IE60387B1 - Hepatitis B virus vaccine - Google Patents

Abstract

A DNA fragment capable of coding for HBsAg and DNA derivatives of Dane particles is fused to the region for controlling the arg3 gene of yeast and cloned in a yeast vector. The recombinant vector is employed for transforming the competent yeast cells in which the HBsAg gene is expressed.

Description

HEPATITIS B VIRUS VACCINE The invention relates to recombinant DNA molecules which code for the hepatitis 3 surface antigen, to their preparation and to the vaccines derived therefrom.

In other words, the invention relates to the cloning of a gene which codes for the hepatitis 3 surface antigen in yeast,· by application of the so-called recombinant DNA techniques t, as well as to the preparation of a vaccine against the hepatitis 3 virus from the antigen produced by the yeast.

The hepatitis 3 vims (HBV) infection constitutes a serious and universal health problem. The infection can reveal itself in the acute or chronic stage. The number of cases of acute hepatitis in the United. States is estimated to be at least 100,000 a year with a level of clinical mortality of 1 to 2%. In the United States, the prevalence of chronic carriers of HBV among healthy adults ranges between 0.1 and 1% depending on age and social class. In South America, the prevalence of chronic carriers is about X to 3%, in the USSR and in Southern Europe about 3 to 6%, and in Asia and Africa more than 10%.

In the developed countries, the populations at high risk of exposure require a vaccine,, especially patients and staff of medical units where blood is handled, military staff, the partners of chronic carriers, travellers visiting areas of high HBV endemicity, the newborn babies of chronic carriers, homosexuals, prostitutes and those abusing drugs. In third world countries, there is a need for a cheap vaccine designed for the immunisation of the general population. Mass immunisation programmes can have an influence, in the final analysis, not only on the incidence of acute hepatitis and that of the group of chronic carriers, but they can also reduce the morbidity and mortality resulting from evolutive chronic hepatitis and hepatocellular carcinoma.

Dane particles, which are thought to be hepaticis 3 virions and which can be isolated from contaminated patients, have a diameter of about 42 nm. Each particle is composed of an envelope containing the hepatitis 3 surface antigen (HBsAg), a capsid(HBcAg), an endogenous polymerase and a DMA genome. The genome is circular and is double-stranded, with a single-stranded region containing about 200 bases. The single-stranded region can be synthesised in vitro by the action of the endogenous polymerase. The longest strand contains about 3200 bases.

It proved difficult to prepare HBV vaccines since it was found to be difficult to propagate the virus in a tissue culture and since the only known host is man. Small quantities of authentic HBV antigens have been isolated from contaminated human beings. The ff-P-C Reports, pp 3-4, 19 July 1982, contain a report of clinical studies on the subject of a vaccine recently developed against hepatitis B.

Valenzuela et al., Mature, Volume 298, 347-350 (1982) mention th® synthesis of HBsAg in yeast by means of an expression vector in which th® sequence which codes for HBsAg is a Taql-HpaX fragment of 835 base pairs (pb) and the promoter is th© yeast alcohol dehydrogenase promoter. Several previous brief reports have mentioned the research preceding this reference, namely a report by Valenzuela et al., Arch- Biol. Med. Exp. (Chile) Volume 14(1), 21-22 (1981) which mentions the expression, in yeast, of a DNA fragment containing a sequence which codes for a protein analogous to HBsAg, which sequence is ligated into a yeast alcohol dehydrogenase promoter region; a report in Scrip. Mo. 616, p 14 (12 August 1981) which states that a team of researchers In the United States, comprising P. Valenzuela and W. J. Rutter, has announced the production In yeast of the protein envelope surrounding the hepatitis B virus; and a report by Zuckerman, Nature, Volume 295, 98-99 (1982) which mentions that w. J. Rutter has reported the expression of glycosylated HBsAg in yeast cells,.

Antigenic components of HBV, such as HBsAg, have been prepared in bacteria after the insertion of a recombinant DNA molecule containing a gene which codes for the antigen. Burrell et al., Nature. Volume 279 No. 5708, 43-47 (1979) mention the expression in the strain KB 101 of B. coli of the HBV DNA sequences cloned into the plasmid pBr 322.

Murray et al.t European Patent Application No. 12823, describe th® preparation of a recombinant vector which can code for the HBV antigens, including HBsAg, in the strain KB 101 ox E. coli. The vector is prepared from the DNA of the Dane particle and the plasmid pBR322. The authors state that the useful hosts may include other bacterial hosts, yeasts and other fungi, animal or plant cells and other hosts, although the only demonstrated host is Ξ. coli.

Chamay et al.. Nature > Volume 286, 893-895 (1980) mention the construction of a bacteriophage comprising a fusion of the ^-galactosidase gene and the HBsAg structural gene. The bacteriophage directs the synthesis of a fusion protein comprising the antigenic determinants of the HBsAg and of the ^-galactosidase.

Tiollais et al., United Kingdom Patent Application Ho. 2,034,323, describe th© preparation of a coliphage containing the DNA of HBV. Th© HBV DNA, fused with phage DNA, is transformed in the strain C600 of B. coli.

In Wired Kingdom Patent Application No. 2,070,621, first published in the form of a PCT application W081/00577, there is described a plasmid which comprises a portion of the HBsAg gene and the promoter and the Z gene of the lactose operon and which can be cloned into B. coli.

Rutter et al., European Patent Application No. 20251, describe recombinant vectors comprising a recombinant vector which contains the plasmid p3R322 and BamHI fragments of the HBV DNA which can be used to transform E. coli. Another vector comprising a BamHI fragment of the HBV DNA and a portion of the tryptophan operon was used to obtain expression in the strain HB101 of E. coli.

Edman et al., Nature, Volume 291, Number 5815, 503-506 (1981), describe the construction of plasmids which direct the synthesis of HBcAg and of the fusion protein j3=lactamase=HBsAg, under the control of the regulatory region of the tryptophan operon, in E. coli.

Other references describing the insertion of the HBV DNA in bacteria include those of Charnay et al., Prog. Med. Virol., Volume 27, 88=92 (1981); Mac Kay et al.-, Proc. Natl. Acad. Sci. U.S., Volume 78, Number 7, 4510-4514 (1981); Fritsch et al., C.R. Acad. Sci., Volume 287, Number 16, 1453 (1978); United Kingdom Patent No. 2,034,323 (Derwent No. 46874C) and Pasek et al., Nature, Volume 282 No. 6, 575 (1979).

The HBV DNA has also been cloned into mammalian cells. These comprise cell lines of human origin, those of mice and those of human hepatoma. For example, Dubois et al., Proc. Natl. Acad. Sci. O.S., Volume 77,, Number 8, 4549-4553 (i960), mention the transformation of murine cells by means of a plasmid containing the EQBV genome and the expression of HBsAg; Hirschman st al., Proc. Natl. Acad. Sci. U.S.,, Volume 77, Number 9, 5507-5511 (1980) mention th© production of particles which resemble HBV, by HeLa cells transformed with the HBV DNA.

Methods for the preparation of th© HBV vaccine using HBsAg from human blood, are mentioned by Funakoshi et al., Frog. Med. Virol-, Volume 27, 163=167 (1981) and Maupas et al., Prog. Med. Virol., Volume 27, 185-201 (1981). The vaccine prepared by Funakoshi et al., contains 40 //c of formalin-treated and purified HBsAg, sodium phosphate/chloride, 20 mg of mannitol and 0.1% aluminium hydroxide as adjuvant. In the last document, Ma,upas et al. mention that one dose of vaccine represented 1 ml of foxmaXiu-treated and purified HBsAg containing 2 to 10 pg/ml of protein (Lowry method) and 0.1% of aluminium hydroxide. The procedure used in the study mentioned by Maupas et al. involved three injections at one month intervals with a booster after one year; the authors propose a procedure consisting of two injections of concentrated HBsAg at three month intervals .

Additional references relating to the preparation of HBV vaccines include those of Maupas et al.? pages 3 e 37 and 57 respectively,, and of Hepatitis B Vaccine INSEBM Symposium No. 18,- edited by Maupas and Guesry, 1981, Elsevier/North Holland Biomedical Press.

Yeasts have been used as host organisms for some other DNA sequences, for example, Fraser et al., United Kingdom Patent Application Ho. 2,068,969? describe the preparation of chicken ovalbumin in yeast; Scrip No. 640, p. 11 (4 November 1981) contains a report according to which a type of interferon is reportedly prepared in yeast. In European Patent No. 11562 (Derwent No. 387S2C) ? there are described yeast hybrid plasmids containing the yeast ura3 gene in the plasmid 2 μ.

The invention relates to the preparation of a recombinant DNA molecule comprising a nucleotide sequence which codes for HBsAg and a regulatory region derived from the yeast arqr3 gene which can effect transcription of the HBsAg sequence in the yeast. Saccharomyces cerevisiae. Th® molecule comprises vectors in which the HBsAg sequence and the regulatory region have been inserted? which vectors can be used to prepare a yeast vector or to conserve HBsAg and the regulatory regions. The microorganisms containing the recombinant DNA molecule? especially the microorganisms transformed by the plasmids of the invention? form part of the invention.

The invention also comprises a vaccine designed to stimulate the protection against HBV infection in humans and which contains a vaccinal quantity of HBsAg prepared according to the present invention as well as a suitable excipient.

Furthermore, the invention comprises methods for preparing the recombinant DNA molecule and microorganisms containing the molecules as well as the methods for preparing HBsAg and the vaccine containing HBsAg.

Figure 1 represents a restriction endonuclease cleavage map of pRIT10601.

Figure 2 represents a restriction endonuclease cleavage map of pRIT10616.

Figure 3 represents a restriction, endonuclease cleavage map pMC206.

Figure 4 represents the nucleotide sequence of a portion of the yeast DNA. inserted in the form of a 3300-pb in the Hindi XX fragment into pMC200, which portion contains the Hindi, BlgXI and BcoRX sites.

Figure 5 represents a diagram illustrating the preparation of pRXTOlSzl and of pRIT10673.

The recombinant DNA. molecule of the invention is prepared by fusion of a nucleotide sequence which comprises a HBsAg structural gene with the yeast arg3 regulatory region, which regulatory region can direct transcription of the HBsAg sequence in the yeast, thus bringing about its expression. Recombinant DNA molecule* is understood to mean a DNA fragment containing the sequence which codes for HBsAg and the regulatory region as well as other DNA molecules containing fhe fragment, such as plasmid or phage vectors.

Regulatory region® is understood to mean a sequence containing a promoter region and other sequences required for the transcription. The yeast arg3 regulatory region is particularly advantageous since it can. he a strong promoter for the expression of a sequence which codes for HBsAg"HBsAg" is understood to mean a protein which is structurally identical to th® authentic HBsAg or which effectively possesses the same antigenic determinants as the authentic HBsAg, that is to say a protein capable of stimulating the production of antibodies which specifically recognise the authentic HBsAg and react with it, or a protein specifically recognised by the antiHBsAg antibodies.

The sequence which codes for HBsAg can be isolated from the DNA extracted from Dane particles in human serum by synthesising the fibre complementary to the single-stranded region by means of a DMA polymerase, preferably the endogenous polymerase, followed by a digestion using a restriction endonuclease. The choice of the endonuclease will partly depend on the Dane particles. For example, as illustrated in the examples below, the sequence which codes for HBsAg of the HBV DNA of certain Dane particles of the adw serotype can be isolated from a single BamHI fragment; the sequence which codes for HBsAg of the HBV DMA of certain Dane particles of the ayw serotype can be isolated from an Hhal fragment. The DNA isolated from Dane particles of th© same serotype can also have different, restriction site patterns.

The restriction of the DMA for the purpose of preparing the DNA fragments used in the invention, the ligation ox such fragments in order to prepare the recombinant DNA molecules used in the invention and the insertion into microorganisms are carried out according to known techniques such as the techniques described in the references mentioned previously and subsequently. The conditions are chosen so as to avoid denaturation of the DMA and that of the enzymes. For example, the pH is buffered so as to maintain it between 7 and 11 and the temperature is maintained below 60°C. The restriction is preferably carried out at a temperature of about 30 to and the ligation is carried out at about 0 to 10 "C. The restriction enzymes and the ligases used during the implementation of the present invention are available commercially and should be used in conformity with the directions which are included therein. T4 DMA ligase is the preferred ligase.

The fusion of the HBsAg sequence with the regulatory region can he carried out by means of intermediate vectors, as illustrated in the examples below. The HBsAg sequence can also be inserted directly into a vector containing the regulatory region. This vector is the DNA which can carry and conserve the DNA fragment of the invention, including for example the phages and the plasmids. Techniques for cloning the DNA fragments into the phages are described for example by Charnay et al.. Nature.» Volume 286, 893-895 (1980) and Txollais, United Kingdom Patent Application No. 2,034,323. Preferably, the HBsAg sequence is positioned relative to the regulatory region such that the HBsAg synthesised by expression of the HBsAg sequence is practically free of foreign amino acid residues.

A regulatory region which has been discovered to be particularly useful is derived from the yeast arg3 gene which codes for ornithine carbamoyltransferase (OCT). The use of the ara3 control region is advantageous since Its activity is subject both to specific and general control mechanisms. It has been cloned Into B. coli in th® plasmid bYeura3arcf3 as reported by Crabeel et al., Proc. Natl. Acad. Sci. U.S.A., Volume 78, 5056 (1981). The preferred hosts are the strains of S. cerevisiae, especially the strain lcll697d, ia which the biosynthetic pathway for obtaining arginine is derepxessed. The use of such strains results in an increased expression from the arg3 promoter compared with the other strain, strain DCS, which was used in the examples.

The preferred vector for cloning the fused DNA fragment into the yeast is the plasmid YBpl3 which is capable of being replicated and conserved both in E. coli and S. cerevisiae and is therefor® known by the name of '’shuttle'* vector. Several other yeast vectors are known and are available. HBsAg and the regulatory regions can be sequentially inserted into a yeast vector or., as illustrated in the examples below, simultaneously. Transformation with plasmid vectors will normally result in the incorporation of the DMA molecule of the invention in the form of a plasmid. However, other reactions such as recombination events can result in the incorporation of the DMA molecule into the chromosomal DNA.

The vaccines designed to stimulate protection against HBV infection in humans and which comprise the HBsAg produced by the yeast according to the invention and a suitable excipient can be prepared by known techniques. The use of an adjuvant such as aluminium hydroxide Is desirable. The HBsAg thus produced can also be combined with other antigens In order to prepare vaccine combinations. The HBV vaccines and the vaccine combinations can be administered for example, subcutaneously, Intravenously or intramuscularly- By using DNA hybridisation techniques and various immunological analyses, the DNA fragment ox the invention and the HBsAg thus produced can also be used as probe to detect HBV in biological samples.

EXAMPLES In the following examples of the invention which are illustrative but nonlimitative, all th© percentages are given by weight and all the temperatures are given in degrees Celsius.

Example 1 Preparation of the intermediate plasmid PRIT10601 bv combination of the HBV DMA with PBR322.

HBsAg-positive serum of the avw serotype was defibrinated by addition of CaCl2 up to a final concentration of 0.28% and the mixture was centrifuged for 2 hours at 27,000 rpm In an SW27 rotor, on a 10-20% sucrose gradient prepared in a buffer (pH 7.5) containing Ί0 mM tromethamine-HCl, 1 M NaCl and 1 mM EDTA. A transparent pellet containing the Dane particles was resuspended In the same buffer and it was centrifuged on 20% buffered sucrose arranged in layers on a 65% sucrose cushion. An opalescent band was recovered at the interface of the cushion and it was centrifuged for I hours a 200,000 g in order to pellet the Dane particles.

A. The single-stranded regions of the HBV genome inside the Dane particles were repaired with endogenous DNA polymerase by resuspending the Dane particles In a reaction mixture (pH 8) containing 50 mM dithiothreitol, 50 mM each of dATB, dCTP and dGTP and 8 μΜ [32P]dTTP (350 Ci/mmol) and by incubating the resuspended particles for 5 hours at 37 °C. The re suspension at pH 75 using a buffer containing 10 mM tromethamine-HC!, 10 mM EDTA, 100 M NaCl and 0.02% sodium dodecyl sulphate (pH 7.5) and it was incubated for 1 hour at 37°C with 0.5 mg/ml of pronas® followed by a phenol extraction and an ethanol precipitation.

The digestion of the DNA with restriction endonuclease BamHI provided two radioactive fragments of about 1450 bp and 2600 bp in size as estimated by agarose gel electrophoresis and by gel autoradiography.

B. About 30 ng of the Dan© particle DNA were recovered with the endogenous DNA polymerase In the presence of unlabelled dTTP, they were extracted and recovered as described above. The DNA was mixed with 100 ng of the plasmid pBR322 which was previously digested with the restriction endonuclease BamHI and treated, with alkaline phosphatase. Th© plasmid pBR322 is generally used in th® so-called recombinant DNA techniques and is deposited and accessible without restriction at the American Type Culture Collection under the serial number 37017. The mixture was extracted with phenol, precipitated with ethanol, centrifuged, dried and resuspended In 12 mi of a mixture (pH 7.5) containing 50 mM tromethamine-HCl, 1 saM ATP, 10 mM MgCl2, 10 »M dithiothreitol, 50 pq/ml of gelatin and 2 units/ml of T4 DNA ligase. The suspension was incubated at 10° for 4 h and it was then kept on ice for 18 h.

The mixture of ligated DNA was used fo transform the competent cells of the strain C600 of B. coli K12 which were prepared according to the procedure of Cohen et al., Proc. Natl. Acad. Sci. U.S., Volume 69, 2110 (1972) . The transformants were selected on a solid medium containing ampicillin (200 ^g/xnl). The isolated colonies were screened based on the loss of resistance to the tetracycline, which indicated the insertion of a foreign DNA fragment into the BamHI site of pBR322. Xt was observed that such a transformant clone contains a plasmid pRITlOSOl which, by digestion with the endonuclease BamHI, provided a 4360-bp pBR322 fragment and IS00-bp and 1450-bp HBV DNA fragments. A culture of the strain C600 of B. coli K12 (pRITlOSOl) was deposited on 2 June 1982, in conformity with the provisions of the European Patent Convention (EPC) and the Budapest Treaty, at the American Type Culture Collection, Rockville, Maryland, U.S. A.. under the serial number ATCC 39132» A restriction endonuclease cleavage map of fhe plasmid pRITlOSOl is given In Figure 1.

C. The sizes of the fragments generated by digestion of the Dane particle DNA and of pRITlOSOl with various restriction enzymes were compared as follows. The Dane particle DMA,, that Is to say the HBV DMA, was labelled with 32P by the endogenous polymerase reaction described above or by treating the purified DNA with DNA polymerase I derived from B. coli. The labelled HBV DNA was mixed with pRITlOSOl, fhe mixture was treated with a restriction endonuclease and subjected to agarose gel electrophoresis. The gel was stained with ethidium bromide and photographed under UV radiation In order to localise the DNA. fragments; If was then dried and autoradiographed In order to localise the radioactive HBV DNA fragments. It was found that the following HBV fragments corresponded exactly to the size of the pRITlOSOl fragments: the 1450-bp and ISOO-bp BamHI fragments; a 1330-bp Hpal fragment and a 1130-bp BamHIXhol fragment. The labelled Dane particle DNA also specifically hybridised with fhe two 1450-bp and 1600-bp fragments liberated by the BamHI digestion of pRITlOSOl, on monitoring the transfer of these fragments from an agarose gel onto a nitrocellulose filter according to the Southern technique, J. Mol. Biol., Volume 98, 503 (1975).

These results show that the DNA insert cloned into pRITlOSOl represents the HBV genome and that the relative orientation of the two BamHI fragments in pRITlOSOl is the same as in the virion. pRITlOSOl was used to prepare ORIT10671 in Example 10 below.

Example 2. Preparation of the intermediate plasmid pRITlOSOl by combination of the HBV DNA with PACTC184.

Dane particles, derived from HBsAg-positlve serum of the adw serotype, were isolated as described above. Restriction endonuclease analysis of the 32P~labelled HBV DNA indicated that the DMA contained an EcoRI site.

The HBV DNA, completed by the endogenous polymerase reaction using unlabelled nucleotides, was digested with EcoRI. The DMA digest was mixed with the plasmid pAC7C184 which had previously been digested with EcoRI and treated with alkaline phosphatase. The plasmid pACYC184 is deposited and accessible without restriction at the American Type Culture Collection under the serial number 37033. The mixture was ligated with T4 DMA ligase. The ligated DNA mixture was used to transform the competent cells of the strain CSOO of a,', coli. The transformants were selected on a tetracycline medium (15 /sg/ml) and they were screened based on the loss of resistance to chloramphenicol, which Indicated the Insertion of OACYC184 into th® EcoRI site. It was found that a transformant colony contains a plasmid pRITlOSlS which is composed of pACYCl84 equipped with a 3200-bp insert containing the HBV DNA at the EcoRI site. A restriction map of pRITlOSlS is given in Figure 2. The strain' CSOO of E. coli 02 (pRITlOSlC) was deposited on 2 June 1982, in conformity with the provisions of the EEC and the Budapest Treaty, at the American Type Culture Collection under the serial number ATCC 39131.

Examole 3, Preparation of the intermediate plasmid ORIT10640 containing a nucleotide sequence which codes for HBsAg bv combination of ORIT10S16 with OBR313. pRIT106l6 was purified by centrifugation on a density gradient of a CsCl/ethidium bromide mixture as described by Kahn et al.. Methods in ensymology. Volume 68, 268 (1979).

The DNA was digested with the endonuclease BamHI, it was mixed with the BamHX-digested DNA of the plasmid pBR313 and treated with alkaline phosphatase, it was ligated and used to trans form the competent cells of the strain C600 of E. coli K12 in substance, as described in Example 1. The plasmid pBR313 is deposited and accessible without restriction at the American Type Culture Collection under the serial number 37018.

The transformants were selected on ampicillincontaining agar and they were screened based on the loss of resistance to tetracycline, which indicated the insertion of pBR313 into the BamHI site. It was found that a transformant colony contained a plasmid pRXT10640 which is composed of pBR313 equipped with a 1350-bp insert at the BamHI site. The insert is a nucleotide sequence which can code for HBsAg. It codes for the portion of a protein which may be the precursor of HBsAg, and the total surface antigen contains 565 bp of the 3* noncoding sequences.

Example <- Preparation of the intermediate .plasmid oMC200 containing the arg 3. regulatory region by combination ..of the veast arc3 gene with _pBR322.

A 3300-bp yeast DNA fragment containing the arg3 gene was obtained by digestion of oYeura3arg3 with Hindlll. The plasmid pTeura.3arg3 has been described by Crabeel ©t al., Proc. Natl. Acad.. Sci. U.S. Volume 78, 502S (1981). The 3300-bp fragment was cloned into the Hindi 11 site of pBR322 and it was transformed in the strain MM294 of E., coli K12 in substance, as described above. The transformants were selected on ampicillin medium and they were screened based on resistance to tetracycline., It was found that a transformant colony contained a plasmid pMC2Q0 which comprises p3R322 equipped with an insert at the HindiII site. This plasmid contains the arq3 regulatory region which can effect transcription of an HBsAg nucleotide sequence in yeast cells as described in the following examples. The strain MM294 of Ξ. coli K12 was deposited on 2 June 1982 in conformity with the provisions of EPC and the Budapest Treaty, at the American Type Culture Collection, under the serial number ATCC 39130.

The nucleotide sequence of the arg3 gene portion comprising the portion of th® N-texminal sequences which code for ornithine carbamoyltransferase (OCT) and the portion of the untranslated 5' region has been determined by Huyghen et al., Arch. Inti. Physical. Biochem., Volume 89, B172 (1981). Figure 4 illustrates a 210-bp fragment of the known sequence. The 210-bp fragment contains the unique HincXX, BglXI and EcoRX sites in the yeast DMA inserted, in th® form of Hindi 11 of 3300 bp, into pMC200.

It is thought that the initiation codon for the sequence which codes for the OCT protein corresponds to the boxed ATG codon. It is assumed that the introduction of the sequences which code for HBsAg alone or the precursor of HBsAg plus HBsAg derived from the HBV DMA cloned to the HincII, BglXX or EcoRX sites of the yeast DMA results in a gene fusion and the production of a hybrid protein transcribed and translated from the arg3 regulatory region provided that the gen® fusion is carried out in the correct orientation and that the reading frame is maintained for the translation beyond the fusion site.

Example 5. Preparation of the, intermediate plasmids PRIT10671 _ . and.. PRXT10672 containing. HBsAg and the regulatory regions by combination of _ PRIT10641 with OMC200. jsg of pHC200 are digested with 6.4 units of Bglll for 2.5 h at 37, they were diluted with an equal volume of a buffer (pH 10.5) containing 0.1 M glycine, 0.01 M MgGl2.6H2O and 0 J. aH ZnCl2 asid they were incubated with 0.5 unit of bovine intestine alkaline phosphatase for 30 minutes at 37° so as to remove the 5 terminal phosphate residues. The mixture was extracted twice with buffer-saturated phenol and three times with ether. The DNA was precipitated with ethanol and it was dissolved in 0.01 M tromethamine buffer. pg of pRIT10640 were digested with the endonuclease BamHx in order to fractionate the 1350-bp BamHI fragment, by preparative electrophoresis on agarose gel and by electroelution. The eluted DNA was recovered and it was concentrated by ethanol precipitation and dissolved in 20 pi of 0.01 M tromethamine buffer (pH 7). An aliguote of the BamHx fragment (0.3 pc), containing th® sequence which codes for HBsAg, was mixed with 0.5 pg of the Bgl ϊ I-digested pMC20G and the mixture was ligated by Incubation with T4 DNA ligase.

The ligated DNA was used to transform the competent cells of the strain MM294 of S. coli K12. The transformants were selected on agar plates containing 200 pg/ml of ampicillin. Twelve resistant colonies were isolated by successive passages on amplcillin-based agar and the plasmids were Isolated according to the process described by Bimboim et al., Nucl. Acid. Res. Volume 7, 1513 (1979). Analysis by agarose gel electrophoresis showed that all the plasmids were digested by Hpal,, which indicated the insertion of the BemHl fragment and that both orientations of the Inserted fragment were present among the twelve transformant colonies. Th® plasmids were Isolated by centrifugation on a density gradient of a CsCl/ethidium bromide mixture. A plasmid pRIT1067I contains the BamHI fragment fused to the Bgl 11 site in the correct orientation for transcription of the sequence which codes for HBsAg so as to generate a fusion protein of 286 amino acids comprising 18 N-terminal amino acids of OCT, 4.2 amino acids of the protein which may be the precursor of HBsAg and the 226 amino acids of HBsAg. The fusion protein is HBsAg as Indicated in Example 8 below. The transformants containing pRIT10671 are called strain MM2 9 4 of E. coli K12 (pRIT10571). pRITX0S71 is illustrated in Figure 5.

The use of pRIT10S40 as intermediate plasmid is not essential. For example, the BamHI fragment could have been excised from pRlT10616.

Another plasmid, pRXT10672? containing the BamHI fragment in the incorrect orientation for the transcription of the sequence which codes for HBsAg. The transformants containing this plasmid are called strain MM294 of E. coli K12 (p-RIT10672).

Example 5. Preparation of the plasmids, the Mshuttle vectors PRIT10673 and PRIT10674., by combination of ORIT10S71 and pRIT105?2 with the shuttle vector TBol3.

The vector YEpl3 is a shuttle" vector for S. cerevisiae and B. coli. It has been described by Broach et al., Gene, Volume 8, 121 (1979). It is provided by J. Hicks, Cold Spring Harbor Laboratories# New York# U.S.A. A small Hindlll fragment was excised from the plasmid by digestion with Hindi 11 and the plasmid was religated in order to prepare a derived plasmid, YEpl3HindIII . which contains a unique Hindlll site. Purified YEpl3aHindIII was digested with Hindi 11 and it was treated with alkaline phosphatase in order to prevent religation. The DMA was recovered by phenol extraction and ethanol precipitation.

Purified pRIT10671 and pRITlO672 were digested with BamHI and they were treated with alkaline phosphatase in order to prevent reformation of the pBR322 fragment. The treated DNA was# in addition, digested with Hindlll in order to liberate a 4650-bp Hindlll fragment which contains the fusion of the HBsAg gene with the regulatory region, the samples were extracted with phenol and precipitated with ethanol. 0.4 pg of each of the DNA preparations derived from pRIT10671 and pRIT10672 were separately mixed with 0.4 ^g of HindiII-digested YEpl3AHindIII and the mixtures were Incubated with T4 DMA ligase.

A portion of each of the ligated mixtures was used to transform the competent cells of the strain MM2 9 4 of B» coli X12. The transformants were selected on ampicillin-containing agar» The colonies were isolated and they were examined on the basis of their plasmid content according the procedure described by Birnboim et al., Nucl. Acid. Res., Volume 7, 1513 (1979). A transformant colony, strain MM294 of E. coli K12 (pRxLT10673) , contains a plasmid, pRITxO6/3, which contains the Hindi!! fragment obtained from pRIT10671 and inserted into YEpl3AHindIII, as illustrated in Figure 6. Another transformant colony, strain. MM204 of E. coli K12 (pRXT10674), contains a plasmid, pRlT10S74, which contains the Hindi!! fragment obtained from pRIT10S/2 and inserted in YEplSAEindlll.

Example 7. Transformation of yeast with PRIT10673 and ORIT10674.

The plasmids pRIT10673 and pRIT10674 were isolated from clarified lysates of th© strains >04294 (pRIT10673) and MM294 (pRIT10674) of B, coli K12 by centrifugation on a density gradient of a CsCl/ethidium bromide mixture.

A. Strain DCS of S. cerevisiae.

The strain DCS of S. cerevisiae (leu 2-3, leu 225 112, his 3, can 1-11) described by Broach et al.. Gene, Volume 8, 121 (1979) was provided by J. Hicks, Cold Spring Harbor Laboratories, New York, U.S.A. and was deposited on 2 June 1982 in conformity with the provisions of the EPC and the Budapest Treaty at the American Type Culture Collection at Rockville, Maryland, U.S.A., under the serial number ATCC 20630. The cells of strain DCS were cultured and they were prepared for the purpose of their transformation according to the procedures described by Hinnen et al., Proc. Natl. Acad.

Sci..- _U._S_,_&-Volume 75, 1929 (1978), except that the protoplast formation is carried out in 0.8 M sorbitol, 0.03 M jS-mercaptoethanol and 0.1 M potassium phosphate buffer (pH 7.5), using a mixture of £-glucouronidase (final concentration of 0.24 unit/ml) and aryl sulphatase (final concentration of 1.2 unit/ml)» The yeast protoplasts were incubated separately with 10 of pRIT10673 and 10 pg of pR!T10S74 and the transformants were selected in leucine-free regeneration agar. The colonies which grew in the regeneration agar were recovered and they were streaked on a solid medium containing 0.67% amino acid-free nitrogen basal medium for yeast, 2% glucose, 2% agar and 80 ,«g/ml of histidine and they were cultured at 30®C. A colony of the DCS strain was transformed by pR!T10673 and it is called S. cerevisiae DCS fpRlTlO673) strain. Another colony was transformed by pRIT10674 and it is called S. cerevisiae DC5 (pRIT10674) strain. Cultures of the strains DCS (pRIT10673) and DCS (pRIT10674) were grown In a nitrogencontaining basal medium for yeast supplemented with 80 pg/ml of histidine up to an optical density of 0.33 to 1 at 620 pm. The last strain Is useful as negative control since it contains the sequence which codes for HBsAg fused to the regulatory region and in the Incorrect orientation. The cells were recovered by centrifugation? washed with a solution of phosphate-buffered saline (PBS) and concentrated 20 to 160 fold by suspension in 5 ml of PBS supplemented with 1 mM phenylmethylsulphonyl fluoride (FMSF). The cells were disrupted by two passages through a French press at· 12,000 psi (83 MPa) and the lysate was centrifuged at 7700 x G for 15 minutes and then at 30,000 x G for 30 minutes. The supernatants were recovered and they were filtered on a Hillex GV membrane.

The supernatants were tested on the basis of the presence of proteins which react with the specific antiHBsAg antibodies according to the Ausriae radioimmunoassay methods. The clarified cell extracts of the S. cerevisiae DC5 (pRIT10673) strain, prepared in the same manner, gave positive reactions In this radioimmunoassay even when they were tested at 16- to 256-fold dilutions in PBS. On the other hand. In the case of the presence of proteins which react with the anti-HBsAg antibodies, the extracts of the DC 5 (pRIT10674) strain were negative in this assay.

B „ Strain lc!697d of S. eerevisiae.

The strain lcl697d of S, eerevisiae was deposited on 2 June 1982 in conformity with the provisions of the EPC and the Budapest Treaty, at the American Type Culture Collection, under the serial number ATCC 20631. Using the procedure described above, the strain IcloS'/d which is bradytrophic for arginine (argj*, leu 2-1) was transformed with pRIT10673 and pRIT10674. A leucine-independent colony of the bradytrophic strain was transformed by pRIT10673 and it is called S. eerevisiae lcl697d (pRIT10S73) strain. Another leucine-independent colony which was transformed by pRIT10674 is called S. eerevisiae IclSS/d (©RIT10674) strain. The cultures of the lcl697d (pRIT10673) and lcl697d (pRIT106/4) strains were grown ia a nitrogen-containing basal medium for yeast supplemented with 20 ^g/sal of arginine. Th® cells were recovered and the cell extracts were prepared as described above. The . clarified cell extracts of the lc!697d (pRIT10673) strain gave positive results in the Ausria* radioimmunoassay at dilutions ranging up to 1/2048 whereas tha extracts of the Icl697d (pRXT10674) strain all proved negative In this assay.

From these results, it can be concluded that the yeast cells of the strains DC5 and lc697d transformed with pRIT10673 specifically synthesise HBsAg in the form of a fusion protein possessing the determinants of HBsAg. Example 8.,. Immunisation of rabbits with HBsAg -derived from S. eerevisiae lclS97d /pRIT10€73) strain.

The S. eerevisiae lc!697 (pRIT10673) strain was cultured up to an optical density of 0.60 at 620 and ' it was recovered by centrifugatlon. The cells, concentrated 40-fold, were resuspended in BBS supplemented with 1 mM PMSF. A clarified cell extract was prepared as described In Example 7. A clarified cell extract of the lcl697d (pRITlO674) strain was prepared. These extracts were used to immunise rabbits. A first group of 6 rabbits received parenteral injections of 1 ml of extract of the lcl697d (pRIT10S73) strain, mixed with 1 ml of complete Freund's adjuvant on days 0, S. 15, 30 and 37» On these same days, a second group of three rabbits received parenteral injections of 1 ml of extract of the lclS97d (pRIT10674) strain, mixed with 1 ml of complete Freund's adjuvant. The sera were collected from the two groups on days 0 (preimmune), 23, 51 and 55 and those of the first group on day 44 and they were tested for the presence of anti-HBsAg antibody using the Ausab® radioimmunoassay. The results of these assays, which are presented in Table 1, indicate that four rabbits out of six which received the Injections of extract of the lcl697d (pRIT10S73) strain produced antibodies directed against HBsAg. None of the three control rabbits which received Injections of extract of the lcl697d (pRIT10674) strain showed signs of production of anti-EBsAg antibody. From these results, if was concluded that the extracts of the lcXS97d (pRIT10673) strain contain HBsAg which can be used without serious side effects in a vaccine to stimulate protection against HBV infection in humans.

TABLE 1 PRODUCTION OF ΑΝΤΙ-HBsAg ANTIBODIES BY RABBITS IMMUNISED WITH CELL-FREE EXTRACTS OF S.___CEREVISIAE lcl697d (pRIT10673) AND lc!697d (pRITlO674) STRAINS. souse: of /ίΗϊΊ-ΗΒΕ&δ αΧΧΒΗ (X? TBS sew* BxiascT ss’ ausas® assaY mnem BAY Big MI MX MI 0 23 44 51 S3 Stseia lc!667 1 sfc«S 1/16 1/16 1/64 -(Ρ&ΑΪ10673) 2 ffi!SS VX024 5/6592 1/6192 1/4096 1/33 3 aag 1/64 1/16 1/16 1/16 4 sx©g 1/34 1/2048 1/1024 3./2046 3 aag 1/1024 1/256 1/1024 1/2046 3 »93 VIS 1/25S 1/1024 1/204B icl6®7<5 -(JBXT10674) 7 a®8 SS«® — a®8 — β aag VI — ang aeg S aeg meg — a®3 aog ® She highest SiluZsaa of sssztsa »hicb glvoc saisulz la she aaaey.

Claims (15)

1. Recombinant DNA molecule comprising a nucleotide sequence which codes for HBsAg and a regulatory region which can effect transcription in yeast, the said sequence being expressed as a polypeptide that retains the immunogenicity of HBsAg and comprises the HBsAg polypeptide of 226 amino acids and a polypeptide extension at its N-terminal end, characterised in that the extension is a polypeptide at least a portion of which is not derived from the pre-S region, in which the sequence comprises a sequence which codes for an extension comprising both a polypeptide which is derived from the preS region and a polypeptide which is not derived therefrom, and in which the sequence comprises a sequence which codes for an extension which is derived from the pre-S region and is composed of a sequence containing up to th® 42 Μ-terminal amino acids which naturally precede the HBsAg polypeptide of 226 amino acids

2. Recombinant DNA molecule according to Claim 1, In which th® portion of the extension which Is derived from the pre-S region Is composed of th© 42 N-termlnal acids ' which naturally precede the HBsAg polypeptide of 226 amino acids.

3. Recombinant DNA molecule according to Claim 2, in which the region which is not derived therefrom is the region coding for the 18 N-terminal amino acids of ornithine carbamoyltransferase.

4. Yeast transformed by a plasmid containing a recombinant molecule according to any one of Claims 1 to 3.

5. Yeast according to Claim 4, characterised in that it is S. cerevisiae.

6. ’ . Method for the production of a polypeptide, characterised in that it comprises the culture of a yeast according to Claim 4 or 5 and In that th® peptide produced Is harvested.

7. - Immunogenic derivative of HBsAg comprising the HBsAg polypeptide of 22S amino acids and a polypeptide extents ion at its N-terminal end, characterised in that the extension comprises a polypeptide at least a portion of which is not derived from the pre-S region, and characterised in that the extension comprises both a 5 polypeptide which is derived from the pre-S region and a polypeptide which is not derived therefrom, in which the portion of extension which Is derived from the pr@-S region is composed of a sequence containing up to the 42 amino acids which naturally precede th® HBsAg polypeptide 10 of 226 amino acids.

8. Immunogenic derivative of HBsAg according to Claim 7, characterised in that the portion of the extension which is derived from the pre-S region Is composed ox th® 42 amino acids which naturally precede the HBsAg 15 polypeptide of 22S amino acids.

9. immunogenic derivative of HBsAg according to Claim 8, characterised in that the portion of the extension which is not derived from the pre-s region is composed of the 13 N-termixsal amino acids of ornithine

10. 0 carbaaoyltraas f erase 10. Vaccine, characterised in that it comprises an immunogenic derivative of EBs&g according to any ©ne of Claims 7 to 9 and a suitable excipient.

11. FL Recombinant DNA molecule as claimed in Claim 1 substantially as described herein with reference to the Examples.

12. Method for the.production of a polypeptide as claimed in Claim 6 substantially as described herein with reference to the Examples.

13. A polypeptide whenever prepared by a process as claimed in Claim β or Claim 12.

14. ,, · Imnunogemc derivative of HBsAg as claimed in Claim 7 substantially as described herein with reference to the Examples. 35

15. Vaccine comprising the immunogenic derivative of Claim 14.