHI fragment of M13TG1160, containing the fragment of sequence coding for HA (hydrophobic region), is cloned into pTG186POLY, opened at the same sites. The resulting plasmid is pTG2155. This plasmid makes it possible to clone, into the BamHI site situated downstream from the anchorage region of HA, a foreign gene possessing in phase a BamHI or BglII site (see diagram) (in this case, the GGA or AGA codon is coding).

e *e Soso Pstl BamHI

ATG

pTG2155 anchorage region N-terminal fragment measle HA The BglII fragment of M13TG1153 (described above), containing the F gene of HIV-2, is cloned in the coding orientation into pTG2155, opened with BamHI. The resulting plasmid containing the F gene fused with the anchorage of HA is pTG2157.

'The coding sequence for the hybrid F protein is transferred into vaccinia virus. The recombinant virus thereby created is VV.TG.F.HIV-2.2157.

Example 11 Introduction of the genes coding for the proteins of HIV-2 into the genome of vaccinia virus and isolation of recombinant viruses.

The strategy described by Smith et al., (1983) is based on the genetic recombination which takes place in vivo between the homologous sequences of the genomes of the vaccinia virus, in the infected cell. This phenomenon i makes it possible to transfer a DNA fragment, cloned into a plasmid, to the viral genome. The use of the thymidine 1: kinase (TK) gene of vaccinia virus makes it possible not P i r t

L

17 only to produce the integration of the foreign DNA in the locus of this gene, but also to have a phenotypic marker for the selection of the recombinant viruses which have become TK.

The TK viruses may be selected by plating on a TK cell line, in the presence of (58UDR) (Mackett et al., 1982). A TK virus can replicate its DNA normally therein, and form visible plaques.

The vaccinia virus propagates in the cytoplasm of infected cells rather than in their nucleus. For this reason, it is not possible to make use of the host's machinery for DNA replication and transcription, and it is necessary that the virion should possess the components for the expression of its genome. Purified VV DNA is non- 15 infectious.

In order to generate the recombinants, it is necessary to perform simultaneously the cellular infection with the VV virion and a transfection with the cloned foreign DNA segment which carries a region of homology 20 with the vaccinia DNA. Nevertheless, the generation of 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 temperaturesensitive (ts) mutant of vaccinia, which is incapable of propagating at a non-permissive temperature of 39.5 0

C

(Drillien et Spehner, 1983), decreases the background con- S sisting of the non-recombinant viruses. When the cells are infected with a ts mutant under non-permissive conditions and transfected with the DNA of a wild-type virus, viraL multiplication will take place only in the cells which are competent for the transfection and in 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 fragment of vaccinia DNA is included in the transfection mixture, at the appropriate concentration, with the wild-type DNA, it is also possible to procure i its participation in the homologous recombination with i 1 I; i r 18 the vaccinia DNA, in the competent cells.

Monolayers of chick embryo fibroblast (CEF) primary cells are infected at 33 0 C with VV-Copenhagen ts7 (0.1 pfu/cell), and transfected with a calcium phosphate coprecipitate of the DNA of the wild-type VV-Copenhagen virus (50 ng/10 6 cells) and the recombinant plasmid ng/10 6 cells).

After incubation for 2 hours at a temperature of 330c, the cells are incubated for 48 hours at 39.5 0

C,

a temperature which does not permit the development 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 ug/ml). Various plaques of TK virus are obtained from these cells which have om 15 received the recombinant plasmid, while the control cultures without plasmid do not show visible plaques. The TK viruses are then subcloned by a second selection in the presence of 5 BUDR.

A double reciprocal recombination event between the transfer plasmids and the VV genome leads to the exchange of the TK gene carrying the insert with the TK gene of the virus, the recombinants thus 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 technique described by Southern (1975). The filter is then incubated with the plasmids used for the transfer, Labeled beforehand with the isotope 3 2 P. The filter is washed and autoradiographed. After development, the presence of fragments whose size bears witness to the transfer of the genes in question into the vaccinia genome is observed on the film.

For each of the plasmids, a recombinant virus was selected and designated VV.TG.HIV-2 with the same numbering as the plasmids. The following recombinant viruses were thus generated: VV.TG.HIV-2-2151 (ENV gpl20-gp32) VV.TG.HIV-2-21.52 (ENV gp120-gp40) i: ii -C~ 19 VV.TG.HIV-2-2158 (uncLeaved ENV) VV.TG.HIV-2-2112 (GAG) VV.TG.HIV-2-2111 (P26) VV.TG.HIV-2-2110 (P16) S VV.TG.HIV-2-1198 (F) VV.TG.HIV-2-2157 (membrane F) Example 12 Immunoprecipitation of the proteins synthesized by the recombinant virus VV.TG.F.HIV-2-1198.

In order to demonstrate the expression of the F gene of HIV-2 using the recombinant vaccinia virus, BHK21 rodent cells, which are cultured in G-MEM medium fetal calf serum, are cultured with said recombinant VV.TG.F.HIV-2-1198.

6 e A semi-confluent monolayer (10 cells) is in- 15 fected with 0.2 pfu/cell and incubated for 18 hours.

6geO The medium is then 'emoved, and either a medium having a low methionine content (1 mL for 106 cells) supplemented with 10 4l/m of 35 Slmethionine (5 mCi/ 300 ul), or 100 4Ci/ml of myristic acid labeled witK tritium (Amersham), in MEM medium, is added.

The cells are incubated at 37 0 C and the labeled

C.

Goose proteins are collected by centrifugation. After separation into pellet and supernatant, the proteins are incubated with a serum belonging to patients who are seropositive with respect to HIV-2 antigens.

goThe proteins that react with the serum are recovered by adsorption on a protein A-Sepharose resin, spread by electrophoresis on an SDS-polyacryamide gel and autoradiographed according to a technique described by Lathe et iL. (1980). Of 4 HIV-2(+) sera tested, only one enabled the F protein of HIV-2 to be specifically immunoprecipitated. The immunoprecipitates obtained are apparent in the autoradiograph of Figure 1, wherein: M labeling with C3H3 myristic acid 0 and P labeling with C (S supernatant (P cell pellet The moLecular weights are in kiLodaLtons.

These immunoprecipitates reveaL, in labeing with i~lL-- I_ _i i I 5 S~methionine, 2 specific proteins migrating at 3land 33 kDa, both in the cell pellet and in the culture supernatant. Labeling with tritiated myristic acid reveaLs only a single protein migrating at 31 kDa. The difference between the theoretical weight (28 kDa) and the observed weight of the F protein of HIV-2 had already been observed for the F protein of HIV-1. As in the Latter case, the Lower band corresponds to the myristyLated form.

0* 00 r 21 EXAMPLE 13 Production of the p16 of I11V2 by a t.coli clone The deletion of restriction fragment EaoRl containing the reading of' proteih p26 and p12 of GAG of M413TG1156 generates the 1413TG1991 plasmid.

The synthetic. oligonuclaotide ATACATCTCAATCBGCTACC-3' permits~ the introd-uction of one Irestriction site BglII immediately in Upstream of the *.:ATG initiation of the p16 by directed mutagdn6sis of H13TG1991. The restriction fragment BglII-EcoRI of *e~eM13TG1992 obtained after the mutagenesis is cloned in the expr-ession vector pr~okaryotic pTG959 digested by 0 the enzymes BamHI and 1BcoRI. The plasmid pTG3947 obtained C. .by this cloning is used to transfori, the clone E.coli TGE901 and it's possible to induce the production of the p16 by keeping the bacterial culture of a.

temperature of 42*C.

EXAMPLE 14 Production of the p2 6 of HIV2 byE.eol,i The synthetic oligonucleotide :0 CTAGATCTATCGCACCAGTGCAACAT-31 permits the introduction of a restriction Bite BglII and, methionine and alanine, amino-acids, at the final NH, end of the readinpa frame of the p26 of M13TG1158 to give the M13TG1994. A new mustagenesis with the synthetic oligonucl 'eotide TAATGTOAGAATTCCTGAAACA-3' positions a STOP codon and a site for the restriction enzyme EcoRI at the final COOII end. of the reading frame of the p26 to give the M13TG1993. The fragment BglII-Rcol containing the'teading frame of the p26 is cloned in the expression vector prokaryotic pTG959 treated by the enzymes EgI.Iland EcoRI to generate the plasmid pT(O3948. The clone E.coli TGE9O1 is transformed by this p1 asmid and the production of the p26 is induced in the bacteriums by keeping the culture to a temperature of 42*C.

7' 7 22 EXAMPLE Production of the p5 7 of HIV2 by E,.coli The oligonuciciotide TCGGCTACC-3' permits the introduction of a site for the restriction enzyme BgtI immediatly in upstream of the initiator ATG of the reading frame of the p57 of M13TG1155 by directed muta The M13TC1993 generated from this mutagenesis is troa ted by the enzymes BglIt EcoRI (partial digest). The restriction fragment containing the reading 0O B 1: frame of the p57 is clonod in the expression vector prokaryotic pTG959 treated by the onzymes Bg1IXl:I and EcoRI e g.

to give the plasnid pTG3949. 'The clone, Lr.coli TG~e9O1 is transformed by this pLasmid and the production of the p57 is induced by a bacteriums culture at 4200.

EXAMPLE 16 Expresaion of the protein Q in the vaccinia virus The restriction fragment BamHI-HindIII of plasmid pROD35 (Guyader and al., 1987) is cloned in the bacteriophage M13TG131 opened to the same sites. A site EcoRI is generated in upstream of the initiation site of the translation of the Q gene. This, is performued by easing the oligonucicotide TC1679 of sequence CTCCATAGTCTCGAATTCTcTTGGTTrTCC-3'. The fragment EcoRI obtained and containing the Q gene of HIV2 is sub-cloned in the plasmid pTG186, and in a second time transferd in the vaccinin virus according a previously described method (Kieny and al., 1984). The recombinant obtained is the VV.TG.QHIV2-3142. As we don't dcapOse actunly specifical antibodies of the Q protein of IIIV2, the integration of Q gone in the viral genuie has been tested by Southern Blot.

The obtained results show a correct integration.

Hc~,- 't -23-

REFERENCES

Chakrabarti, Robert-Guroff, Wong-Staal, Gallo, R.C. and Moss, B. Nature 320, 53540 (1986).

Orillien, Spehner, Virology 131, 385-393 (1983).

Guyader, Emerman, Sonigo, Clavel, Montagnier, L. and Alizon, M. Nature 326, 662-669 (1987).

Hu, Kosowski, S.G. and Dabrymple, J.M. Nature 320, 537-- 540 (1986).

Kieny, Lathe, Drillien, Spehner, Skory, S., Schmitt, Wiktor, Koprowski, H. and Lecocq, J.P Naue 1, 6-16(18) Kieny, Rautmann, Schmitt, Oott, Wain-Hobson, Alizon, Girard# Chamaret, Laurent, A., see**:Montagnier, L. and Lecocq, J.P. Biotechnology 4, 790-795 Lathe, Hirth, Dewilde, Harford, N. and Lecocq, J.P. Nature 284, 473-474 (1980).

Mackett, Smith, G.L. and Moss, B. Proc. Natl. Acad. Sci.

USA 79, 7415-7419 (1982).

S. S Montagnier, L. and Alizon, M. Ann. Inst. Pasteur 138, 3-12 Muesing, Smith, Cabradilla, Benton, C.V.o Lasky, L.A. and Capon, D.J. Nature 313, 450-458 (1985).

Panicali, D. and Paoletti, E. Proc. Natl. Acad. Sci. USA 79, 4927-4931 (1982).

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Nati. Acad. Sci. USA 80, 5364-5368.

Ratrier, Haseltine, Patarca, Livak, Starcich, EsJosephs, Doran, Rafaiski, Whitehorn, Baumeister, Ivanoff, Petterway Jr*, S*Rs, Pearson, Lautenbergerf 7.Aoo Papas, Ghrayeb, Chang, Gallo, R.C. and Wong-Staal, F. Nature 313, 277-284 (1985).

Sanchez-Pescador et al., Science 227, 484-492 (1985).

Smith, Mackett, moss, V. Nature 302, 490-495 (1983).

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USA 80, 7155-7159 (1983).

Starcich, Hahn, Shaw, Mc~eely, Modrow, Wolf, Parks, Parks, Josephs, S.F., Gallo, R.C. and 'Wong-Staal, F. Cell 45, G37-648 (1986).

Wain-Hobson, Sonigo, Danos, Cole, S. and Alizon, M. Cell 40, 9-17 (1985).

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Claims

1. A viral or plasmid vector for the expression of a protein of the HIV-2 virus in a cell infected or transformed by this vector, which contains at least: a portion of the genome of a heterologous virus or of a plasmid a nucleotide sequence coding for one of the proteins of the HIV-2 virus selected from env, gag and F proteins as well as the elements providing for the expression of the selected protein in eukaryotic or prokaryotic cells.

2. The vector as claimed in claim 1, in which the portion of the genome of a heterologous virus is a portion of the genome of a virus chosen from poxviruses, adenoviruses, herpes viruses and baculoviruses.

3. The vector as claimed in claim 2, in which the poxvirus is the vaccinia virus.

4. The vector as claimed in claims 1 to 3, which contains the nucleotide sequence which codes for an envelope glycoprotein of the HIV-2 virus (envelope gp). The vector as claimed in claim 4, in which the sequence coding for one of the envelope gp's is the sequence coding for the

6. The vector as claimed in claim 4, in which the sequence coding for one of the gp's is the sequence coding for the

7. The vector as claimed in claim 4, in which the sequence coding for one of the gp's is the sequence coding for the gp40 or gp32.

8. The vector as claimed in claims 4 to 6, in which the env gene has undergone a mutation in order to eliminate the site of cleavage by proteases between the gpl20 and the

9. The vector as claimed in claims 1 to 3, which contains the nucleotide sequence coding for at least one of the proteins encoded by the gag gene of the HIV-2 virus.

10. The vector as claimed in claim 9, in which the sequence coding for at least one of the proteins of the gag gene is the sequence coding for the P57, the P26 or the P16. F~ YV J:i i I i i: -:1 i F:i _iii1ZITo mwspe#7633 91 11 26 -I U~

26- 11. The vector as claimed in claims 1 to 3, which contains the nucleotide sequence coding for the F protein of the HIV-2 virus. 12. The vector as claimed in claim 11, in which the sequence coding for the F protein is fused in phase with an N-terminal anchorage sequence. 13. The vector a- claimed in one of claims 1 to 12, in which the DNA sequence coding for the protein or proteins of the HIV-2 virus is under the control of a vaccinia virus gene promoter. 14. The vector as claimed in one of claims 3 to 13, in which the promoter is a promoter of a vaccinia virus gene. S 10 15. The vector as claimed in one of claims 3 to 14, in which the DNA sequence 6 coding for the protein or proteins of the HIV-2 virus is under the control of the promoter of the gene for the 7.5 K protein of vaccinia. 16. The vector as claimed in one of claims 3 to 15, in which the sequence coding for the protein or proteins of the HIV-2 virus is cloned into the TK gene of vaccinia. 15 17. A recombinant DNA corresponding to a vector as claimed in one of claims 1 to 16. 6. 18. Cells infected or transformed by a viral or plasmid vector as claimed in one of claims 1 to 16, or by the DNA as claimed in claim 17. 19. A culture of mammalian cells infected by a viral vector as claimed in one of claims 1 to 16. A method for preparing proteins of the HIV-2 virus, wherein cells as claimed in one of claims 18 and 19 are cultured and wherein the proteins produced are recovered. 21. Proteins or envelope glycoproteins of the HIV-2 virus, obtained by carrying out the method as claimed in claim 22. A diagnostic kit containing a protein or glycoprotein as claimed in claim 21. 23. A vector according to any one of claims 1 to 16 substantially as hereinbefore LU jT mwspe#7633 9111 26 -27 described. 24. DNA according to claim 17 substantially as hereinbefore described. Cells according to claim 18 substantially as hereinbefore described. 26. A culture according to claim 19 substantially as hereinbefore described.

27. A method according to claim 20 substantially as hereinbefore described.

28. Protein according to claim 21 substantially as hereinbefore described.

29. A diagnostic kit according to claim 22 substantially as hereinbefore described. DATED this 26 November 1991 CARTER SLIITH BEADLE Fellows Institute of Patent Attorneys of Australia Patent Attorneys for the Applicant: TRANSGENE S.A. INSTITUT PASTEUR S Se S S V 0055 0 *s S OSS q .1. a ij i-J i i g!, i! L mwspe#7633 91 11 26