DK166357B - PARTICLES WITH THE IMMUNOGENIC PROPERTIES RESPONSIBLE FOR THE HBS ANTIGEN AND VECTORS AND ANIMAL CELLS FOR PRODUCING SUCH PARTICLES - Google Patents

Abstract

The invention relates to particles possessing the essential features of particles based on HBs antigen, these particles being formed from a major polypeptide characteristic of the HBs antigen into which has been incorporated a foreign polypeptide sequence provided with an immunogenic site different from those normally borne by the S gene. The invention also relates to recombinant DNAs coding for the major polypeptide transformed in the manner described above, and to cell lines transformed by this recombinant DNA and capable, in particular when the foreign amino acid sequence contains not more than 16 amino acids, of being excreted into the culture medium of the abovementioned cell line.

Description

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The present invention relates to polypeptide particles which are preferably substantially spherical, at least for the most part, which have immunogenic and immunological properties that are characteristic of the surface antigen (often termed the abbreviation HBs antigen) of hepatitis B virus. The particles in question are characterized in that they additionally carry at least one foreign peptide sequence of a polypeptide normally encoded by the S gene in hepatitis B virus. The invention further relates to recombinant DNAs and eukaryotic cell lines, preferably of animal origin, which in their culture medium are capable of secreting polypeptide particles of the aforementioned kind.

It is well known that serum from chronic hepatitis B virus (HBV) carriers contains empty viral sheaths in the form of particles or filaments with a diameter of 22 nm and sometimes complete infecting virions which are spherical particles of diameter 42 nm.

Once the blank caps are purified on the basis of serum from chronic virus carriers, they are used to prepare vaccines against hepatitis B. It is now known that it is now also possible to obtain particles of 22 nm diameter in large quantities. by other methods. The genetic manipulations of the gene encoding the main protein of the particles (the gene S) have made it possible to produce the particles in the cell lines of the culture (M.F. Dubois et al., (1980),

Proc. Natl. Acad. Sci. USA, 77, 4549-4553), in yeast (P. Va-lenzuela et al., (1982), Nature, 298, 347-350) or under the aid of recombinant virus (GL Smith et al., (1983) , Nature, 302, 490-495). One method of producing the particles comprises a transformation of eukaryotic cells by an appropriate vector containing the gene S under the control of an efficient promoter, a cultivation of the transformed cells, and an extraction of the produced particles, either from advance illuminated 2

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cells or from the culture medium when the particles have been separated by the cell lines used (especially in the case where monkey cells are used, for example the VERO type).

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The main polypeptide encoded by the gene S, which is part of the constitution of the subject particles, is composed of 226 amino acids and has a molecular weight of 25400 daltons.

In addition, it has been shown that certain natural particles in the major polypeptide may consist of a higher molecular weight polypeptide, in particular of the order of 34000 daltons, which contains the polypeptide sequence of the above major polypeptide having the same C-terminal end as the major polypeptide. and in addition, a complementary sequence of 55 amino acids at the N-terminal end (Stibbe X. and Gerlich WH, (1983), J. Virology, 46, 626-628) encoded by the pre-S region of the genome in hepatitis. B.

This additional sequence at the N-terminal position does not appear to be particularly stable in the natural particles, and therefore does not appear to play any significant role in the constitution and cohesion of HB particles. The HBs particles are known to be composed of organized aggregates which are only insensitive to proteases, and they comprise about 100 such major polypeptides and other constituents, in particular lipidic constituents. A method enabling compositions containing a significant portion, more specifically up to a total of 35% of the polypeptides formed, of more stable particles containing said supplemental sequence has been previously described (Michel, ML et al., (1984), Proc. Natl. Acad. Sci. USA, 81, 7708-7712). This known process utilizes eukaryotic cell lines, more specifically a culture of human or animal cells, which is transformed in advance by vectors containing a DNA sequence encoding the S and pre-S regions of the hepatitis B virus. the genome, which sequence is located. in the interior of the vector and is under the direct control of an exo-3

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gene promoter. This promoter has a known ability to ensure effective initiation of the genetic transcription under direct control in the eukaryotic cells, in particular human or animal cells, to which the aforementioned 5 vectors are intended. For example, reference may be made to the article by Galibert et al. (1979), Nature 281, pp. 646-650), as to said DNA sequence.

When the cell lines used are derived from monkeys, it is an advantage to have a promoter derived from SV40 virus, which virus is known to have the ability to initiate an efficient transcription of the neighboring genes in the monkey cells. This promoter may advantageously correspond to the "previous" SV40 virus promoter, which normally controls the expression of the "small T antigen" and likewise the "large" T antigen. ").

The natural variability of the N-terminal end of the polypeptides in the envelope of hepatitis B virus has given rise to the assumption that the protein fragments that are separate from said supplemental sequence may be substituted and fused with said major polypeptide. lypeptid. This idea has been realized by Valenzuela et al., Which, in transformed yeast types, obtained particles 25 on the basis of hybrid proteins consisting essentially of the above-mentioned major polypeptide modified at the N-terminal end by a complementary polypeptide comprising about 100 amino acids derived from glycoprotein D from herpes virus. Valenzuela et al. have reported that the transformed particles are capable of inducing antibodies against both hepatitis B virus and herpes virus at once (P. Valenzuela et al., (1982), Nature, 298, 347-350, and P. Valenzuela (1984), "Proceedings of the Twelfth International Conference on Yeast Genetics 35 and Molecular Biology", Edinburgh, (1984), 16).

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The object of the present invention is to provide polypeptide particles which have basic immunogenic properties similar to those of HBs antigen, and which contain at least one other peptide sequence which is preferably also immunogenic, in other words polypeptide particles which is capable of being utilized to build mixed vaccines having optimum stability, which second peptide sequence must be present each time the main polypeptide, either the major polypeptide itself or preferably the major glycoprotein of HBs the antigen must be synthesized without any significant effect on the particular and characteristic structure of the envelope antigens of hepatitis B virus1.

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It is also an object of the invention to provide particles of this type, which may also contain, as appropriate, the complementary sequence normally encoded by the pre-S region of the genome in hepatitis B virus in intact condition. However, this is to be understood so that the supplementary sequence can also be modified, e.g. according to the cases described by Valenzuela et al. However, the maintenance of the intact nature of said supplemental sequence can be explained by the enhanced immunogenicity which can occur in the modified polypeptides of the invention.

Particles of the invention which contain such a large portion of the characteristic amino acid sequences of the major peptide of the hepatitis B virus surface antigen (HBs antigen) that this portion is sufficient to impart to the particles a structure similar to the structure of the natural particles. which are characteristic of HBs antigen are characterized in that the particles on their surface 35 and embedded in the main polypeptide carry at least one foreign amino acid sequence which preferably itself contains an immunogenic region and which is inserted into one of the main polypeptide.

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its hydrophilic regions, which are usually exposed to the outer surface of the particle or replace one or more of the amino acids in one of the hydrophilic regions of the main polypeptide.

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Specifically, the foreign amino acid sequence can be introduced into one of the regions comprising amino acids 32-74 or amino acids 110-156 of the major polypeptide and whose general formulas are reproduced in the article by P. Tiollais 10 et al., (1981) Science , 213, pp. 406-411. These formulas are shown below:

A

t With Clu Asn Ile Thr Ser Cly Phe Leu Cly Pro Leu Leu Fat Lcu 6ln Atå Cly Phe Ph · 21 Leu Leu Thr Arg Ile Leu Thr Ile Pro Cln Ser Leu Asp Ser Trp Trp Thr Ser Leu Atn

Ser Pro Thr t LI Phe Leu Cly Cly Thr Thr Val Cys Leu Cly Cln Asn Ser Cln Ser Pre Thr SerlAsn His Thr Thr I le - Ile 6t Seri Pro Thr Ser CyS Pro Pro Thr Cys Pro Cly Tyr Arg Trp With Cys Leu Arg Arg Phe

Thr 20 8l Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu Val Leu Leu Asp Tyr

Thr Pro 101 Cln Cly With Leu Pro Val Cys Pro Leu Ile Pro Cly Ser Ser Thr Thr Ser Thr Cly Pro

Ser * «r

Lys Thr Pro Asn Phe 121 Cys Arg Thr Cys With Thr Thr Ala Cln Cly Thr Ser Mat Tyr Pro Ser Cys Cys Cly Thr Thr Arq Thr Pro I le Tyr

Eel"

M | ir «fro Ser (up dr» * Cr «Thr C, * Ile Three Ile Bel Ser Ser Trp Μ» The dr Lr * Ser - e, T

t6l ph £ Leu Trp Clu Trp Ala Ser Ala Arg Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Phe Ala

Thr Ala 30 ll Cln Trp Phe Val Cly Leu Ser Pro Thr Val Trp Leu Ser Val Ile Trp net With Trp Tyr

Ile Val

Val Ile 201 Trp Cly Pro Ser Leu Tyr Ser Ile Leu Ser Pro Phe Leu Pro Leu Leu Pro IU Phe Phe

Lcu Leu

Val 221 Cyt Le «Trp Val Tyr Ile ΛΙλ 35 6

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It should be noted that the formula for the main polypeptide may undergo variations. This is especially true with regard to the differences between the constitutive amino acids observed by Valenzuela et al. (1980) "Animal Virus 5 Genetics", B. Fields, R. Jaenisch, C.F. Fox, Ed .: Academic Press, New York, p. 57, and shown above the main lines of the above formula and the constitutive amino acids observed by Pasek et al., Nature (London) 282, 575 (1979) , and shown under the main lines mentioned.

More particularly, the invention relates to useful compositions for the preparation of vaccines containing substantially spherical polypeptide particles (or consisting of such particles), at least with respect to the majority of the particles, which vaccines have immunogenic and immunological properties characteristic of HBs antigen. The particles have a size of 18-25 nm, especially 20-22 nm, and densities which enable them to be isolated in a density zone of 1.20-1.22 g / ml in a density gradient based on CsCl. The particles have a level of purity such that there is complete absence of Dane particles and HBs antigen, including HBc, and the particles are more specifically characterized by the presence of said foreign sequences in the above states.

The size of the foreign sequences which can be introduced into the interior of the main polypeptide, as just defined, can be modified within wide limits. It is possible to introduce amino acid sequences, which may comprise, for example, up to 100 amino acids, sometimes more. However, it is an advantage that the foreign peptide sequence has a size not exceeding 16 amino acids, and it is preferably 5 to 16 amino acids, e.g. 6-13 amino acids, especially in those instances where it is introduced into the major polypeptide without suppression of a substantially

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equal equivalent number of amino acids to which it has previously encompassed. The present invention provides a remarkable result which consists in the possibility of producing cellular systems which allow the secretion of modified particles encompassed by the invention from the cells in question when transformed in advance by a suitable vector. containing a DNA sequence encoding the modified polypeptide of the invention.

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Of course, the invention also relates to the recombinant DNAs encoding said modified polypeptides included in the composition of the above particles. In this regard, the recombinant DNAs, and preferably the vectors containing these DNAs, which contain a DNA sequence encoding the S region and optionally the pre-S region of the genome in hepatitis B virus , characterized in that said DNA sequence is locally modified by at least one nucleotide sequence encoding the aforementioned foreign sequence and at least one of the zones of the S region corresponds to the hydrophilic regions of the above-mentioned major polypeptide. The S region, and optionally also the pre-S region, is located within the interior of the recombinant DNA, placed under the direct control of an exogenous promoter, which has a known ability to effect an efficient transcription of genes under direct control. in the eukaryotic cells, especially human or animal cells, but also in yeast cells to which the vectors in question are particularly suitable.

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The exogenous promoter used is distinct or foreign to the "endogenous" promoter, which is normally associated with the S genes and the pre-S genes in the genome of the hepatitis B virus. When these cells originate from monkeys, it is an advantage to have one of the SV40 virus promoters, as discussed above.

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However, the invention is not limited to the use of this particular promoter, although it provides particularly favorable results with respect to the production of hybrid polypeptides of the invention due to the 5 transformed cells, HBs antigen and a pHSA receptor. characteristics and the excretion of the substances produced in the culture medium used. For example, one can also use the later SV40 promoter (which controls the expression of proteins VP1, VP2 and VP3).

In this connection, reference can be made to the SV40 virus restriction diagram (J. Tooze, Ed., DNA Tumor Viruses,

Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1980, chapters 2-5), which indicate the relative positions of these promoters and genes encoding the various 15 associated antigens.

It goes without saying that the SV40 promoters can be replaced with any other promoter type known to possess (or which may be conferred) an ability to promote transcription in the cell lines used, the sequences of which encode the aforementioned S regions and possibly pre-S regions as soon as they are placed under the appropriate control. Hereby it is achieved that these sequences with said promoter are incorporated into the genomes of the receiving cells, and / or that the thus-transformed receiving cells are imparted to the ability to synthesize and secrete substantial amounts of hybrid polypeptides of the invention, the capacity thus obtained can then be transmitted to subsequent generations of the cells.

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Said transformed cell lines are referred to as "stable" when the property obtained by the cell lines of the invention for synthesizing the above polypeptides can be transferred from one generation of cells 35 to the next over at least 10 generations.

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With respect to other useful promoters, e.g. mention the early polyom promoter or LTR promoters of various retroviral types, as well as the EA adenovirus promoter as well as the effective promoters for genes of cellular origin.

As is well known, the promoters taken from the viral genomes from which they originate are preferably accompanied by activating "sequences" which normally precede the promoters (relative to the transcriptional direction of the gene sequences that are normally placed under their control). For examples of such activating sequences, reference can be made to the articles in Science, 1983, vol. 219, pages 626-631, and Nature, 1982, vol. 295, pages 15. 568-572.

Advantageously, the aforementioned DNA sequence encoding said pre-S and S regions is located immediately after a DNA fragment made up of the promoter and the activating sequence allowing a normal transcription of the pre-S or S sequence. Preferably, said fragment comprises 300-400 base pairs depending on the promoter type and activating sequences used.

The invention thus also relates to cell lines which are transformed by vectors such as the vectors defined above and which are capable of secreting the aforementioned immunogenic particles into their culture medium.

The preferred cell lines of the invention are composed of mammalian cells, in particular CHO cells or VERO cells.

Such cell lines that can be maintained in the culture can be produced by transforming these cell lines with a vector as defined above, and then isolating the cells from the culture expressing sequences which

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10 encodes the hybrid protein of the invention.

Further characteristics of the present invention will become apparent from the following description which provides 5 examples of constructs illustrating the basic principle of the invention. In the following description, reference is further made to the drawing, in which fig. 1 shows the schematic structure of plasmid pLAS used in the constructs of the invention; and FIG. Figure 2 shows the schematic structure of plasmid pPAP derived from plasmid pLAS, which additionally comprises a synthetic DNA fragment encoding a sequence of 11 15 amino acids from the VP1 protein of type I poliovirus (Mahoney strain).

Construction of transfected plasmids 20 Plasmid pLAS

This plasmid comprises (see Fig. 1) the coding portion of the S gene (P. Charnay et al. 1979) with the natural site of the polyadenylation (fragment Stul (43) BglII (1984)) where the Bam HI site (1400) is omitted and replaced by a klenow enzyme.

This gene without a promoter is brought under control by the early promoter of SV40 virus (an SV40 virus fragment 30 between the PvuII site (250) and HindIII site (5154)).

Furthermore, the plasmid comprises the large BamHI (375) -Sall (650) fragment of plasmid pML2 (lusky, M. and Botchan, M.

1981).

The fragment of the S gene is ligated at its BglII end to the BamHI end of plasmid pML2 (the BglII and BamHI ends are mutually compatible). The fragment of the S gene is modified in its 11

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Strain end with a binding nucleotide sequence ("linker") obtained by chemical synthesis and containing a HindIII site to produce a ligation with the HindiII end of the SV4 virus fragment containing the said promoter. Finally, the Sall end of the fragment, derived from plasmid pLM2, is replaced before ligation with the PvuII (free end) end of the SV40 virus fragment.

The plasmids pLASj. and pLAS ^ 10

These two plasmids are derived from plasmid pLAS described above by introducing into the sole BamHI site (488) one or two BamHI fragments of DNA consisting of 24 base pairs derived from pSKS104 (Shapiro et al. 1983). This 15 fragment of 24 nucleotides encoding eight amino acids and whose introduction into the S gene does not modify the read-out phase carries a cleavage site for the restriction enzymes PstI and two cleavage sites for the enzymes HindIII (SalI, Accl).

EXAMPLE 1

Generation of cell lines from mice producing Hbs protein after transfection and selection of the producing clones LMTK ~ cells (clone ID, mouse cells derived from clone L929 lacking thymidine kinase grown in Eagle medium modified with Dulbecco (medium DMEM) supplemented with 10% calf serum and 4 mM glutamine) was subjected to co-transfection (following the technique described by Grahåm and Van der Eb, 1973, modified by Wigler et al., 1979) with DNA from one of three vectors (pLAS, pLAS ^, pLAS ^) and with plasmid pW DNA carrying the aminoglycoside 3'-phosphorus transferase gene 35 APH3 'resistant to neomycin (Colbére-Garapin et al., 1981). The transfected cells expressing enzyme APH3 'were selected in the presence of 400 µg / ml amine.

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noglycoside G418 (Colbére-Garapin et al., 1981). The clones obtained from this selection were then tested 5 - for HBs protein production. Thus, 5 x 10 5 LMTK cells were co-transfected with 10 µg DNA from the first 5 plasmid and with 2 µg DNA from plasmid pW. Four days after transfection, the selective G418 medium was applied.

The viable clones obtained were isolated, cultured and tested for their ability to produce HBs antigen in the medium.

The presence of HBs antigen was detected by the radioimmunological test AUSRIA. II (Abbott Laboratories). Among the cellular clones that gave positive response, 3 clones (LAS, LASI and LASII) were selected corresponding to plasmids pLAS, pLASI and pLASII, which were then characterized.

Characterization of the detected particles in the medium of the cellular clones

The clones are brought to flow and the nutrient medium of the cells is changed and allowed to accumulate for 48 hours. Then the supernatant is cleared at 2000 rpm.

25 and centrifuged to sediment the particles (Smith et al., 1983). The compressed precipitate is resumed in buffer, treated with a CsCl gradient, centrifuged and collected, and the fractions tested by RIA (Moriarty et al., 1981). The HBs antigenic activity of the modified and unmodified proteins is seen to be concentrated around a single peak located between the densities of 1.18 3 and 1.24 g / cm, which corresponds to the densities of purified serum particles (Pillot et al. , 1984). A sample is then deposited on a sucrose rose gradient (Moriarty et al., 1981). After collecting the fractions, HBs anti-gene activity accumulates around a single peak with a sedimentation coefficient that appears to be the same among 13

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the three types of polypeptides.

Characterization of the S gene sequences integrated into the genome of the cellular clones LAS, LASI and LASII 5

The cellular DNA of clones LAS, LASI and LASII is prepared by the method described by Gross-Bellard et al. (1973) and digested with the restriction enzymes HindIII and PstI. Following agarose gel electrophoresis, the DNAs 10 are transferred to a layer of nitrocellulose (Soutern, 1975), which is hybridized with a radioactive probe made from a DNA fragment containing the S gene, by so-called "nick translation" ( Rigby et al., 1977).

After autoradiography of the copies on nitrocellulose, it appears that one or more of the genes have been integrated into the three clones. In addition, a single Pst I site exists at the level of the modified S gene in the cellular clones LASI and LASII. This PstI site does not exist in the natural sequence of the S gene used (P. Charney et al., 1979), whereas it exists in the foreign DNA fragments introduced into the plasmids pLASI and pLASII used. .

25 Immunoprecipitation with an anti-HBs antigen serum of proteins secreted by clones LAS, LASI and LASII

The confluent cellular clones are labeled with methionine 35 [S] for 48 hours, and the supernatants are immunoprecipitated with antibodies to rabbit anti-HBs antigen (Behring) and then with protein A Sepharose (Pharmacia) in the presence of 0.5% NP40. After waxing, the immunoprecipitates are applied to a 15% polyacrylamide gel according to the technique described by Laemmli (1970) in the presence of molecular weight markers (Pharmacia). The treated and dried gels are then subjected to autoradiography.

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For each supernatant, 2 significant bands appear. Their molecular weights are 23000 and 27000 for the LAS clone, 24750 and 28500 for the LASI clone, and 26000 and 29000 for the LASII clone, respectively.

5 Plasmid pLAS has been shown to be effective in promoting the expression of HBs antigen in L cells. In addition, plasmid pLASI carries additional restriction sites in the region where the S gene is encoded, which may facilitate introduction of new sequences.

The fact that RIA can detect the structures corresponding to HBs antigen particles in the cellular supernatants makes it possible to predict that the 15 structures induced by plasmids pLASI and pLASII retain at least one antigenicity , which partially corresponds to the antigenicity of the particles in human serum. The introduction of the 8 or 16 amino acids used prevents the secretion of the modified HBs antigenic proteins into the cytoplasm facing the outer medium of the L cells.

The choice of the BamHI site for carrying out the introductions into the S gene seems justified. In fact, it corresponds to the beginning of the major hydrophilic region of the protein (P. Tiollais et al., 1981) and respects the fact that the hydrophobic Sequences must be in contact with the lipid membrane of the particles. The intermembrane region of the HBs antigen, which is responsible for the structure of the particles with a size of 22 nm, apparently undergoes only minimal conformational changes.

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The analyzes of supernatants from cellular clones with cesium chloride and sucrose gradients do not make it possible, within the experimental limits used, to distinguish the differences between the modified and unmodified structures.

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An analysis of the cellular DNAs shows that the integrated S genes always carry the modifications contained in the plasmids used.

5 The proteins that emerge after immunoprecipitation show the expected differences in molecular weight. However, the measured molecular weight of the modified proteins appears to be higher than the real molecular weight (the molecular weight of a fragment of 8 amino acids is actually 957). On the other hand, the modifications always allow partial glycosylation of the HBs antigen protein. The two bands that appear on the polyacrylamide gel are characterized by a glycosylated polypeptide and a non-glycosylated polypeptide. The residue most likely to be glycosylated, namely asparagine residue 146 (Machida et al., 1983), is included in the sequence that participates in the major antigenic assay (Pillot et al., 1984). Thus, the conformation of this part of the protein must be relatively identical to the conformation of modified or natural proteins.

EXAMPLE 2

Preparation of particles carrying surface antigens 25 for hepatitis B modified by introduction of sequences of diphtheria toxin

Plasmid pTD134 DNA containing the diphtheria toxin gene (M. Kaczorek et al., 1983) is cut by the enzyme 30 Haelll, treated with NAL31 nuclease and ligated in the presence of T4 DNA ligase with BamHI adapters. After cleavage with the enzyme BamHI, the fragments are ligated with plasmid pSKS105 DNA (Shapiro et al., 1983) cut with the same enzyme. This DNA is then used to transform a 35 strain of E. coli. The colonies are transferred to layers of nitrocellulose where they are lysed. The nitrocellulose layers are hybridized with a radioactive probe produced by

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16 "nick translation" based on a purified fragment on an acrylamide gel after digestion of plasmid pTD134 with HaellI endonuclease (fragment Haelll 597-HaeIII 746). The plasmids from the colonies forming hybrids with this probe are partially sequenced using the method described by Maxam and Gilbert (Maxam et al., 1980). One of the plasmids containing a BamHI fragment encoding amino acids 201-231 of the diphtheria toxin gene is selected (Kaczorek et al., 1983). This fragment 10 is then reintroduced at the BamHI site in plasmid pLAS.

The new plasmid pTAS is purified and transfected into mouse L cells. After selection of the cellular clones resistant to G418 (according to the method described in the first section of Example 1), the presence of HBs antigen in the supernatants is tested. Of the 20 clones studied, none are positive.

The cells from 10 clones are treated with trypsin, washed 2 times with PBS and lysed by 3 times freezing and thawing in 250 µl Tris (10 mM) pH 7.4, EDTA (1 mM). The lysate is cleared at 2000 rpm and tested. All the lysates of the clones contain HBs antigen.

25 One of the clones is lysed under the same conditions. Subsequently, samples for treatment with cesium chloride clay sucrose gradients are transferred together with HBs antigen-purified human serum (IPP) particles and a lysate of clones transfected by plasmid pLAS producing unmodified particles. It turns out that no difference can be detected between the particles of human serum and the HBs antigen signals from the lysates studied. This seems to prove that, although not expressed, the modified proteins, after lysing the cells, are in a conformation that is relatively identical to the conformation of the "natural" particles. An introduction to the BamHI site (amino acids 112-113) of HBs antigen

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17 32 amino acids encoding a portion of diphtheria toxin no longer allow a secretion of the protein when translated into the L cells. This is evidenced by the experience of plasmid pTAS. However, the fact that the modified protein is recovered in the form of non-secreted particles makes it possible to assume that it will be possible to modify HBs antigen and express it in a non-secretory system (e.g. yeast). The maintenance of certain structures gives the protein 10 a high degree of stability, which facilitates its purification.

Example 3 Preparation of Particles Carrying the Surface Antigen of Hepatitis B Modified by Insertion of Poliovirus Sequences

The 2 strands of a 47 base pair DNA fragment encoding 20 for 11 amino acids of the poliovirus type I protein VP1 (amino acids 93-103) and for 2 sites recognized by the enzyme BamHI are chemically synthesized by a automated synthesizer (Applied Biosystem). The 2 strands are purified separately on a denaturing polyacrylamide gel and then hybridized. The fragment is cut by BamHI endonuclease and introduced into the BamHI site in plasmid pLAS. The new plasmid pPAP (Fig. 2) is partially sequenced (Maxam and Gilbert, 1980), amplified and introduced into the L cells by the method previously described; see the first 30 sections of Example 1. The clones resistant to G418 are isolated and their supernatants tested for the presence of HBs antigen. 14 out of 20 clones were found positive.

For the purification of particles of 22 nm size, the cellular clones are grown in DMEM and after 8 days the cell supernatants are subjected to a clearance where 18

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after adding 45% of an ammonium sulfate solution (pH 7.5).

The precipitate is collected by centrifugation, after which the granules obtained are dissolved in 10 mM Tris-HCl, pH 7.5, 150 mM NaCl and 1 mM EDTA (TNE) and dialyzed against the same buffer.

0.3 mg / ml CsCl is added and then centrifuged for 72 hours at 4 ° C at a speed of 40000 rpm. in a Beckman 60 Ti rotor.

After centrifugation, 1 ml fractions are collected which are tested for HBs antigen by RIA.

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The fractions containing HBs antigen are regrouped and centrifuged again in CsCl at 4 ° C for 48 hours at 47000 rpm. in a Beckman 50 Ti rotor.

From the supernatant, fractions of 0.33 ml are harvested, which is tested again for HBs antigen present.

The fractions corresponding to a maximal HBs antigenic activity are regrouped and dialyzed against TNE, after which the 25 HBs antigen is precipitated by centrifugation for 24 hours at 28000 rpm. in a SW41 rotor. The precipitate is kept in suspension in 0.5 ml TNE and placed in a linear 10-30% (w / w) sucrose gradient in TNE with 0.5 ml 66% sucrose.

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Centrifuge for 4.5 hours at 40 ° C and a speed of 35000 rpm. in a SW41 rotor, after which fractions of 0.33 ml are taken from the supernatant.

The fractions corresponding to a maximal HBs antigenic activity are regrouped and dialyzed against TNE. The purified sheath particles are analyzed by SDS-polyacrylamide gel

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19 trophoresis followed by a staining with silver.

The concentration is determined by the BioRad method.

The particles purified from the culture medium containing the cellular clones (PAP and LAS) and transfected by pPAP and pLAS, respectively, have approximately the same density in CsCl. They do not differ significantly from human HBs antigen particles, as demonstrated by sedimentation experiments in sucrose, but they appear to have more variable diameters.

The polypeptides HBs antigen and HBspolioAg (HBs polio antigen), which are immunoprecipitated by an anti-serum anti-HBs antigen obtained from LAS and PAP particles, respectively, are in glycosylated and non-glycosylated form.

The difference of 1.5 kDa (kilodalton) between the apparent molecular weights of HBs antigen and HBspolioAg corresponds to 20 the molecular weight of the introduced sequence.

These results show that such introduction does not prevent the specific interactions between proteins and lipids needed to collect the envelope particles, nor impede the glycosylation and secretion of particles in the culture media.

In order to determine whether the sequence of the introduced polio virus is expressed on the surface of the particles, and to investigate whether changes in the conformation have been induced, studies have been conducted on the susceptibility to protease of HBs antigen and HBspolioAg particles.

35 Cellular clones (LAS, PAP) were grown under confluence, washed twice with DMEM without methionine and incubated for 2 hours in the same medium, to which 420 was further added.

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mM glutamine and 1% fetal calf serum.

The incubation was extended to 24 hours in a fresh medium containing 2 x 10 6 cells and 100 µCi / ml S-Met 5 (100 Ci / mmol; Amersham).

After 6 hours of incubation in the presence of 30 µg / ml of unlabeled Met, the sheath particles were partially purified from the supernatant of the culture medium and centrifuged for 10 24 hours at 28000 rpm. and at 4 ° C (SW41 rotor), then centrifuged against a CsCl gradient (1.1-3.6 g / cm) for 24 hours at 4 ° C at 35000 rpm.

0.5 ml fractions were collected and the top fractions were dialyzed against PBS. 100 µl samples were mixed with 50 µl trypsin (300 µg / ml Worthington) in PBS with or without 3% β-mercaptoethanol and incubated for 2 hours at 37 ° C. Then 50 µl of a solution of 300 µg / ml of a soy trypsin inhibitor in PBS (Worthing / 20 ton) was added. The volume was increased to 400 µl with PBS and 1% bovine serum albumin, 1% sodium deoxycholate and 0.1% SDS were added. Immunoprecipitation was obtained after standing overnight at 4 ° C in the presence of rabbit antiserum directed against human HBs antigen particles (Behring) at a dilution of 1: 100. 50 µl of Sepharose Protein A was added which was kept in suspension in a volume of 25 mM Tris-HCl (pH 7.2), 2.5 mM EDTA and 2 mM PMSF.

After 1 hour of gentle shaking at 4 ° C, the Sepha-30 rose phase was washed 3 times with 10 mM Tris-HCl (pH 7.2),

NaCl, 1% Triton X-100, 0.1% SDS and 1% sodium deoxycholate and 2 times with 25 mM Tris-HCl (pH 6.8).

Finally, the proteins were eluted, allowing the sepharose to boil in 40 µl of a buffer solution for gel electrophoresis.

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For the gel electrophoresis, a 15% polyacrylamide gel was used and the method described by Laemli was used.

After treatment by fluorography, the gel was dried and exposed to a film (Kodak XAR-5) at -70 ° C.

In this way, it was found that HBs antigen particles are highly resistant to trypsin under non-reducing conditions, whereas HBspolioAg is completely cleaved at a single site (or at several nearby sites) to produce polypeptides of apparent molecular weight at 17.4 and 14.3 kDa.

The size of the fragments corresponds to the cleavage 15 of the inserted sequence.

In the presence of a reducing agent, the HBs antigen is cleaved only at the Arg-122 level (Peterson, DL), forming fragments of 16.6 and 13.2 kDa, while 20 HBspolioAg is cleaved into 2 fragments of 17.4 and 13, 2 kDa.

This indicates that the 14.3 kDa fragment obtained under non-reducing conditions from HBspolioAg contains Arg122 and is 10-12 amino acids longer at the N-terminal than the 13.2 kDa fragment. This also confirms that the cleavage of HBspolioAg particles under non-reducing conditions takes place around one or more Lys residues in the introduced sequence (Fig. 2).

These results show that the introduced peptide sequence becomes readily accessible to proteases, that is, it is exposed on the surface of the envelope hybrid particles. In type 1 (Mahoney) polioviruses, the peptide sequence corresponds to a less exposed structure, which renders the Lys residue inaccessible to trypsin under the reducing conditions (Fricks et al.).

22

DK 166357 B

The other potential cleavage sites in the hybrid HBspolioAg particles are inaccessible to trypsin, which also indicates that these portions of the HBs antigen molecule remain in a highly structured scheme.

5

However, a number of conformational changes may occur in the main antigenic region of HBs. This is shown by a reduction (about 20-fold) of the binding of anti-HBs antigen antibody to HBs antigen particles as determined by radioimmunology, using HBs antigen particles as a reference and determining the proteins by staining with silver after SDS-PAGE.

Experience with immunoprecipitation of HBs antigen and 15 HBspolioAg with different serum types indicates that the 2 particles react with anti-HBs antigen antibody and that HBspolioAg particles are specifically immunoprecipitated by monoclonal C 2 different antiserum types against the 20 synthetic oligopeptides containing the introduced sequence.

In order to assess the immunogenic properties of the hybrid particles, mice were immunized with HBs antigen 25 or HBspolioAg (Table 1). Ascites were prepared intraperitoneally by means of tumorigenic cells which do not produce antibodies to obtain an ascetic fluid which is immunologically similar to mouse serum (Anacker et al.).

30

A vaccination with HBs antigen leads to a high concentration of antibodies that react with human HBs antigen particles (mouse # 1). However, the mice immunized with HBspolioAg respond only weakly to the HBs antigens (mouse # 2 and 4, Table Ib).

23

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This is consistent with the observation that the determinants of HBs antigen are partially displaced in the hybrid particles.

On the other hand, the introduced poliovirus VP1 sequence is immunologically active, and in all mice it induces antibodies that recognize the synthetic peptides carrying this sequence (Table Ic) as well as the entire VP1 protein in type 1 poliovirus (Western blot). In addition, the antiserum obtained has a specific efficacy against the infecting virus as well as against the virus types denatured by heat, as evidenced by the immunoprecipitation experiments listed in Table Id. Furthermore, all of the tested antiserum types possess a significant content of antibodies which neutralize poliovirus (Table 1c).

Preliminary results have shown that HBs antigen particles also become immunogenic in rabbits. Following injection of 2 20 doses of HBspolioAg (10-40 µg per dose), 3 out of 4 animals form antibodies that can immunoprecipitate with the infecting polio virus.

The ability of HBspolioAg particles to produce neutralizing antibodies that recognize a common rare epitope on the infecting poliovirus and are denatured by heat (Emini et al.) Shows that the binding activity to the antibody and immunogenicity of the corresponding amino acid sequence at least in part 30 is expressed on the surface of the HBV envelope particles.

This short sequence forms a peak on the capsid of the poliovirus (Hogle et al.) And, as a result, may have an autonomous structure.

In addition, in the HBspolioAg particles, the adjacent HBsAg sequences can maintain stability or flexibility.

DK 166357 B

of the introduced poliovirus sequence.

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DK 166357 B

The studies on the immunogenic properties of HBs antigen and HBsPolioAg, which are consistent with the previous commented results from Table I, were performed as follows: 5 a) Mice of the Balb / C strain, 8 weeks old, were immunized with either 2 µg HBs antigen (# 1) or 30 µg HBsPolioAg (# 2-4), which was purified as previously described, by intraperitoneal injection in combination with an emulsion 10 of 50% complete Freund's adjuvant supplemented 2 weeks later with incomplete Freund's adjuvant.

After 3 weeks, myeloma cells were injected from mice of strain sp2 / 0-Agl4 (Couillin et al.) Followed by a 15 post-injection without adjuvant.

Mice # 5 were treated without antigen. The ascites fluid was collected after 2 weeks and analyzed using the procedures described below.

B) The concentrations of anti-HBs antigen were determined by a radioimmunological test called AU5AB (Abott) and expressed in international units (I.E.).

C) The binding to the peptide corresponding to amino acids 93-104 in poliovirus VP1 was determined by the ELISA method (Voller et al.).

The wells were coated with this peptide (0.5 µg 30 in PBS), and after standing overnight, the vacant sites were blocked with BSA (1% in PBS containing 0.05% Tween-20).

After repeated washings with Tween-20 (0.05% in PBS) 35, the ascites liquors were added at a dilution of 1:80 to PBS containing 1% BSA and 0.05% Tween-20 and incubated for 2 hours at 37 ° C.

DK 166357 B

27

The wells were washed and goat anti-mouse IgG antibody (Cappell), which was labeled with peroxidase and diluted to 1: 1000.

After leaching, o-phenylenediamine (Merck; 0.5 ag / ml) was added in 50 mM citrate / phosphate buffer, pH 5, and after 10 minutes at room temperature, the reaction was quenched by the addition of 2.5% 2 SO 2.

The positive serum samples were those that exhibited an absorption value (DO) that was at least 3 times the control value (mouse # 5).

D) Type 1 poliovirus (Mahoney) was labeled with S-Met 15 and purified by centrifugation in a CsCl gradient.

Heat-denatured virus was prepared by incubating infecting virus for 1 hour at 56 ° C.

20 Samples of 50 µl containing 15000 cpm particles labeled 35 with S-Met (Emini et al) in solution in 150 mM NaCl, 5 mM EDTA, 50 mM Tris (pH 7.4), 0.02% NaN , 05% Nonid and P40 were incubated in the presence of 50 µl ascites fluid from mice. Incubate for 1 hour at 37 ° C and overnight at 4 ° C.

The immune complexes were precipitated with Staphylococcus aureus (strain Cowan I) (Kessler) and their radioactivity tested. The figures in Table 1D show the percent 30 values of the immunoprecipitated activity.

e) The concentration of neutralizing antibodies in each serum sample was measured by a standard reduction test with VERO cells, adding an amount of 10 stain-forming units of type 1 poliovirus (Mahoney).

DK 166357B

28

The inverse serum dilutions (log 2) yielding 5% stain reduction were calculated from regression curves for the mean values obtained on 3 trials.

5 The experiments with the modified gene of plasmid pPAP show that the foreign sequences introduced into the protein can be expressed on the surface of the particles. If the original conformation of the exogenous sequences is modified, it is reasonable to assume that the introduction of larger structures 10 or an exclusion of certain portions of the HBs antigen protein may solve this problem. A research into other places of introduction is also possible. An insertion of 8 amino acids at the residue 50 into the HBs antigen (at the site Ball in the S gene) enables the production of particles and their secretion.

Thus, the invention enables the preparation of mixed vaccines.

Inserting the determining sequence of the foreign antigens into the HBs antigen makes it possible, if the HBs antigen particles are expressed on the surface, to produce mixed vaccines against another subtype of HBV virus, against virus determinants (HBcAg, HBeAg). (AM

25 Prince et al., 1983, S. Iwarson et al., 1984), against antigenic determinants of virus affecting the same populations as hepatitis B (AIDS, herpes) and against antigenic determinants of other virus types (TM Shinnick et al. , 1983). The 8 amino acids contained in the major antigenic determinant of the poliovirus type 3 VP1 protein (D.M.A. (Evans et al., 1983)) can be introduced into the HBs antigenic protein via an intermediate DNA fragment synthesized chemically.

35 This type of manipulation may also allow expression of biologically active sequences which have pharmaceutical interest.

29

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The particles produced by animal Hepadna virus (P.L. Marion et al., 1983) can be used under the same conditions.

For this reason, the invention encompasses any vaccine composition against viral hepatitis B which contains an effective dose of particles of the invention, especially 3-6 µg protein per day. ml, e.g. 5 µg protein per ml (unit dose), in combination with a suitable pharmaceutical carrier selected according to the route of administration selected. This is preferably parenteral.

The list below lists the literature references referred to above.

15 - Anacker, R.L. and Munoz, J.J. Immunol. 87, 426-432 (1961).

- Cattaneo, R., Will, H., Hernandez, N. and Schaller, H., (1983) Nature, 305, 336-338.

20 - Colb'ere-Garapin, F., Horodniceanu, F., Kourilsky, P.

and Garapin, A.C. (1981), J. Mol. Biol., 150, 1-14.

- Couillin, P., Crainic, R., Cabau, N., Horodniceanu, F.

& Boué, A., Ann Virol. (Inst. Pasteur) 133E, 315-323 (1982).

25 - Dubois, M.F., Pourcel, C., Rousset, S., Chany, C. and

Tiollais, P. (1980) Proc. Natl. Acad. Sci. USA 77, 4549-4553.

- Emini, E.A., Bradford, A.J. and Wimmer, E., Nature 304, 699-703 (1983).

30 - Evans, D.M.A., Minor, P.D., Schild, G.S. and Almond, J. W., (1983) Nature 304, 460-462.

- Fricks, C.E., Icenogle, J.P. and Hogle, J.M., J. Virol.

54, 856-859 (1985).

- Graham, F.L. and Van der Eb, A. J., (1973) Virology, 52, 35 456.

- Gross-Bellard, M., Oudet, P. and Chambon, P., (1973)

Europ. J. Biochem. 36, 32.

30

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- Hogle, J. M., M. and Filman, D. J., Science 229, 1358-1365 (1985).

- Hollinger, F.B., Sanchez, Y., Troisi, C., Dressman, G.R. and Melnick, J.L., (1984) The 1984 International Symposium on Viral Hepatitis, San Francisco, USA.

- Iwarson, S., Tabor, E., Thomas, H., Snoy, P. Gerety, R. J., (1984) The 1984 International Symposium on Hepatitis Virus, San Francisco, USA.

- Kaczorek, M., Delpeyroux, F., Chenciner, N., Streeck, 10 R.E., Murphy, J.R., Boquet, P. and Tiollais, P. (1983),

Science 221, 853-855.

- Kessler, S.W., J. Immunol. 115, 1617-1624 (1975).

- Laemmli, U.K., (1970) Nature, 227, 680-685.

- Lusky, M. and Botchan, M. (1981) Nature, 293, 79.

15 - Machida, A., Kishimoto, S., Ohnuma, H., Miyamoto, I.,

Baba, K., Oda, K., Nakamura, T., Funatsu, G., Mijakawa, Y. and Mayami, M., (1982) Mol. Immunol. 19, 1087-1093.

- Marion, P.L., Knight, S.S., Feitelson, M.A., Oskiro, L.S. and Robinson, W.S. (1983) Journal of Virology, 48, 20 534-541.

- Maxam, A. and Gilbert, W. (1980), Methods Enzymol. 65, 499.

- Michel, M.L., Pontisso, P., Sobczak, E., Malpi'ece, Y., Streeck, R.E. and Tiollais, P., (1984), Proc. Natl. Acad.

Sci. USA 81, 7708-7712.

- Moriarty, A.M., Hoyer, B.H., Wai-Kuo Shih, J., Gerin,

JL and Hamer, D.H., (1981) Proc. Natl. Acad. Sci. USA

78.

- Neurath, A.R., Kent, S.B.H. and Strick, N., (1984) The 30th 1984 International Symposium on Hepatitis Virus, San

Francisco, USA.

- Newmark, P. (1984), Nature 311, 510-511.

- Pasek, M. et al., Nature (London) 282-575 (1979).

- Peterson, D. L., Paul, D. A., Gavilanes, F., and Achord, 35 D. T., in: Advances in Hepatitis Research (ed. Chisari, F. V.) 30-39 (Mason Publishing U.S.A., Inc. 1984).

- Pillot, J. and Petit, M.A. (1984) Molecular Immunology, 31

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21, 53-60.

- Prince, A.M., Vnek, J. and Stephan, W., (1983) Develop.

Biol. Standard. 54, 13-22 (S. Kargel, Basel).

- Rigby, P.W.J., Dieckmann, M., Rhodes, C. and Berg, P.

5 (1977) J. Mol. Biol. 113, 237.

- Shapiro, S.K., Chou, J., Richaud, F.V. and Casadaban, M.J., (1983) Gene, 25, 71-82.

- Smith, G. L., Mackett, M. and Moss, B. (1983) Nature, 302, 490-495.

10 - Southern, E.M. (1975), J. Mol. Biol. 98, 503-517.

- Stibbe, W. and Gerlich, W. (1983) Journal of Virology 46, 626-628.

- Tiollais, P., Charnay, P. and Vyas, G.N. (1981) Science 212, 406-411.

15 - Valenzuela, P., Medina, A. and Rutter, W.J. (1982)

Nature, 298, 347-350.

- Valenzuela, P., in Proceedings and the Twelfth International Conference on Yeast Genetics and Molecular Biology, Edinburgh 1984, 16.

20 "Van der Werf, S., Wychowski, C., Bruneau, P., Blondel, B., Crainic, R., Horodniceanu, F., and Girard, M. (1983),

Proc. Natl. Acad. Sci. USA 80, 5080-5084.

- Voller, A., Bedwell, D.E. & Berlett, A. ϊ A Guide with Abstracts of Microplate Applications, p. 1 (Dynatech, 25 Guernsey 1979).

- Wigler, H., Pellicer, A., Silverstein, S., Axel, R.,

Urlaub G. and Chasin, L., Proc. Natl. Acad. Sci. U.S.A.

76, 1373-1376 (1979).

30 35

Claims (14)

1. Particles containing such a high proportion of the characteristic amino acid sequences of the major polypeptide of the hepatitis B virus surface antigen (HBs antigen) that this portion is sufficient to impart to the particles a structure similar to the structure of the natural particles Characteristic of HBs antigen, characterized in that the particles on their surface and embedded in the main polypeptide carry at least one foreign amino acid sequence, which preferably itself contains an immunogenic region, and which is inserted into one of the hydrophilic regions of the main polypeptide. usually exposed to the outer surface of the particle or replacing one or more of the amino acids in one of the hydrophilic regions of the main polypeptide. Particles according to claim 1, characterized in that the foreign amino acid sequence is inserted into the region comprising amino acids 32-74 of the polypeptide. Particles according to claim 1, characterized in that the foreign amino acid sequence is inserted into the region comprising amino acids 110-156 of the polypeptide. Particles according to any one of claims 1-3, characterized in that the foreign polypeptide sequence has a size not exceeding 16 amino acids, preferably a size of 5-16 amino acids, especially 6-13 amino acids. Particles according to any one of claims 1-4, characterized in that they also contain the polypeptide sequence encoded in the pre-S region of the hepatitis B virus genome. DK 166357 B Recombinant DNA containing a DNA sequence encoding the S region and optionally also the pre-S region of the genome of the major polypeptide of hepatitis B virus, characterized in that it comprises at least one nucleotide sequence encoding for said foreign amino acid sequence, in at least one of the zones in the S region corresponding to the hydrophilic regions of said major polypeptide of hepatitis B virus, and that the S region and optionally also the pre-S region which is located itself in the DNA is under the direct control of an exogenous promoter known to effectively activate the transcription of the genes under direct control of the promoter in eukaryotic cells, especially human or animal cells or yeast cells, to which the vectors is calculated. 15 Recombinant DNA according to claim 6, characterized in that the nucleotide sequence encoding said foreign sequence is located in the region of the S gene encoding the polypeptide extending between the 20 amino acids 32 and 74 of the main polypeptide. , which is part of the constitution of HBs antigen. Recombinant DNA according to claim 6, characterized in that the nucleotide sequence encoding said foreign sequence is located in the region of the S gene encoding the polypeptide extending between amino acids 110 and 156 of the major polypeptide. , which is part of the constitution of HBs antigen. A recombinant DNA according to any one of claims 6-8, characterized in that said foreign nucleotide sequence encodes a polypeptide comprising at most 16 amino acids, preferably 6-13 amino acids. Recombinant DNA according to any one of claims 6-9, characterized in that the aforementioned exogenous promoter is one of the promoters found in SV40 virus. DK 166357 B A cell line derived from a host organism, especially a human or animal host organism or yeast, capable of recognizing the exogenous promoter of a recombinant DNA according to any of claims 6-10, characterized in that it comprises a recombinant DNA according to any of claims 6-10 incorporated in the genome. Cell line according to claim 11, characterized in that it is composed of cells that recognize the promoter of SV40 virus. A method for producing particles according to any one of claims 1-5, characterized in that a cell line, in particular a human or animal cell line, is transformed with a recombinant DNA according to any one of claims 6-10, promoter is selected according to the nature of the host cell used, and to culture the thus-transformed microorganism, after which the particles produced are recovered. 20 Process according to claim 13, characterized in that the foreign sequence incorporated in the main polypeptide comprises at most 16 amino acids and that the generated particles are recovered from the culture medium of the host organism in which they are secreted. 30 35