FR2552776A1 - Vectors for expressing a antigenic rabies protein in eucaryotic cells and their application in the preparation of a vaccine - Google Patents

Abstract

The present invention relates to vectors for expressing a antigenic rabies protein in eucaryotic cells, characterised in that they comprise at least: - a DNA sequence 1 encoding the said antigenic rabies protein, and - elements for expressing this sequence in a eucaryotic cell. These vectors may be used for transforming eucaryotic cells so as to make them produce a antigenic rabies glycoprotein.

Description

 The present invention relates more particularly to the development of a rabies vaccine.

Rabies is a disease1 caused by the rabies virus, which presents in the form of a very serious encephalomyelitis which is almost always fatal in the absence of treatment before the onset of symptoms "
Rabies is a zoonosis. All warm-blooded animals, whether mammals or birds, are receptive. The main vector is generally constituted by biting mammals, whether domestic or wild.

 Effective preventive measures have made it possible to limit cases of human rabies, which are reduced to a few accidental cases in highly industrialized countries.

 However, rabies is still a very important disease in some countries of South America, Africa and Asia.

 The essential weapon in the fight against rabies could be prevention through vaccination.

 The vaccines currently used consist of inactivated viruses.

 However, to be reproduced industrially, these viruses must be cultured on media such as the brains of newborn mice, on bird embryos, on animal renal cells or on human diploid cells, after which they are completely inactivated. by deâ-propio lactone action for example.

 The use of inactivated vaccines always poses the problem of the risk of insufficient inactivation. Finally, the particular nature of the culture media leads to numerous difficulties at the industrial stage which mean that the rabies vaccine currently remains a very expensive vaccine.

 The TRANSGENE Company has already provided a first solution to this problem using vectors which ensure the expression of the antigenic protein of rabies in bacteria such as E. coli (see in particular FR-A 82 08401).

 The object of the present invention is the preparation of a vaccinating antigenic protein free of risk of accidental infection which, moreover, can be prepared under industrial conditions such that it ensures a very low cost of the vaccine by no longer using bacteria as production agents, but eukaryotic cells.

One of the potential advantages of eukaryotic cells as an element of expression is that they are capable of producing antigens with a structure closer to that of rabies and more complete than bacteria.

 The rabies virus is a rhabdovirus.

 This rhabdovirus consists of a helicotdale nucleocapdite containing an RNA included in a lipoprotein envelope having the shape of a shell.

 This virus comprises five proteins which are coded by the genome: G (glycoprotein), N (nucleocapdite), M1 and M2 (matrix) and L (large, transcriptase).

Like the membrane covering originating from the host, the only effective antigen present on the surface is a glycoprotein which is a transmembrane protein of molecular weight of approximately 65,000 daltons which has hemagglutinating activity.

 The purified glycoprotein is approximately six times more active in hemagglutination tests than the intact virus.

 As its name suggests, the glycoprotein of the rabies virus is glycosylated, although the role of this glycosylation is unknown.

The DNA sequence complementary to the mRNA of the rabies virus glycoprotein was described in the article by A. Anilionis et al., Nature 294; 275-278
(nineteen eighty one).

 This gene was used in the context of the experiments which will be described below and is shown diagrammatically in FIG. 1 appended.

The present invention relates to a vector for the expression of an antigenic protein of rabies, in eukaryotic cells, characterized in that it comprises at least
a DNA sequence (1) coding for said rabies antigenic protein,
the expression elements of this sequence in a eukaryotic cell.

 By DNA sequence (1) coding for an antigenic protein of rabies is meant a sequence which when expressed in eukaryotic cells leads to an antigenic protein of rabies.

 It may be all or part of the effective DNA sequence coding for the rabies antigenic protein or else a DNA sequence which, when expressed, results in a protein having identical immunological properties or very similar to that of the rabies antigenic protein.

It is also advantageous to use the complete DNA coding for the antigenic protein of rabies in the form of a fragment -PstI - PstI. In such a fragment, it is possible, for example, to delete all or part of the
G / C end without harming the quality of the expressed glycopro tein.

 The expression elements of DNA (1) vary according to the nature of the eukaryotic host cells. We can distinguish two cases which are currently of industrial interest: animal cells in particular mammalian cells, and yeasts, although the other types of eukaryotic cells should not be ruled out a priori.

Expression in animal cells
In the case where the host cells are animal cells, the expression elements will generally be taken from a virus, for example viruses of the Papovaviridiae family such as SV40 (simian virus 40, polyomavirus) or BPV- (papilloma virus bovine, papillomavirus). But we can use other viruses
such as adenoviruses. One of the advantages of the SV40 virus
is that its replication in host cells leads
to a considerable amplification, several thousand copies.

The elements of expression of the vectors according to
the invention will comprise at least the promoter of a virus which can be one of the promoters of the vector virus, thus in SV40 one can use the promoter of late genes, but this promoter can come from a virus thus diffuse in BPV the herpes simplex virus thymidine kinase gene promoter will be used below.

In order to improve the expressive vectors
according to the invention can also include introns and polyademylation sites located in this order a
the end of the DNA sequence (1), These elements can
also come from the vector virus or be exogenous.

Thus in the BPV vector the polyadenylation sites
will be the early or late SV40 sites. Among the introns that can be used, mention should be made of intron I of the rabbit globin gene, although this is in no way limiting.

Finally, an autonomous expression vector must be capable of replicating in host cells, the vector must therefore contain an origin of replica
tion, for example origin of replication of SV40 or
BPV. In addition, the vector will eventually have to be capa
ble to express the proteins necessary for its maintenance
and its replication in said cells as anti
T gene for SV40.

In some cases the vectors comprising
all of these may become too important
to be usable, in this case some of the elements
necessary for the maintenance and replication of the vector
can be provided via another vector or else be produced by the cells themselves, making it possible to delete the corresponding parts in the vector.

 Thus, COS cells which carry an integrated SV40 genome and which produce the T antigen necessary for the replication of SV40, are capable of ensuring the replication of an SV40 vector which has an origin of replication but which does not include the functions SV40 early.

 In the example which will be described below, the complementation of the expression vector proper (that is to say the vector carrying the DNA sequence (1) will be ensured by another vector called the complement vector "ensuring expression of missing elements in the expression vector.

In certain cases it may be advantageous to introduce into the expression vector a marker, a positive or negative selection gene in host cells, for example a gene for resistance to a particular chemical compound. In the case of construction
BPV this gene is the gene coding for dhfr (dihydrofolatereductase) which gives cells resistance to methotrexate
This selection gene allows the manipulation of vectors in host cells and can, in some cases, ensure their maintenance.

 Expression in yeasts.

In the case where the host cells are yeasts, the expression elements essentially consist of a promoter and optionally a terminator. These elements are preferably, although not necessarily, promoters and terminators of host yeast genes. So when the host cell is Saccharomyces cereviseae, we can use the promoter and the terminator of the URA3 gene or the gene
PGK (phosphoglycerate kinase).

 As in the previous case, the autonomous expression vector must be capable of replicating in yeasts, to do this it will include an origin of yeast replication such as the origin of replication of plasmid 2, but it is possible to use other origins of replication, for example origins of replication of chromosomal origins.

The vectors according to the invention will also preferably comprise a selection gene, for example the LEU2 or URA3 gene which complements a leu or ura strain.
The vectors according to the invention may be in the form of an autonomous vector, that is to say in the form of a plasmid or else be integrated into the chromosomes of eukaryotic cells, both yeast and mammalian cells in this case it is possible to integrate the fragment coding for DNA (1), for example the promoterXgene / terminator fragment in one or more copies into the chromosomes (Weawer & coll. 1981).

 In general, the vectors according to the invention will also comprise elements allowing their maintenance in bacteria and in particular will comprise an origin of functional replication in E. coli (ori-eco) and a selection gene for example a resistance gene a an antibiotic, for example Ampicillin (Ampr).

 These elements are useful during the construction of vectors, when bacteria are used as host cells; during the expression of DNA (1) in eukaryotic cells these elements may be lacking.

 The vectors thus obtained are used to transform or transfect the corresponding eukaryotic cells according to known processes.

 Thus for yeasts, the transfection technique described by H. Ito & coll 1983 can be used.

In the examples, the strain used is a particular segregant of Saccharomyces cereviseae (TG Y 1 spl), but other strains of the same species or of other species
(Schizosaccharamyces pombes, S. uvanum) can also be suitable hosts.

 In the case of mammalian cells, the transfection can be carried out for example by the method described by Wigler et al 1978 for cellular DNA, by techniques of fusion of protoplasts or microinjections, other techniques which are quite simple, as vector and in viral form. the. The mammalian host cells can be varied but adapted to the vector used.

 The transformed or transfected cells are cultured on an appropriate medium ensuring their growth.

 After culture, the cells are harvested and the rabies antigenic protein can be isolated by techniques known in the field of purification of proteins and antigens.

 The antigens thus prepared are useful both for the treatment and prevention of rabies like the antigens prepared by the "conventional" methods, but can also be used in the in vitro diagnostics of this disease.

 The examples below are intended to demonstrate other characteristics and advantages of the present invention without, however, limiting it.

In the attached drawings, FIG. 1 represents the structure of the gene encoding
for the rabies glycoprotein.

FIG. 2 represents the structure of the SV40 virus FIG. 3 diagrams the synthesis of pal 47 FIG. 4 diagrams the synthesis of pTG150 FIG. 5 diagrams the synthesis of pSVTGl5l FIG. 6 diagrams the synthesis of the vector of
Complementation pTGl 52 Figure 7 shows schematically the synthesis of pTG155 Figure 8 explains the technique for
remove the G / C ends in 5 '
of a cDNA of the glycoprotein in pTGl55 FIG. 9 diagrams the synthesis of SVTG171 FIG. 10 diagrams the synthesis of M13TG140 FIG. 11 represents the simplified structure of BPV FIG. 12 represents the simplified structure
of the plasmid pMOP FIG. 13 represents the detail of the structure of
pMOP at the level of the expression block of the
embarrassed dhfr Figure 14 represents the synthesis of pTG158 - SAL Figure 15 represents the synthesis of pTG172 Figure 16 represents the synthesis of pTG856 Figure 17 represents the modification of the end
of the PGK promoter Figure 18 represents the elements and the structure
final of the junction between the promoter
yeast and the rabies gene Figure 19 shows the specific immunoprecipitates
of the glycoprotein polypeptide
rabies virus in extracts of
yeast figure 20 represents an immunofluorescence picture
obtained from 3T3 cell extracts
The following materials and methods are used in the examples, unless otherwise indicated
Strains and plasmids
The strain of bacteria used is a strain
E. coli 1106 (SupE hSdS met supF). TGY1spl is a segregeant obtained by crossing the GFR18 strain (MatO (His3-11-15
Leu2-3-12) (FINK et al.) With the strain TGY10-1 (Mata
Ura3-251-373-328). The TGY10-1 strain is an isogenic derivative of the FL100 strain (ATCC 2283).

Enzymes and chemicals
All enzymes used come from Bethesda
Research Laboratories, New England Biolab or Borhienger
Manhein.

The oligonucleases used come from either
New England Biolab, Hana Biologics and Worthington Biochemical, or are synthesized by techniques Kohli et al.

 All radioactive products were obtained from Radio Chemical Center Amerscham.

Transformation
The transformation of E. coli 1106 by phsmids is carried out as described by Dagert and Ehrlich 1979.

Use of restriction enzymes
Restriction enzymes are used under the conditions specified by the manufacturer. The other enzymatic treatments are carried out according to the techniques described by Maniatis et al. (1982).

 After each enzymatic treatment, the DNA is extracted with phenol, twice with octanol-chloroform (1: 8) then precipitation with ethanol. In the case of small fragments (<100 bp), triple extraction with diethyl ether replaces precipitation with ethanol.

 The analysis of the digestion products is carried out by electrophoresis on agarose gel according to known techniques.

The insertion of non-phosphorylated "linkers" was carried out as follows
The linkers are taken up in TE buffer (10 mM Tris.HCl pH 7.5, 1 mM Na2EDTA) at a concentration of 1 mg / ml and prehybridized by cooling from 650C to 40C. The binding is carried out in a concentration buffer 30 mM NaCl, 30 mM Tris.HCl pH 7.5, 1 mM spermidine, 0.25 mM ATP, 2 mM DTT, 0.2 mM Na2EDTA and 0.1 mg / ml serum albumin bovine. In general, the binding (10 to 50 ssl is carried out with a molar excess of the linker on the DNA ends of 80 times, and a target DNA concentration of 0.1 FM. The ligase is used at the concentration of 100 at 200 units / ml and the binding is carried out at 40C for 16 hours. The excess linkers are eliminated by precipitation with spermine (Hoopes and Mc Clure, 1981). The DNA is then taken up in 10 mM Tris.HCl pH 7, 5.1 mM Na2EDTA, then diluted in hybridization buffer (100 mM Tris.HCl pH 7.5, 5 mM Na2EDTA, 100 mM NaCl). Rehybridization is carried out as prehybidation of linkers, and aliquots are transformed directly in E. coli. The principle of the method is described by
LATHE et al., 1983.

Example 1: Expression using the SV40 virus
1 - The expression of the gene coding for a rabies antigen protein (hereinafter the rabies gene) in monkey cells via SV40 is based on the complementation between two vectors, one of which carries the gene for rabies and part of the SV40 genome and the other carries the complementary part of the SV40 genome this in order to obtain two vectors of reduced dimensions of the order of 5200 bp (the size of SV40 without insertion) capable of complementing each other and get wrapped up.

 The genome of the simian virus shown diagrammatically in FIG. 2 according to Griffin (1981) consists of a circular double-stranded DNA molecule comprising 5243 bp.

This genome includes, for our purposes, an origin of OR replication, an early region coding for two antigens, the T and t antigens and a late region comprising VP1, VP2 and VP3.

Construction of pTG149 - Figure 4
The plasmid pBR327 (Soberon et al 1980) is digested with HindIII, the cohesive ends are complemented by treatment with DNA polymerase
Klenow, and the plasmid is linked with a "linker1, BglII octamer 5 'dCAGATCTG-3'. After rehybridization of the" linker "ends and transformation in E. coli, the correct plasmids are demonstrated by restriction with
BglII.

Construction of pTG147
The construction of this vector is shown diagrammatically in FIG. 3 appended.

 The plasmid pK 14 of ddpart comprises an EcoR1-BamH1 fragment originating from SU40, inserted between the EcoRI-BamH1 sites of pBR322.

 The SV40 fragment comprises an EcoR1 HpaI fragment linked via a "linker" KpnI oligonucleotide to a Hpal-BamH1 fragment. Thus the polyadenylation sites (A) for the immediate and late transcription of SV40 are flanked by a KpnI site and a BamH1 site.

To clone this plasmid in the form of an EcoR1 fragment
Sal1, it is necessary to discard the part between the BamH1 and SalI sites (this fragment already being present in the vector virus), in addition, a direct repetition of this fragment would produce a deletion by homologous recombination in vivo.

 This is why, pK14 is treated with BamH1 and the nuclease S1 followed by digestion with SalI and a treatment with DNA polymerase, in order to fill the 5 ′ extensions produced by the cleavage with SalI. The fusion of the two ends by DNA ligase leads to a reconstruction of the SaiI site in pTG142.

 As it is desired to insert the cDNA of the glycoprotein, either into the EcoR1 sites or into the KpnI site, it has been envisaged to use the EcoR1 site of pBR328, this site is an internal site of the plasmid gene which is found located in the chloramphenicol resistance gene. Having encountered problems of lethality associated with the expression of the glycoprotein gene in bacteria, the EcoR1-Sal1 fragment was transferred to the corresponding sites of pBR322 to obtain pTG143. A Bg1II linker is then inserted between the two EcoR1 and Kpn1 sites and adapter technology is used to insert the glycoprotein gene.

The insertion of the rabies glycoprotein gene in the form of a Pstl - Pstl fragment from the plasmid pRG described by Anilianis 1981, into the BglII site was made possible using a 5 'octamer adapter
GATCTGCA 3 'as intermediary (Lathé et al 1983).

Construction of pTG150 - Figure 4
Plasmids pTG147 and pTG149 are digested completely with NI bu, mixed in equimolar amount, lids, and the ligation mixture is used to transform
E. coli. The plasmid pTG150 is identified as the plasmid comprising the complete cDNA of the rabies glycoprotein inserted into the BglII site of pTGl49.

Construction of pTG15î and SVTG151 - Figure 5
The plasmid pTG150 is introduced entirely in the form of a Clai digestion fragment into the HpaII restriction site of the SV40 virus (hereinafter referred to as SV401) by subjecting pTG150 to Cla, SV40 to HpaII respectively and putting the restriction pieces in the presence ligase.

 This gives the plasmid pTG151 of 10.1 kb.

 This plasmid is too large to be able to be introduced into mammalian cells. This is why it is used in the form of a fragment linearized with BamHI.

Indeed, the BamH1 fragment comprises, in addition to the gene for the rabies glycoprotein, the part of the SV40 genome which can be complemented by the plasmid pTG152, the construction of which will be explained below.

Construction of pTG152 - Figure 6
The plasmid pTG152 is obtained from pBR327 D by digestion with BamH1 and ClaI in order to isolate a fragment comprising an origin of functional replication in
E. coli (ori-eco) and the ampicillin resistance gene.

This fragment is linked to a fragment obtained by digestion of
SV40 by Bcl1 and Tagal. This gives the plasmid pTG152 of 5.3 kb which comprises the complementary part for SV40 of the plasmid SVTG151.

Example 2: Synthesis of the SVTG160 vector - fig. 7
Synthesis of the plasmid pTG155
From the plasmid pTG150, a partial digestion is carried out with Bglil and then a complete digestion with MstII. The plasmid is recircularized by ligation in the presence of a synthetic double-strand adapter whose formula is given in B figs 8, in order to merge the BglII and MstII sites to delete the G / C polymer at the 5 'end of the element cDNA.This plasmid pTG155 contains a site of initiation of the reconstituted translation in which the nucleotide adenine is located in position -3 to be in conformity with the generally accepted sequence for the initiation of translation in eukaryotic cells.

 This plasmid can be treated as pTG150 to give the vector SvTG160.

Example 3: Synthesis of the SVTG171 vector - FIG. 9
This vector differs from the previous ones in that it comprises an intron following the rabies gene.

This intron is intron I from pSXBETA + (rabbit ssglobin). This intron is taken in the form of a PvuII / BamH1 fragment and inserted into an M13 phase, in this case M13 TG120, between the HindII and BamH1 sites. The phage M13 TG140 (FIG. 10) is thus obtained which comprises the intron I, which is taken in the form of a BglII / BamH1 fragment to be inserted between the corresponding sites of pTG149 (see above)
The plasmid pTG170 is thus obtained.

 By digestion of pTG170 and pTG151 (see above) with BglII and SalI and placing in the presence of the digestion fragments, the plasmid pTG171 is obtained which, as before, is linear with BamHI in order to provide the vector SVTG171.

 It is possible, under analogous conditions and using pTG160, to obtain a plasmid comprising an intron.

 The different plasmids SVTG171, SVTG151 and SVTG160 complemented by pTG152 in mammalian cells, make it possible to produce an antigenic protein of rabies.

 Example 4: Preparation of a vector from PVB.

1) The plasmid pMOP - Figures 11 - 12
The BPV virus is divided into two parts limited by two unique restriction sites HindIII and BamH1 which delimits two fragments said to be "69%" and "31%" respectively. (Figure 11 after Chen et al. 1982).

 The vector derived from BPV used in this example is called pMOP. it is obtained by introduction into the BamH1 site of BPV of the dhfr marker coding for dihydrofolate reductase under the control of the SV40 early promoter (see FIG. 13).

 This pMOP vector further comprises a fragment of pBR322 carrying the ampicillin resistance gene which comes from the plasmid pML2 described by Lusky and Botchan (1981). FIG. 13 represents the detail of the structure of pMOP between the BamH1 sites, these elements do not constitute essential elements in the context of the present invention since they are intended to ensure the expression of the dhfr gene.

2) The plasmid pTG158SAL - Figure 14
The gene coding for the rabies antigenic protein comes from the plasmid pTG147, the construction of which has been explained previously.

This gene is taken in the form of a fragment
BglII / SalI by partial digestion of pTG147 to be introduced between the corresponding sites of the plasmid pTG301 treated with BglII / SalI. This plasmid pTG301 comprises the thymidine kinase gene (tk) and its promoter (ptk), it is obtained by insertion into the BamH1 site of pBR322 of a BamH1-BamH1 fragment of the Herpes virus simplex, the restriction treatment eliminates the tk gene.

 The ligation product pTG156 contains, in its interesting part, the rabies gene under the control of the thymidine kinase promoter, the gene being followed by the polyadenylation site of SV40 (A).

The introduction of the globin intron is carried out from the phage M13TG140 (see above) in the form of a BglII / BamH1 fragment which is inserted into the BglII site of pTG156 after deletion of the fragment
BhlII / BglII corresponding.

 This deletion having deleted the rabies gene, it is reintroduced into the unique BglII site of pTG156 in the form of a BglII / BglII fragment of pTG147.

The plasmid pTG158 is thus obtained.

The essential part of this plasmid having to be taken in the form of a SalI / SalI fragment (one of the rare unique sites of pMOP) is introduced upstream of the promoter at the BamHI site, a small fragment originating from
M13TG120 and comprising a SalI site. M13TG120 is digested with BglII and BamHI and pTG158 with BamHi the whole is linked to give pTG158 SAL.

3) Synthesis of the vector - figure 15
By digestion of pTG158 and pMOP with SalI and ligation, the plasmid pTG172 is obtained which comprises, in addition to all the elements mentioned for pMOP, the rabies gene under the control of the Ptk promoter and terminated by the globin intron and a polyadenylation site.

 By transfection of mammalian cells this vector produces an antigenic protein of rabies, as will be demonstrated below.

Example 5 Transformation of NIHA 3 T 3 Cells (Mouse Fibrolasts)
The original pMOP plasmid and the pTG172 plasmid are used for these transformations.

 The cells are cultured in a proportion of 8.105 cells / Petri dish 10 cm in diameter in Dulbecco medium + 10% SVNN + antibiotic. (SVNN newborn calf serum).

 The transfection with the vectors is carried out after 24 hours of culture by the technique of Wigler et al. 1978.

 The medium is changed 24 hours after transfection. Then transplanting is carried out on selective medium containing methotrexate.

 After 2 to 3 weeks, the cells can be fixed in a colony and the antigens produced can be demonstrated using immunofluorescence.

Cells of the 3T3 line were transfected with DNA purified from
a): plasmid pTG172-O (pMOP; vector BPV dhfr without additional insert).

b): plasmid pTG172-rage (BPVTG172 t vector
BPV dhfr with an additional insert which includes the different elements allowing the expression of the rabies glycoprotein).

 Colonies derived from cells which have incorporated a plasmid and therefore a resistance to methotrexate (dhfr +) were fixed by treatment with acetone. The expression of rabies glycoprotein was visualized by successive treatments with a monoclonal antibody (nO 509-6) directed against the glycoprotein and a second fluorescent anticoprs directed against this first antibody.

 Magnification: approximately 1,000 times.

 Example 6: Construction of the plasmid pTG 856 comprising the gene for the rabies glycoprotein (FIG. 16).

The yeast PGK gene was isolated from a chromosomal DNA library produced by integration of fragments cut at random (partial digestion with SaU3A) at the site.
BamHI of a plasmid (pFL 1) capable of replicating both in yeast and in E. coli (Chevallier et al, 1980). The detection of a fragment containing the PGK gene was carried out by in situ hybridization of bacterial colonies, transformed by this library, with a specific probe (5'-TGTTTAGTTTAGTAGAACCTCGTGAAACTT-3 ') (Maniatis et al, 1982). The 5 'region of the PGK gene was recloned as a HindIII-SalI fragment (2.3. Kb) in pBR 322.

 This fragment includes all of the sequences involved in controlling the initiation of transcription (promoter) of the PGK gene, plus the start of the coding sequence (Hitzeman et al, 1982>.

An ATG-destroying BglII site opening the PGK coding sequence was created by in vitro mutagenesis (Smith and
Gillam, 1979) (Figure 17).

On the other hand, an EcoRI-HindIII fragment (0.5. Kb) comprising the end (3 ') of the coding sequence and the transcriptional termination signals of the PGK was treated with Klenow polymerase and ligated to the PvuII site. unique of the plasmid described in the previous paragraph, so that the EcoRI site thus reconstituted is upstream of the destroyed Hindili site. This new integrated fragment comprises a BglII site located in the coding part of the PGK gene. The deletion of the fragment between the two BglII sites, that newly created downstream of the promoter
PGK and that upstream of the terminator, leaves a unique BglII site where one can clone the genes that one wants to express.

 The HindIII - Pst 1 fragment comprising, in order, the promoter part of the PGK gene, the BglII site, the terminator part of the PGK gene, the origin of replication of pBR 322 and part of the sequence of the Ampr gene conferring resistance to the ampicillin was ligated with the largest fragment resulting from the double digestion HindIII - Pst 1 of the plasmid pJDB 207 so as to reconstitute the Ampr gene (deggs, 1981) (cf. FIG. 16). The resulting plasmid (pTG 841) therefore has: the yeast LEU2 gene, the E. coli Ampr gene, the yeast plasmid 2 X REP3 gene, the origin of replication of the yeast plasmid 2 p, the origin of replication of bR 322, the promoter and the terminator of the yeast PGK gene.

A 1.1 kb Hindili fragment comprising the gene
Yeast URA3 (European Patent No. 78 3210) was cloned at the HindIII site of pTG 902, a pBR 322 derivative lacking the Pstl and NdeI sites, to form the plasmid pTG 802. The DNA of this plasmid was linearized by unique Pstl site - located in the 5 'non-coding region of the URA3 gene -, treated with Klenow polymerase to make blunt the ends freed by digestion pstl, and recircularized by the action of DNA ligase from phage T4. The 1.1 kb HindIII fragment thus modified (devoid of PstI site) was integrated into the unique HindIII site of the plasmid pTG 841 so that the direction of transcription of the URA3 gene is divergent compared to that of the PGK (plasmid pTG 849). The plasmid PTG 849 therefore has the same characteristics as pTG 841, with the addition of the modified URA3 gene.

The rabies glycoprotein cDNA was isolated in the form of a BglI fragment comprising the entire coding sequence plus the polydGdC ends. An MstII site is located at the beginning (5 ′) of the coding sequence, the enzyme cutting between the seventh and the eighth nucleotide following the
From the initiating ATG. A synthetic sequence was designed to substitute the BglII - MstII fragment in the 5 'region of the gene, so as to replace the 5' polydGdc end with a sequence closer to the untranslated sequences of the yeast messenger RNAs (FIG. 17). The BqlII fragment thus modified was integrated into the unique BglII site of pTG 849, so that the direction of transcription of the rabies glycoprotein gene is the same as that of the PGK.

 Example 7: Expression of rabies virus glycoprotein polypeptide in yeast.

Plasmid PTG856 is introduced into the strain
S.Cerevisiae TGYlspl using the lithium salt technique (Ito & coll 1983).

 The same strain was transformed with PTG849 which is the same plasmid as PTG856 but which does not contain the glycoprotein gene. These strains were cultivated in 25 ml of medium without uracil allowing the plasmid to be stabilized.

 Radioactive cysteine S35 is added to label the yeast proteins.

The cells are crushed in the following buffer
250 µl: 25 mM Tris HCl pH 7.5
10 mM Na2 EDTA pH 8.

250 pl: 1M.KCl
0.1 M Tris / SCl pH 8.8
2% Triton X 100
5 r1: PMSF Phenyl-methyl-sulfunyl-fluoride
5 pl: "Soybean Trypsin inhibitor" 10 mg / m
Iodoacetamide 100 mM
10mM N-ethylmaleimide
1,10 - phenanthroline 10 mM 5 5 pl: DNAse I (0.5 mg / ml)
The supernatants of these ground materials containing the radioactive proteins are incubated overnight at 40 with 5 μl of rabbit serum. Two sera were directed against the glycoprotein (lots 2726 and 2527) and one against the total virus (lot 2725). 50 μl of protein A-sepharose (Pharmacia CL-4 B) are added. After incubation, the pellet is washed 4 times with a buffer (0.5 M Kcl; 10 mM Tris.Hcl pH 8.8; 0.5% Triton X100) and three times with a second buffer (10 mM Tris / Cl pH 8.8). The pellet is incubated for 10 minutes at 1000C in denaturation buffer (62 mM Tris.HCl pH 6.8; 2% SDS; 5% 6-mercaptoethanol; 0.01% bromophenol blue). The extract is then analyzed on polyacrylamide gel according to the technique of Laemmli (1970).

After exposure of the gel to a photographic film and development, the different bands corresponding to the proteins are visible (FIG. 19).

One can see in particular in the extracts of strains carrying PTG 856 (a) and (b) a strongly marked band of molecular weight approximately 65000 indicated by an arrow which is absent from the extracts of strain carrying
PTG 849 (c) and (d). This molecular weight is that expected for the rabies virus glycoprotein.

The following strains were deposited on September 30, 1983 in the National Collection of microorganism cultures (CNCM) of the Institut Pasteur
E.Coli 1106 / pTG 152 nO I 247
E.Coli 1106 / pTG 171 nO I 248
E.Coli 1106 / pTG 172 nO I 249
S.Cereviseae TGYlspl / pTG 856 nO I 250
The plasmids pK14, pMOP and pSXBETA were kindly provided by R. Breathnach.

REFERENCES * ANILIONIS, WH WUNNER & PJ CURTIS, Nature, vol.294
pp 275-278 (1981) * BEGGS J. (1981) in Genetics Engineering, vol. 2, 175-203
Ed. R. WILLIAMSON, Academic Press * CHEN et al. Nature vol. 299, pp 529-534 (1982) * CHEVALLIER MR, BLOCH JC, LACROUTE F., Gène, 11-19
(1979) * DAGERT M. and ERHLICH SD, Gene, 23-28 (1979) * FINK and Coll. Natl. Sci. USA, 75, (1978) * GILLAM S. & M. SMITH, Gene 8, 81-97 (1979) * GRIFFIN BE, DNA tumor viruses (1981) * HARDISON et Coll. Cell, Vol. 18, 1285-1297 (1979) * RA HITZEMAN, FE HAGIE, JS HAYFLICK, CHEN,
PHSEEBURG, R.DERYNCK, Nucleic Acides Research 10,
7791-7808 * HOOPES et al., Nucleic Acid Research 9, 5493-5504 (1981) * IS <-HOROWITZ et al., Nucleic Acids Research 9, 2989-2998
(1981) * M.ITO, Y. FUKUDA, K. MURATA, A.KIMURA (1983) J. Bacteriol,
153, 1 p.163-168 * LAEMMLI UK, Nature 227, 680-685 (1970) * LATHE et al., Genetic Engineering Ed. Bob Williamson vol .4
p.1-51 (1983) * LUSKY et al., Nature 293, 79-81 (1981) * MANIATIS T., E F. FRlTSCH, J.SAMBROOK (1982) in molecular
Cloning, p.309 Ed. CSH laboratory * MIYANOHACA, A. TOH-E, C. NAZAKI, F.NAMADA, N.OKTOMO
& K. MATSUBARA (1983) Proc. Nat. Acad. Sci.USA 80, 1.5 * ORR. WEANER TL, SZOSTAK JW, ROTHSTEIN RJ (1981) Proc.

Natl. Acad. Sci. (USA) 78 (10) 6354-6358 * SOBERON et al., Gene 9, 287-305 (1980) * VALENSUELA P., A. MEDINA, W.RUTTER, G.AMMERER #
BD HALL, Nature 298, 347-350 (1982) * WIGLER et Coll., Cell, vol.14,725-731 (1978)

Claims (26)

 1 - Vector for the expression of an antigenic protein of rabies in eukaryotic cells, characterized in that it comprises at least  a DNA sequence (1) coding for said rabies antigenic protein, and  - the elements of expression of this sequence in a eukaryotic cell.  2 - Vector according to claim 1, characterized in that it is an autonomous vector comprising at least one origin of replication in eukaryotic cells  3 - Vector according to one of claims 1 and 2, characterized in that the DNA sequence (l) is between the PstI-PstI sites.  4 - Vector according to one of claims 2 and 3, characterized in that it is an autonomous vector comprising at least one origin of virus replication.  5 - Vector according to claim 4, characterized in that the origin of replication is that of the SV40 virus or BPV.  6 - Expression vector according to one of claims l to 5, characterized in that the expression elements come from the DNA of a virus.  7 - Expression vector according to claim 6, characterized in that the expression elements comprise at least all or part of a promoter of the SV40 virus.  8 - Expression vector according to claim 6, characterized in that the expression elements comprise at least all or part of the thymidine kinase promoter of the Herpes simplex virus.  9 - Expression vector according to claims 1 to 8, characterized in that it further comprises a marker gene expressing in eukaryotic cells.  10 - Expression vector according to claims 1 to 9, characterized in that the DNA sequence (1) is followed by an intron.  II - Expression vector according to Claims 1 to 10, characterized in that the DNA sequence (1) is followed by a polyademylation site.  12 - Expression vector according to claim 6, characterized in that the expression vector consists of two complementary vectors each comprising an origin of replication of SV40, one of the vectors carries the DNA sequence (1) under the control of an SV40 promoter and part of the SV40 genome, the other vector comprises the complementary part of said genome.  13 - Expression vector according to claim 12, characterized in that one of the complementary vectors comprises at least in addition to the origin of replication of SV40, the entire region of the late genes of SV40, the expression vector proper comprises at least in addition to the origin of replication of SV40 and the DNA sequence (1) the entire region of the SV40 early genes and the elements ensuring the expression of the DNA gene (1).  14 - Expression vector according to claim 12, characterized in that the vectors used are the vector SVTGl5l and the complementary vector pTG152, or else the vector SVTG160 and the complementary vector pTG152.  15 - Expression vector according to claim 12, characterized in that the vectors used are the vector SVTG171 and the vector pTG152.  16 - Expression vector according to claim 4, characterized in that it comprises all or part of the BPV genome and a sequence comprising in order all or part of a virus promoter, the DNA sequence (1 ), an intron and a polyadenylation site.  17 - Vector according to claim 16, characterized in that the promoter is a fragment of the promoter of the thymidine kinase gene from simple Herpes, the intron is the intron of a globin, the polyadenylation site is the SV40 early polyadenylation site.  18 -. Vector according to claim 1, characterized in that it is the vector pTG172.  19 - Vector according to one of claims 2 and 3, characterized in that it is a plasmid comprising an origin of replication in yeasts.  20 - Vector according to claim 19, characterized in that it is the origin of replication of the plasmid 2 ,.  21 - Vector according to claims 1 to 3, and 19 and 20, characterized in that the expression elements come from a yeast.  22 - Vector according to claim 21, characterized in that the expression elements are at least made up of the URA3 promoter or the phosphoglycerate kinase promoter.  23 - Vector according to one of claims 21 and 22, characterized in that the expression elements comprise a terminator of the URA3 gene or of the phosphoglycerate kinase.  24 - Vector according to claims 19 to 23, characterized in that it comprises a labeling gene.  25 - Vector according to claims 19 to 24, characterized in that it is the plasmid pTG856.  26 - Vector according to claims 1 to 25, characterized in that it comprises an origin of replication in bacteria and a resistance gene to an antibiotic expressing itself in bacteria.  27 - Process for the preparation of an antigenic protein of rabies, characterized in that cultured cells transfected according to claim 26 and that the protein is isolated.  28 - Rabies antigenic protein obtained by the implementation of the method according to claim 27.