NZ204952A

NZ204952A - Hepatitis b virus vaccine

Info

Publication number: NZ204952A
Application number: NZ204952A
Authority: NZ
Inventors: T Cabezon; M Crabeel; Wilde M De; N Harford
Original assignee: Smith Kline Rit
Priority date: 1982-09-08
Filing date: 1983-07-19
Publication date: 1986-05-09
Also published as: ES8600938A1; IE60387B1; EP0106828A2; IL69202A; ES532730A0; MY102920A; ES8600939A1; DE3382704T2; EP0106828B1; FI833205A0; DK406483D0; ES532732A0; AU9001491A; DK406483A; CA1222707A; JP2702899B2; EP0106828A3; CY1932A; AU1569288A; PT77265A

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

<div class="application article clearfix" id="description"> 204 952 Priority Ctete(s): :%?. Complete Specification Filed: /9; class: MiksaJj&k. Publication Date: .... P.O. Journal, Wo: /S&k No: Date: NEW.ZEALAND PATENTS ACT, 1953 COMPLETE SPECIFICATION HEPATITIS B VIRUS VACCINE We,j SMITH KLINE - RIT, a Belgian company, of rue du Tilleul, 133 B-1320 Rixensart (Genval), Belgium^Marjolene Groboo^ a..Belgi'an-■citizen, of Dworsbos, '-32, D-1G30 Linkebeek-,- Belgium— hereby declare the invention for which we pray that a patent nay be granted to us3 and the method by which it is to be performed3 to be particularly described in and by the following statement: -1- (foil owed by page -la-) la- 2049S2 5 10 * ;15 ;TITLE ;Hepatitis B Virus Vaccine FIELD OF THE INVENTION The invention relates to cloning of a gene which codes for hepatitis B surface antigen in yeast by use of 20 recombinant DNA techniques and preparation of a hepatitis B virus vaccine from antigen produced by the yeast. ;BACKGROUND OF INVENTION Inreccion with hepatitis 3 virus (HBV) is a serious, widespread health problem. Infection can. be manifested in 25 acute or chronic phases. The number of cases of acute hepatitis in the United States is estimated to be at least 100,000 per year with a fatality rate of 1 to 2 per cent. In the United States, the prevalence of chronic carriers of HBV among healthy adults varies between 0.1 and 1% 30 depending on age and social class. In South America, the prevalence of chronic carriers is about 1 to 3%; in the U.S.S.R. and southern Europe, about 3 to 6%; and in Asia and Africa, more than 10%. ;35 vaccine for people at high risk of exposure, such as ;In developed countries, there exists a need for a ;- 2 - ;20495 ;patients and personnel in medical units where blood is handled, military personnel, spouses of chronic carriers, travelers to areas of high HBV endemicity, newborns of chronic carriers, homosexuals, prostitutes and drug 5 abusers. In third world countries, there exists a need for an inexpensive vaccine for mass immunization. Mass immunization programs may ultimately affect not only the incidence of acute hepatitis and the pool of chronic carriers but may also reduce the morbidity and mortality 10 from chronic active hepatitis and hepatocellular carcinoma. ;Dane particles, which are believed to be hepatitis B virions and which are isolatable from infected patients, have a diameter of about 42nm. Each consists of an . envelope, comprising the hepatitis B surface antigen 15 (HBsAg), a capsid (HBcAg), an endogenous polymerase and a DNA genome. The genome is circular and double-stranded with a single strand region consisting of about 200 bases. The single strand region can be filled in, i_n vitro, by the action of the endogenous polymerase. The longer strand 20 contains approximately 3,200 bases. ;It has been difficult to prepare HBV vaccines because it has proven difficult to propagate the virus in tissue culture and because the only known host is man. Small amounts of authentic HBV antigens have been isolated from 25 infected humans. F-D-C Reports, pp. 3-4, July 19, 1982, carries a report of clinical studies of a recently developed Hepatitis B vaccine. ;Valenzuela et al., Nature, Volume 298, 347-350 (1982), report synthesis of HBsAg in yeast using an expression 30 vector wherein the HBsAg coding sequence is an 835 bp Taql-Hpal fragment and the promotor is the yeast alcohol dehydrogenase I promotor. Several earlier brief reports noted research preceding this reference. These are Valenzuela et al., Arch. Biol. Med. Exp. (Chile), Volume 35 14(1), 21-22 (1981), which reports expression in yeast of a ;- 3 - ;204952 ;DNA fragment containing a sequence which codes for a protein similar to HBsAg ligated to - a yeast alcohol dehydrogenase promotor region; a report in Scrip No. 616, p. 14 (Aug. 12, 1981) (available on request), which states that a team of U.S 5 researchers including P. Valenzuela and W. J. Rutter have announced production in yeast of "the protein-coating surrounding hepatitis B virus;" and Zuckerman, Nature, ;Volume 295, 98-99 (1982), which reports that W. J. Rutter ■ has reported expression of glycosylated HBsAg in yeast 10*cells. Antigenic components of HBV, such as HBsAg, have been prepared in bacteria following insertion of a recombinant DNA molecule containing a gene which codes for the . antigen., Burrell et al., Nature, Volume 279, Number 5708, 15 43-47 (1979), report expression in E. coli strain HB101 of HBV DNA sequences cloned in plasmid pBR3220 Murray et al., European Patent Application 13,828, disclose preparation of a recombinant vector which can code for HBV antigens, including HBsAg, in E.coli strain HB101. 20 The vector is prepared from Dane particle DNA and plasmid pBR322. The authors state that useful hosts may include other bacterial hosts, yeasts and other fungi, animal or plant cells and other hosts, although the only demonstrated host is E. coli. 25 Charnay, et al., Nature, Volume 286, 893-895 (1980), report construction of a bacteriophage carrying a fusion of the B-galactosidase gene and the HBsAg structural gene. The bacteriophage directs synthesis of a fusion protein comprising antigenic determinants of both HBsAg and 30 fl-galactosidase. Tiollais et al., United Kingdom Patent Application 2,034,323, disclose preparation of a coliphage containing HBV DNA. Fused phage-HBV DNA is transformed into E. coli strain C600. H ; 2049 - 4 - In United Kingdom Patent Application 2,070,621, first published as PCT application W081/00577, a plasmid which comprises a part of the HBsAg gene and the promotor and the Z gene of the lactose operon and which can be cloned in E^ 5 coli is disclosed. Rutter et al., European Patent Application 20,251, disclose recombinant vectors including a recombinant vector comprising plasmid pBR322 and BamHI fragments of HBV DNA, which can be used to transform E. coli. Another vector, p 10 comprising a BamHI fragment of HBV DNA and a portion of the tryptophan operon, was used to obtain expression in E. coli strain HB101. Edman et al., Nature, Volume 291, Number 5815, 503-506 . (1981), describe construction of plasmids which direct 15 synthesis of HBcAg and a B-lactamase-HBsAg fusion protein, under the control of the tryptophan operon regulatory region, in E. coli. Other references disclosing insertion of HBV DNA into bacteria include Charnay, et al., Prog. Med. Virol., Volume 20 27, 88-92 (1981); MacKay 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); U. K. Patent Specification 2,034,323 (Derwent No. 46874C). and Pasek et al., Nature, Volume 282 No. 6, 575 (1979). 25 HBV DNA has also been cloned in mammalian cells. These include, human, mouse, and human hepatoma cell lines. For example, Dubois et al. , Proc. Natl. Acad. Sci. U. S., Volume 77, Number 8, 4549-4553 (1980), report transformation of mouse cells with a plasmid containing the 30 HBV genome and expression of HBsAg; Hirschman et al., Proc. Natl. Acad. Sci. U.S., Volume 77, Number 9, 5507-5511 (1980), report production of HBV-like particles by HeLa cells transformed with HBV DNA. Procedures for preparing HBV vaccine using HBsAg from 35 human blood are reported by Funakoshi et al., Prog. Med. - 5 - 204952 Vi_rol. , Volume 27, 163-167 (1981), and Maupas et al., Prog. Med. Virol., Volume 2 7,"", 185-201 (198JL). The vaccine prepared by Funakoshi et al. contains 40 yug of purified, formalin-treated HBsAg, phosphate sodium chloride, 20 mg of 5 mannitol; and 0.1% of aluminium hydroxide as adjuvant. In the latter paper, Maupas et al. report that one dose of vaccine was 1 ml of purified, formalin-treated HBsAg containing 2-10 yug/ml of protein (Lowry's method) and 0.1% •of aluminum hydroxide. The protocol used in the study 10 "reported by Maupas et al. called for three injections at one month intervals with a booster after one year; the authors propose a protocol consisting of two injections of concentrated HBsAg at three month intervals. Additional references to preparation of HBV vaccines 15 include Maupas et al., Adamowicz et al., and Funakoshi et al. at pages 3, 37 and 57, respectively, of Hepatitis B Vaccine INSERM Symposium No. 18, edit. by Maupas and Guesry, 1981, Elsevier/North-Holland Biomedical Press. Yeasts have been used as host organisms for certain 20 other DNA sequences. For example, Fraser et al. , U„ K. Patent Application 2,068, 969-, disclose preparation of chicken ovalbumin in yeast; Scrip No. 640, p. 11 (Nov. 4, 1981).(available on request) contains a report that a type of interferon is beinc prapared in yeast. In European Patent 11,562 (Derwent No. 25 38762C) are reported hybrid yeast plasmids containing the urat yeast gene in the 2 yu plasmid. SUMMARY OF THE INVENTION The invention relates to the preparation of a 30 recombinant DNA molecule comprising a nucleotide sequence which can code for HBsAg and a regulatory region derived from the yeast arg 3 gene which can effect transcription of the HBsAg sequence in the yeast, Saccharomyces cerevisiae. The molecule includes vectors in which the HBsAg sequence 204952 can be used to prepare a yeast vector or to maintain the HBsAg and regulatory regions. Microorganisms containing the recombinant DNA molecule, such as microorganisms transformed with plasmids of the invention, are included 5 within the invention. The invention also includes a plasmid having the arg3 regulatory region and a restriction site proximate thereto for insertion of a coding sequence, such as an HBsAg sequence, such that . the protein synthesized by expression of the coding sequence is devoid 6 ■ 10 of extraneous amino acid residues. The invention also includes a vaccine for stimulating protection against HBV infection in humans comprising a vaccinal amount of HBsAg prepared in accordance with this . invention and a suitable carrier. 15 Further, the invention includes processes for preparing the recombinant DNA molecule and microorganisms containing the molecules as well as processes for preparing HBsAg and HBsAg containing vaccine. BRIEF DESCRIPTION OF THE FIGURES 20 Figure 1 is a restriction endonuclease cleavage map of pRIT10601. Figure 2 is a restriction endonuclease cleavage map of pRIT106l6. Figure 3 is a restriction endonuclease cleavage map of 25 pMC200. Figure 4 is the nucleotide sequence of a portion of the 3300 bp Hindlll yeast DNA insert in pMC200 which portion contains HincII, Bglll and EcoRI sites. Figure 5 is a flow sheet illustrating preparation of 30 pRIT10671 and pRIT10673. Figure 6 is a flow sheet illustrating preparation of pRIT10749. Figure 7 is a flow sheet illustrating preparation of pRITl0761 and pRIT10764. - 7 - 204952 DETAILED DESCRIPTION OF THE INVENTION The recombinant DNA molecule of the invention is prepared by fusing a nucleotide sequence which includes a structural gene for HBsAg with the yeast arg3 regulatory 5 region, which regulatory region can direct transcription of the HBsAg sequence in yeast, thereby effecting expression thereof. By "recombinant DNA molecule" is meant a DNA fragment containing the HBsAg coding sequence and the regulatory region as well as other DNA molecules containing C' 10 the fragment, such as plasmid or phage vectors. By "regulatory region" is meant a sequence which contains a promotor region and other sequences necessary for transcription. The yeast arg3 regulatory region is • especially advantageous because it can be a powerful 15 promotor for expression of an HBsAg coding sequence. By "HBsAg" is meant a protein which is structurally identical to authentic HBsAg or has substantially the same antigenic determinants as authentic HBsAg, that is, is capable of stimulating production of antibodies which 20 specifically recognize and react with authentic HBsAg or is specifically recognized by anti-HBsAg antibodies. The HBsAg coding sequence can be isolated from DNA extracted from Dane particles in infected human serum . by filling in the single strand region with a DNA polymerase, 25 preferably the endogenous polymerase, followed by digestion with a restriction endonuclease. The choice of endonuclease will depend, in part, on the particular Dane particles. For example, as illustrated in the Examples below, the HBsAg coding sequence of HBV DNA of certain Dane 30 particles of the adw serotype can be isolated on a single BamHI fragment; the HBsAg coding sequence of HBV DNA of certain Dane particles of the ayw serotype can be isolated on a Hhal fragment. HBV DNA of Dane particles of the same serotype may also exhibit different patterns of restriction 35 sites. 204952 Restriction of DNA to prepare DNA fragments used in the invention, ligation of such fragments to prepare recombinant DNA molecules used in the invention and insertion into microorganisms are carried out by Known 5 techniques such as techniques disclosed in the previously and subsequently cited references. Conditions are selected to avoid denaturation of the DNA and enzymes. For example, the pH is buffered to remain at about 7.0 to 11.0 and the temperature is kept below about 60°C. Preferably 10 restriction is carried out at a temperature of about 30 to 40°C and ligation is carried out at about 0 to 10°C. Restriction enzymes and ligases used in carrying out this invention are commercially available and should be used in . accordance with instructions included therewith. T4 DNA 15 ligase is the preferred ligase. Fusing of the HBsAg sequence to the regulatory region can be accomplished by use of intermediate vectors as illustrated in the Examples, below. Alternatively, the HBsAg sequence can be inserted directly into a vector which 20 contains the regulatory region. A vector is DNA which can carry and maintain the DNA fragment of the invention, including, for example, phages and plasmids. Techniques for cloning DNA fragments in phages are disclosed, for example, by Charnay et al. Nature, Volume 286, 893-895 25 (1980) and Tiollais, United Kingdom Patent Application 2,034,323. Preferably, the HBsAg sequence is positioned relative to the regulatory region such that the HBsAg synthesized by expression of the HBsAg sequence is devoid of extraneous amino acid residues. 30 A regulatory region which has been found to be especially useful is derived from the yeast arg3 gene which codes for ornithine carbamoyltransferase (OCT). Use of the arg3 regulatory region is advantageous because its activity is subject to both specific and general control 35 mechanisms. It has been cloned in E. coli on plasmid - 9 - 204952 pYeura3arg3 as reported by Crabeel et al., Proc. Natl. Acad. Sci. U.S.A., Volume 78, 5026 (1981). Preferred hosts are cerevisiae strains in which the arginine biosynthetic pathway is derepressed, such as strain 5 lcl697d. Use of such strains results in increased expression from the arg3 promotor as compared to the other strain which was used in the Examples, strain DC5. The preferred vector for cloning the fused DNA fragment into yeast is the plasmid YEpl3, which is capable 10'of replication and maintenance in both E. coli and S. cerevisiae and is, therefore, known as a shuttle vector. Several other yeast vectors are known and available. The HBsAg and regulatory regions can be inserted into a yeast . vector sequentially or, as illustrated in the Examples, 15 below, simultaneously. Transformation with plasmid vectors will normally result in incorporation of the DNA molecule of the invention as a plasmid. However, other reactions, such as recombination events, can result in incorporation of the DNA molecule into chromosomal DNA. 20 Vaccines for stimulating protection against HBV infection in humans comprising HBsAg produced by yeast in accordance with the invention and a suitable carrier can be prepared by known techniques. Use of an adjuvant, such as aluminum hydroxide, is desirable. The HBsAg so produced 25 can also be combined with other immunogens to prepare combination vaccines. The HBV or combination vaccines can be administered, for example, by the subcutaneous, intravenous or intramuscular route. The DNA fragment of the invention and the HBsAg produced thereby, can also be 30 used as a probe for detection of HBV in biological samples by DNA hydridization techniques and various immunoassays. EXAMPLES In the following examples of the invention, which are illustrative and not limiting, all percentages are by 35 weight and all temperatures are in degrees Celsius. 2049b Example 1. Preparation of Intermediate Plasmid, pRIT10601, by Combining HBV DNA with pBR322 HBsAg positive serum of ayw serotype was defibrinated by addition of CaCl^ to a final concentration of 0.28% 5 and centrifuged for 2 h at 21,000 rpm in a SW 27 rotor on 10-20% sucrose gradients made up in a buffer (pH 7.5) containing 10 mM tromethamine-HCl, 1 M NaCl and 1 mM EDTA. A transparent pellet containing Dane particles was resuspended in the same buffer and centrifuged on buffered 10 20% sucrose layered . on a 65% sucrose cushion. An opalescent band at the cushion interface was recovered and centrifuged on a similar 20-65% gradient at 200,000 x G for 4 h to pellet the Dane particles. A. 'Single strand regions of the HBV genome within the 15 Dane particles were repaired using the endogenous DNA polymerase by resuspending the Dane particles in a reaction mixture (pH 8.0) containing 50 mM tromethamine-HCl, 10 mM MgCl^, 500 mM NaCl, 0.5 mM dithiothreitol, 50 mM each of dATP, dCTP and dGTP and 8 jM of 32P-dTTP (350 Ci/mmole) 20 and incubating the resuspended particles for 5 h at 37°. The resuspension was diluted to pH 7.5 with a buffer containing 10 mM tromethamine-HCl, 10 mM EDTA, 100 mM NaCL and 0.02% sodium dodecyl sulfate (pH 7.5) and incubated with 0.5 mg/ml of pronase for 1 h at 37° followed by 25 phenol extraction and ethanol precipitation. Digestion of the DNA with BamHI restriction endonuclease, yielded two radioactive fragments with sizes of about 1450 bp and 1600 bp as judged by agarose gel electrophoresis and autoradiography of the gel. 30 B. About 30 ng of Dane particle DNA was repaired with the endogenous DNA polymerase in the presence of non-labelled dTTP, and was extracted and recovered, as described above. The DNA was mixed with 100 ng of plasmid pBR322 which had been previously digested with BamHI 35 restriction endonuclease and treated with alkaline - 11 - 204952 phosphatase. Plasmid pBR322 is commonly used in recombinant DNA procedures and is on deposit, without restriction on availability, in the American Type Culture Collection under accession number 37017. The mixture was 5 extracted with phenol, precipitated with ethanol, centrifuged, dried, resuspended in 12 ul of a mixture (pH 7.5) containing 50 mM tromethamine-HCl, 1 mM ATP, 10 mM MgCl^, 10 mM dithiothreitol, 50 yjg/ml g.elatin and 2 units/ml T4 DNA ligase. The suspension was incubated for 4 o 10 h at 10 and then held on ice for 18 h. The ligated DNA mixture was used to transform competent cells of E. coli K12 strain C600 prepared according to the procedure of Cohen et al., Proc. Natl. Acad. Sci. U. S., Volume 69, 2110 (1972). Transformants 15 were selected on a solid medium containing ampicillin (200 /Ug/ml). Isolated colonies were screened for loss of tetracycline resistance, indicative of insertion of a foreign DNA fragment in the BamHI site of pBR322. One such transformant clone was found to contain a plasmid, 20 pRIT10601, which on digestion with BamHI endonuclease gave a pBR3 22 fragment of 4360 bp and HBV DNA fragments of 1600 bp and 1450 bp. A culture of E. coli K12 strain C600(pRIT1060x) was deposited in accordance with regulations of the European Patent Convention (EPC) and the 25 Budapest Treaty in the American Type Culture Collection, Rockville, Maryland U.S.A. on June 2, 1982 under accession number ATCC 39132. A restriction endonuclease cleavage map of 'plasmid pRIT1060l is shown in Figure 1. C. The sizes of fragments generated by digestion of 30 the Dane particle DNA and pRIT10601 with various restriction enzymes were compared as follows. Dane particle DNA, that is, HBV DNA, was labelled with ^P by the endogeneous polymerase reaction described above or by treating purified DNA with DNA polymerase I from E. coli. 35 The labelled HBV DNA was mixed with pRIT10601 and the 204952 mixture was treated with a restriction endonuclease and electrophoresed on an agarose gel. The gel was stained with ethidium bromide and photographed under UV light to locate the DNA fragments and was then dried and 5 autoradiographed to locate the radioactive HBV DNA fragments. The following labelled HBV fragments were found to match exactly the size of pRIT10601 fragments: 1450, and 1600 bp BamHI fragments; a 1330 bp Hpal fragment; and a 1130 bp BamHI-XhoI fragment. Labelled Dane particle DNA 10 also hydridized specifically to both the 1450 and 1600 bp fragments released by BamHI digestion of pRIT10601 following transfer of these fragments from an agarose gel onto a nitrocellulose filter by the technique of Southern, J. Mol. Biol., Volume 98, 503 (1975). 15 These results show that the cloned DNA insert on pRITl0601 represents the HBV genome and that the relative orientation of the two BamHI fragments on pRIT10601 is the same as in the virion. pRIT10601 was used to prepare pRIT10671 in Example 10, below. 20 Example 2. Preparation of Intermediate Plasmid, PRIT10616, by Combining HBV DNA with pACYC184 Dane particles were isolated from HBsAg positive serum of adw serotype as described above. Restriction 32 25 endonuclease analysis of P-labelled HBV DNA indicated that the DNA contained one EcoRI site. HBV DNA, filled in by the endogenous polymerase reaction using non-labelled nucleotides, was digested with EcoRI. The restricted DNA was mixed with plasmid pACYC184 30 which had been previously digested with EcoRI and treated with alkaline phosphatase. Plasmid pACYCl84 is on deposit, without restrictions on availability, in the American Type Culture Collection under accession number 37033. The mixture was ligated with T4 DNA ligase. - 13 - 204952 The ligated DNA mixture was used to transform competent cells of E. coli K12 strain C600. Transformants screened for loss of chloramphenicol resistance which is 5 indicative of insertion in the EcoRI site of pACYC184. A transformed colony was found to contain a plasmid, pRIT10616, which consists of pACYC184 with a 3200 bp insert, comprising the HBV DNA, at the EcoRI site. A restriction map of pRIT10616 is shown in Figure 2. E. coli 10 K12 strain C600 (pRIT10616) was deposited in accordance with EPC regulations and the Budapest Treaty in the American Type Culture Collection on June 2, 1982 under accession number ATCC 39131. 15 Example 3. Preparation of Intermediate Plasmid, pRIT10640, Containing a Nucleotide Sequence Coding for HBsAg, by Combining pRIT10616 with pBR313 pRIT106l6 was purified by CsCl-ethidium bromide density gradient centrifugation substantially as described 20 by Kahn et al, Methods in Enzymology, Volume 68, 268 (1979). The DNA was digested with BamHI endonuclease, mixed with BamHI-digested, alkaline phosphatase-treated DNA of plasmid pBR313, ligated and used to transform competent cells of E. coli K12 strain C600 substantially as described 25 in Example 1. Plasmid pBR313 is on deposit, without restrictions on availability, in the American Type Culture Collection under accession number 37018. Transformants were selected on ampicillin-containing agar and screened for loss of tetracycline resistance, 30 indicative of insertion at the BamHI site of pBR313. A transformed colony was found to contain a plasmid, pRIT10640, which consists of pBR313 with a 1350 bp insert at the BamHI site. The insert is a nucleotide sequence which can code for HBsAg. It codes for part of a putative were selected on tetracycline agar medium and 20495 - 14 - HBsAg precursor protein and the complete surface antigen and includes 565 bp of 3' non-coding sequences. Example 4. Preparation of Intermediate Plasmid, 5 pMC200, Containing the arq3 Regulatory Region, by Combining Yeast arg3 Gene with pBR322 A 3300 bp yeast DNA fragment specifying the arg3 gene was obtained by digestion of pYeura3arg3 with Hindlll. Plasmid pYeura3arg3 has been described by Crabeel et al., 10 Proc. Natl. Acad. Sci.. U.S., Volume 78, 5026 (1981). The 3300 bp fragment was cloned into the Hindlll site of pBR322 and transformed into E. coli K12 strain MM294, substantially as described above. Transformants were selected on ampicillin medium and screened for tetracycline 15 resistance. A transformed colony was found to contain a plasmid, pMC200, which consists of pBR322 with an insert at the Hindlll site. This plasmid contains the arg3 regulatory region which can effect transcription of a HBsAg nucleotide sequence in yeast cells as described in 20 following examples. E. coli K12 strain MM294(pMC200) was deposited in accordance with EPC regulations and the Budapest Treaty in the American Type Culture Collection on June 2, 1982 under, accession number ATCC 39130. The nucleotide sequence of part of the arg3 gene, 25 including part of the N-terminal coding sequences for ornithine carbamoyltransferase (OCT) and part of the 5' non-translated leader region has been determined by Huyghen et al. , Arch. Intl. Physical. Biochem., Volume 89, B172 (1981). Figure 4 illustrates a 210 bp fragment of the 30 known sequence. The 210 bp fragment contains unique HincII, Bglll and EcoRI sites on the 3300 bp Hindlll yeast DNA insert in pMC200; the initiation codon for the OCT protein coding sequence is believed to be the boxed ATG codon. Introduction into the HincII, Bglll or EcoRI sites 35 of the yeast DNA of coding sequences for HBsAg alone or 204952 - 15 - HBsAg precursor plus HBsAg derived from cloned HBV DNA is expected to result in a gene fusion and production of a hybrid protein transcribed and translated from the arg3 regulatory region provided that the gene fusion is in the 5 correct orientation for continued translation beyond the site of fusion. Example 5. Preparation of Intermediate Plasmids, pRIT10671 and pRIT10672, Containing HBsAg and Regulatory 10 Regions, by Combining pRITl0641 with pMC200 pMC200, 5ug, was digested with 6.4 units of Bglll for o 2.5 h at 37 , diluted with an equal volume of a buffer (pH 10.5) containing 0.1 M glycine, 0.01 M MgCl^ 6H20 and 0.1 mM ZnCl^, and incubated with 0.5 units of calf 15 intestine alkaline phosphatase for 30 minutes at 37 to remove 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 dissolved in 0.01 M tromethamine buffer (pH 7.5). 20 pRITl0640, 25 yug, was digested with BamHI to excise the 1350 bp BamHI fragment of HBV DNA. The 1350 bp fragment was purified by preparative agarose gel electrophoresis a.nd electroelut ion. The eluted DNA was recovered and concentrated by ethanol precipitation and 25 dissolved in 20 yul of 0.01 M tromethamine buffer (pH 7.0). A 0.3 yag aliquot of the BamHI fragment, which contains the HBsAg coding sequence, was mixed with 0.5 ^jg of the Bglll-digested pMC200 and the mixture was ligated by incubation with T4 DNA ligase. 30 The ligated DNA was used to transform competent cells of E. coli K12 strain MM294. Transformants were selected on agar plates containing 200 yjg/ml ampicillin. Twelve resistant colonies were isolated by serial passage on ampicillin agar and plasmids were isolated by the procedure 35 described by Birnboim et al., Nucl. Acid. Res. Volume 7, 204952 16 - 1513 (1979). Analysis by agarose gel electrophoresis showed that all of the plasmids were restricted by Hpal, indicative of insertion of the BamHI fragment and that both orientations of the inserted fragment were present among 5 the twelve transformed colonies. The plasmids were isolated by CsCl-ethidium bromide density gradient centrifugation. One plasmid, pRIT10671, contained the BamHI fragment fused at the Bglll site in correct orientation for transcription of the HBsAg coding sequence 10 to generate a 286 amino acid fusion protein consisting of 18 N-terminal amino acids of OCT, 42 amino acids of the putative HBsAg precursor protein and the 226 amino acids of HBsAg. The fusion protein is HBsAg as shown in Example 8, below. Transformants containing pRIT10671 are designated 15 E. coli K12 strain MM294(pRIT10671). pRIT10671 is illustrated in Figure 5. Use of pRIT10640 as an intermediate plasmid is not essential. For example, the BamHI fragment could have been excised from pRIT10616. 20 Another plasmid, pRIT10672, contained the BamHI fragment in the incorrect orientation for transcription of the HBsAg coding sequence. Transformants containing this plasmid are designated E. coli K12 strain MM294(pRIT10672). Example 6. Preparation of Plasmids, pRIT10673 and 25 pRIT10674 Shuttle Vectors, by Combining pRITl0671 and pRIT10672 with Shuttle Vector YEpl3 Vector YEpl3, is an E. coli-S. cerevisiae shuttle vector. It has been described by Broach et al, Gene, Volume 8, 121 (1979). It was supplied by J. Hicks, Cold 30 Spring Harbor Laboratories, New York, U.S.A. A small Hindlll fragment was excised from the plasmid by digestion with Hindlll and the plasmid was religated to prepare a derivative plasmid, YEpl3 Hindlll which contains a single Hindlll site. Purified YEpl3 Hindlll was digested with 35 Hindlll and treated with alkaline phosphatase to inhibit 2 04952 - 17 - religation. The DNA was recovered by phenol extraction and ethanol precipitation. Purified pRIT1067l and pRIT10672 were digested with BamHI and treated with alkaline phosphatase to inhibit reformation of the pBR3 22 moiety. The treated DNA was further digested with Hindlll to liberate a 4650 bp Hindlll fragment, which contains the regulatory region-HBsAg gene fusion, and the samples were extracted with phenol and precipitated with ethanol. The DNA preparations derived from pRIT1067l and pRIT10672, 0.4 yug of each, were separately mixed with 0.4 yag of the Hindlll-digested YEpl3 Hindlll and the mixtures were incubated with T4 DNA ligase A portion of each of the ligated mixtures was used to transform competent cells of E. coli K12 strain MM294. Transformants were selected on ampicillin agar. Transformant colonies were isolated and examined for their plasmid content by the procedure described by Birnboim et al-. , Nucl. Acid. Res., Volume 7, 1513 (1979). One transformant colony, E. coli K12 strain MM294(pRIT10673), contained a plasmid, pRITl0673, which contains the Hindlll fragment from pRIT1067l inserted on YEp 13HindIII, in the correct orientation, as illustrated in Figure 6. Another transformant colony, E. coli K12 strain MM294(pRIT10674), contained a plasmid, pRIT10674, which contains the Hindlll fragment from pRIT10672 inserted on YEpl3 Hindlll in the incorrect orientation. Example 7. Transformation of Yeast with pRIT10673 and pRIT10674 Plasmids pRIT10673 and pRIT10674 were isolated from cleared lysates of E. coli K12 strains MM294(pRIT10673) and MM294(pRIT10674) by CsCl-ethidium bromide density gradient centrifugat ion. A. S. cerevisiae strain DC5. 2049 - 18 - The S. cerevisiae strain DC5 (leu 2-3, leu 2-112, his 3, can 1-11) described by Broach et al., Gene, Volume 8, 121 (1979), was obtained from J. Hicks, Cold Spring Harbor Laboratories, New York, U.S.A. and was deposited in 5 accordance with EPC regulations and the Budapest Treaty in the American Type Culture Collection in Rockville, Maryland, U.S.A., on June 2, 1982 under accession number ATCC 20630. Cells of strain DC5 were grown and prepared for transformation by the procedures described by Hinnen et 10 al., Proc. Natl. Acad. Sci. U.S.A., Volume 75, 1929 (1978) except that protoplasting was done in 0.8 M sorbitol, 0.03 M fi-mercaptoethanol, and 0.1 M potassium phosphate buffer (pH 7.5) using a mixture of 13-glucouronidase (0.24 units/ml final concentration) and arylsulphatase (1.2 units/ml final 15 concentration). Yeast protoplasts were separately incubated with 10 yug of pRIT10673 and 10 ug of pRIT10674 and transformants were selected in regeneration agar lacking leucine. Colonies which grew in the regeneration agar were recovered and streaked on solid medium containing 20 0.675% Yeast Nitrogen Base medium lacking amino acids, 2% glucose, 2% agar and 80 yjg/ml histidine and were grown at 30°. One colony of strain DC5 had been transformed with pRIT10673 and is designated £3. cerevisiae strain DC5(pRIT10673). Another colony had been transformed with 25 pRIT10674 and is designated £. cerevisiae strain DC5(pRIT10674). Cultures of strains DC5(pRIT10673) and DC5 (pRIT10674) were grown in Yeast Nitrogen Base medium plus 80 yug/ml histidine to optical densities of 0.33 to 1.0 at 620 yum. 30 The latter strain is useful as a negative control because it contains the HBsAg coding sequence fused in incorrect orientation to the regulatory region. Cells were recovered by centrifugation, washed with phosphate buffered saline (PBS) and resuspended in 5 ml PBS plus 1 mM phenyl methyl 35 sulphonyl fluoride (PMSF) at 20 to 160-fold 2049 concentrations. Cells were broken by two passages through a French Pressure Cell at 12,000 psi (83 MPa) and the lysate was centrifuged at 7700 x G for 15 minutes and then for 30 minutes at 30,000 x G. The supernatants were 5 recovered and filtered over a Millex GV membrane. Supernatants were tested for the presence of protein reacting with specific anti-HBsAg antibodies by the . ® Ausna^ radioimmunoassay procedures. Clarified cell extracts of S. cerevisiae strain DC5(pRITl0673) prepared in 10 this manner gave. positive reactions in this radioimmunoassay even when tested at 16 to 256—fold dilutions in PBS. In contrast, extracts of strain DC5(pRIT10674) were negative in this assay for the presence of proteins reacting with anti-HBsAg antibodies. 15 B. !S. cerevisiae strain lcl697d j3. cerevisiae strain lcl697d was deposited in accordance with EPC regulations and the Budapest Treaty in the American Type Culture Collection, on June 2, 1982 under accession number ATCC 20631. Using the procedure described 20 above, the arginine bradytrophe strain lcl697d (argJ leu 2-1) was transformed with pRIT10673 and pRIT10674. One leucine-independent colony of the bradytrophe strain had been transformed with pRIT10673 and is designated cerevisiae strain lcl697d(pRITl0673). Another 25 leucine-independent colony, which had been transformed with pRITl0674, is designated S_. cerevisiae strain lcl697d(pRITl0674) . Cultures of strains lcl697d(pRIT10673) and lcl697d (pRITl0674) were grown in Yeast Nitrogen Base medium 30 supplemented with 20 pq/ml arginine. The cells were recovered and cell extracts were prepared as described above. Clarified cell extracts of strain lcl697d is\ (pRITl0673) gave positive results in the Ausria^ radioimmunoassay at dilutions of up to 1/2048 whereas 204952: - 20 - extracts of strain lcl697d(pRIT10674) were uniformly negative in this assay. From these results, it is concluded that yeast cells of strains DC5 and lcl697d transformed with pRIT10673 5 specifically synthesize HBsAg in the form of a fusion protein having HBsAg determinants. Example 8. Immunization of Rabbits with HBsAg from S. cerevisiae strain lc!697d(pRIT10673) 10 cerevisiae strain lcl697d (pRIT10673) was grown to an optical density at 620 urn of 0.60 and collected by centrifugation. The cells were resuspended at a 40-fold concentration in PBS plus 1 mM PMSF. A clarified cell ■ extract was prepared as described in Example 7. A 15 clarified cell extract of strain lcl697d(pRIT10674) was similarly prepared. These extracts were used to immunize rabbits. A first group of six rabbits received parenteral injections of 1 ml of extract of strain lcl697d(pRIT10673) mixed with 1 ml of Freund 1 s complete adjuvant on days 0, 9, 20 15, 30 and 37. A second group of three rabbits received parenteral injections of 1 ml of extract of strain lcl697d(pRIT10674) mixed with 1 ml of Freund's complete adjuvant on the same days. Sera was obtained from both groups on days 0 (pre-immune) 23, 51 and 65 and, from the 25 first group, on day 44, and tested for the presence of R anti-HBsAg antibodies using the Ausab radioimmunoassay. The results of these assays, which are given in Table 1,. show that four of six rabbits receiving injections of extract from strain lcl697d(pRIT10673) produced antibodies 30 directed against HBsAg. None of the three control rabbits receiving injections of extract from strain lcl697d(pRIT10674) showed evidence of production of anti-HBsAg antibodies. From these results, it is concluded that extracts of strain lcl697d(pRIT10673) contain HBsAg, 35 which can be used in a vaccine to stimulate protection 2 04 95 - 21 - against HBV infection in humans without serious side effects. 10 table i PRODUCTION OF HBsAg ANTIBODIES BY RABBITS IMMUNIZED WITH CELL-FREE EXTRACTS OF S. CEREVISIAE STRAINS lcl697d(pRIT10673) and lcl697d (pRIT10674) SOURCE OF EXTRACT RABBIT ANTI-HBsAg SERUM TITRE BY Ausa NUMBER DAY 15 Strain lcl697d -(pRIT10673) 20 Strain lcl697d 25 -(pRiri0674) 1 2 3 4 5 6 7 8 9 0 neg neg neg neg neg neg neg neg neg DAY 23 DAY 44 neg 1/16 1/1024 1/8192 1/64 1/16 1/64 1/2048 1/1024 1/256 1/16 1/256 neg 1/1 neg DAY 51 1/16 ASSAY ■DAY 65 1/64 1/8192 1/4096- 1/8192 1/16 1/16 1/1024 1/2048 1/1024 1/2048 1/1024 1/2048 neg — neg neg neg neg 30 highest dilution of serum giving a positive result in the Ausab®aSSa^ 35 - 22 - 20495 Example 9. Preparation of Plasmid, pRIT10749, Containing the arq3 Regulatory Region, from pMC200 Purified pMC200 was digested with HincII and electrophoresed on a 10% acrylamide gel. A 1940 bp 5 fragment containing the arg3 regulatory region was recovered from the gel by electroelution and ethanol precipitation. About 4 yjg of the fragment was incubated with 0.2 units of Bal31 exonuclease, for 1 to 3 seconds at 30° in 100 yul of buffer (pH 8.0) containing 12.5 mM 10 "MgCl2, 12.5 mM CaCl2, 200 mM NaCl, 1 mM EDTA and 200 mM tromethamine-HCl. The treated DNA was extracted with phenol and precipitated with ethanol. A 1 p.g aliquot was incubated with T4 DNA polymerase in the presence of deoxynucl,eotide triphosphates to repair any single strand 15 extremities and was then digested with EcoRI endonuclease. This procedure produced DNA fragments of varying lengths due to the resection with Bal31. Each fragment had a fixed EcoRI extension at one end with the other extremity being blunt ended and located at some distance from the original 20 HincII site and the OCT protein initiation codon. Fragments of about 1480 bp containing the arg3 regulatory region were isolated by electrophoresis on a 7.5% acrylamide gel followed by electroelution and ethanol precipitation. This cut-back regulatory region is 25 preferred because it can promote transcription leading to synthesis of a protein devoid of OCT amino acid sequences, as described in Example 10, below. In a second series of reactions, pMC200 was digested with Xbal and the 5" single strand extremities were filled 30 in or rendered blunt-ended, by incubation with T4 DNA polymerase and deoxynucleotide triphosphates. This DNA was digested with EcoRI and a large EcoRI-T4/Xbal fragment of about 5700 bp, containing yeast DNA sequences located 3' to the Xbal site, plus pBR322 sequences, was purified by 35 electrophoresis, electroelution and ethanol precipitation. - 23 - 2049 This fragment was mixed with and ligated to the 1480 bp promotor-containing fragments. The ligation mixture was used to transform competent cells of E. coli K12 strain MM294. Transformants were 5 selected on ampicillin agar. Plasmid DNA was isolated from transformant colonies by the procedure described by Birnboiin et al. , Nucl. Acid. Res., Volume 7, 1513, (1979) and were screened for presence of an Xbal site by digestion with Xbal. An Xbal. site was present only in those plasmids e 10 in which the filled-in Xbal site on a fragment from the second series of reactions was ligated to another fragment from the first series which terminated in a 3' T residue so as to restore an Xbal recognition sequence, TCTAGA, in a . new location. A plasmid possessing an Xbal site was 15 identifed as pRIT10749. A flow sheet illustrating these manipulations is provided in Figure 6. E. coli K12 strain MM294 (pRIT10749) was deposited in accordance with EPC regulations and the Budapest Treaty in the American Type Culture Collection, Rockville, Maryland, U.S.A. on June 2, 20 1982 under accession number ATCC 39133. A sample of pRIT10749 was digested with a combination of BstEII and Xbal. The size of the deleted arg3 regulatory region was estimated to be about 210 bp . by comparison with pMC200 which had been similarly digested 25 with BstEII and Xbal and by reference to the known molecular weights of fragments of phage (j) xl74 DNA described by Fuchs et al. Gene, Volume 4, 1, (1978). To determine the DNA sequence of the BstEII-Xbal fragment of pRIT10749, plasmid DNA was digested with 32 30 BstEII, labelled with y - P-ATP by exchange kination and digested with BamHI endonuclease to liberate a 2000 bp fragment which was purified by electroelution and ethanol precipitation. DNA sequencing of the labelled fragment was performed according to the chemical modification method 35 described by Maxam et al., Methods in Enzymology, Volume - 24 - 204 952 65/ 499 (1980). A part of this sequence was determined to be CCCATCAACTTGTACACTCGTCTAGA. The underscored nucleotides were derived from the 5700 bp EcoRI-T4/Xbal fragment. Comparison to the appropriate region of the DNA sequence 5 shown in Figure 4 indicates that the restored Xbal site is located in the 5' untranslated leader region nine nucleotides upstream of the original HincII site in the pMC200 yeast DNA insert. I'■ 10 Example 10. Preparation of Plasmids, pRIT10759 and pRIT1076l, Containing HBsAg and Regulatory Regions, by Combining pRIT10749 with pRITl0601 pRIT10601 was digested with Hhal endonuclease, and . electorphoresed on a 7.5% acrylamide gel. A 1100 bp 15 fragment was recovered by electroelution and ethanol precipitation. The DNA sequence of part of this fragment contains sequences corresponding to the known N-terminal amino acid sequence of HBsAg of ayw serotype as described by Peterson et al., Viral Hepatitis, G. N„ Vyas, S. N. 20 Cohen and R. Schmid, Eds., Franklin Institute Press, Philadelphia, U.S.A., 1978, p.569. The fragment contains 6 nucleotides located 5' to the HBsAg initiation codon, the complete HBsAg coding sequence and about 390 3' non-translated nucleotides. 25 DNA of plasmid pRITl0749 was digested with Xbal and about 400 ng of this DNA was mixed with about 280 ng of the purified Hhal fragment. The mixture was incubated with 0.5 units of T4 DNA polymerase for 30 minutes at 37° in 20 yul buffer (pH 7.5) containing 20 mM tromethamine-HCl, 10 mM 30 MgCl^, 1 mM dithiothreitol, and 33 mM each of dATP, dCTP, dGTP and dTTP. To this reaction mixture was added 2.5 yul of 1 mM ATP, 2.5 ^al of 10 mM EDTA and 2 ^1 (2 units) of T4 DNA ligase and the mixture was incubated for 16 h at 15°. 204 9 5 - 25 - The ligated mixture was used to transform competent cells of E. coli K12 strain MM294 with selection being made for ampicillin resistant transformants. Plasmid preparations were made from several of these transformants 5 and analyzed by restriction endonuclease digestion and gel electrophoresis. The results showed that the blunt-ended HBV fragment had been inserted in both possible orientations into the Xbal-digested pRIT10749„ One such plasmid, pRIT10761 contained the 1100 bp insert in the 10 correct orientation for transcription of the HBsAg sequence from the arg3 promotor and gave a 271 bp fragment after sequential digestion with BstEII and Xbal. It is illustrated in Figure 7. A second plasmid, pRlT10759 ■ contained the insert in the incorrect orientation and gave 15 rise to an estimated 1175 bp fragment on digestion with a combination of BstEII and Xbal endonucleases. The nucleotide sequence at the arg3 regulatory region-HBsAg insert junction was determined for pRIT10761, by sequencing the 271 bp BstEII-Xbal fragment using the chemical 20 modification methods of Maxam et al., Methods in Enzymoloqy, Volume 65, 499 (1980), after exchange kination 32 labelling of the Xbal terminus with P-ATP. The sequence determined for the arg3 regulatory region - HBsAg junction is TACACTCGTCTACTGAACATG. It can be seen that the 25 Xbal restriction site was not completely repaired by the T4 DNA polymerase and that one guanine residue was lost. The arg3 non-translated leader region is immediately followed by 6 nucleotides of HBV origin, CTGAAC, followed by the HBsAg initiation codon, ATG, and the coding sequence of the 30 HBsAg structural gene. The HBsAg initiation codon is the first initiation codon encountered downstream of the arg3 promotor. Therefore, translation of the mRNA transcribed from the DNA will initiate at that codon and HBsAg synthesized will be devoid of extraneous amino acid 35 residues. The HBsAg synthesized is not a fusion protein? 20495 - 26 - it is substantially structurally identical to authentic HBsAg. Example 11. Preparation of Plasmid pRITl0764 and 5 pRIT10765 by Combining pRIT10761 with YEpl3 Hindlll. pRlT10761 and pRIT10759 were separately digested with PstI and BamHI to destroy the pBR322 replicon moiety and then with Hindlll to release the DNA fragment carrying the inserted HBV DNA and the regulatory region. The DNA1s were b- 10 extracted with phenol, precipitated with ethanol and dissolved in buffer (pH 7.5) containing 0.01 M tromethamine-HCl and 0.001 M EDTA. A 0.6 yug portion of each DNA preparation was separately mixed with 0.3 yug . Hindlll-digested, alkaline phosphatase-treated DNA of 15 YEpl3 Hindlll prepared as described above and the mixture was ligated. The ligated mixture was used to transform competent cells of E. coli K12 strain MM294 with selection being made for ampicillin resistant transformants. One transformant 20 was shown to contain a plasmid, pRITl0764, consisting of the Hindlll fragment from pRITl0761 inserted on YEp 13HindIII. A further transformant was shown to contain a plasmid, pRIT10765, consisting of the Hindlll fragment from pRIT10759 inserted on YEp 13HindIII. A flow sheet 25 illustrating the preparation of pRIT10761 and pRIT10764 is provided in Figure 7. Example 12 - Transformation of Yeast with PRIT10764 and pRIT10765 30 pRIT10764 and pRIT10765 were isolated from cultures of the transformants prepared in Example 11 by CsCl-ethidium bromide density gradient centrifugation and 10 ^g of each was used to transform cells of yeast strain lcl697d as described in Example 7 with selection being made for 35 leucine-independent transformants on regeneration agar. 204952 - 27 - One leucine-independent colony arising from these transformations is designated strain lcl697d(pRIT10764). Another transformant is designated S. cerevisiae strain lcl697d(pRIT10765). Cultures of these strains were grown 5 at 30° in Yeast Nitrogen Base medium supplemented with 2% glucose and 20 yjg/ml arginine to an optical density at 620 jim of 0.80 to 0.88. Cells were harvested and cell-free extracts were prepared and assayed for HBsAg by the Ausria radioimmunoassay as described in Example 7. 10 Extracts of lcl697d(pRITl0764) gave positive results in this assay at dilutions in PBS of up to 1/1024 whereas undiluted extracts of strain lcl697d(pRIT10765) were uniformly negative in this assay. From the genetic and .radiomimmunoassay evidence, it is concluded that cells of 15 S. cerevisiae strain lcl697d(pRIT10764) synthesize HBsAg which can be used in a vaccine for stimulating protection against HBV infection in humans without serious side effects. </div>

Claims

<div class="application article clearfix printTableText" id="claims"> 204952 - 28 - WHAT WE CLAIM IS:

1. A recombinant DNA molecule comprising a nucleotide sequence which can code for HBsAg and a regulatory region derived from the yeast arg3 gene which can effect 5 transcription of the HBsAg sequence in yeast.

2. The molecule of claim 1 wherein the HBsAg sequence is positioned relative to the regulatory region such that the HBsAg synthesized by expression of the HBsAg sequence is substantially devoid of extraneous amino acid residues. t- ■ 10

3. The molecule of claim 1 wherein the regulatory region is derived from pYeura3arg3.

4. The molecule of claim 1 wherein the regulatory region is derived from a 3300 bp yeast DNA fragment specifying the . arg3 gene obtained by digestion of pYeura3arg3 with Hindlll 15 restriction endonuclease.

5. The molecule of claim 1 wherein the HBsAg sequence is derived by treatment of HBV DNA with BamHI restriction endonuclease.

6. The molecule of claim 1 wherein the HBsAg sequence is 20 inserted in the yeast regulatory region at the HincII, Bglll or EcoRI site.

7. The molecule of claim 1 wherein the HBsAg sequence is derived from HBV DNA extracted from Dane particles of adw serotype. 25

8. The molecule of claim 1 wherein the HBsAg sequence is derived from HBV DNA extracted from Dane particles of ayw serotype.

9. The molecule of claim 7 wherein the HBsAg sequence is a BamHI fragment of the HBV DNA. 30

10. The molecule of claim 8 wherein the HBsAg sequence is a Hhal fragment derived from a fused pair of BamHI fragments of the HBV DNA.

11. The molecule of claim 9 wherein the HBsAg sequence is inserted in the Bglll site of the yeast regulatory region. 2049 - 29 -

12. The molecule of claim 10 wherein the regulatory region is a 1480 bp fragment of pMC200 having an EcoRI extension at one extremity and terminating in a 3" T residue at the other extremity which is blunt-ended. 5

13. The molecule of claim 12 wherein the HBsAg sequence is inserted in the Xbal site of a molecule having the 1480 bp fragment fused to a 57 00 bp Xbal-EcoRl fragment of pMC200 after polymerase repair of the Xbal site in the 5700 bp fragment. 10

14. The molecule of claim 1 which is plasmid pRIT10671.

15. The molecule of claim 1 which is plasmid pRIT10673

16. The molecule of claim 1 which is plasmid pRIT10761.

17. The molecule of claim 1 which is plasmid pRIT10764. .

18. A microorganism containing the molecule of claim 1. 15

19. A microorganism containing the molecule of claim 2.

20. coli transformed with the plasmid of claim 14.

21. coli transformed with the plasmid of claim 15.

22. E. coli transformed with the plasmid of claim 16.

23. E^ coli transformed with the plasmid of claim 17. 20

24. The microorganism of claim 18 which is S_. cerevisiae.

25. The microorganism of claim 19 which is £3. cerevisiae.

26. The microorganism of claim 24 which is strain DC5 or lcl697d.

27. The microorganism of claim 24 in which the arginine 25 biosynthesis pathway is derepressed.

28. The microorganism of claim 24 which is cerevisiae strain DC5(pRITl0673).

29. The microorganism of claim 24 which is S_^ cerevisiae strain lcl697d(pRIT10673). 30

30. The microorganism of claim 24 which is S^_ cerevisiae strain lcl697d(pRIT10764).

31. The microorganism of claim 25 which is strain DC5 or lcl697d.

32. The microorganism of claim 25 in which the arginine 35 biosynthesis pathway is depressed. - 30 - 204^

33. The microorganism of claim 27 which is strain lcl697d„

34. The microorganism of claim 32 which is strain lcl697d„

35. A vector comprising the yeast arg3 regulatory region and a restriction site proximate thereto such that a 5 protein synthesized by expression of a coding sequence inserted in the restriction site is devoid of extraneous amino acid residues.

36. The vector of claim 35 which is a plasmid comprising the regulatory region, which is a 1480 bp fragment of t 10 pMC200 having an EcoRI extension at one extremity and terminating in a 31 T residue at the other exremity which is blunt-ended, fused to a 5700 bp Xbal-EcoRl fragment of pMC200 after polymerase repair of the Xbal site of the 5700 . bp fragment. 15

37. The vector of claim 35 which is pRIT10749.

38. A vaccine for stimulating protection against HBV infection in humans without serious side effects comprising a vaccinal amount of HBsAg produced by the microorganism of claim 24 and a suitable carrier. 20

39. A vaccine for stimulating protection against HBV infection in humans without serious side effects comprising a vaccinal amount of HBsAg produced by the microorganism of claim 25 and a suitable carrier.

40. A vaccine for stimulating protection against HBV 25 infection in humans without serious side effects comprising a vaccinal amount of HBsAg produced by the microorganism of claim 26 and a suitable carrier.

41. A vaccine for stimulating protection against HBV infection in humans without serious side effects comprising 25 a vaccinal amount of HBsAg produced by the microorganism of claim 31 and a suitable carrier.

42. A process for preparing a recombinant DNA molecule containing a nucleotide sequence which can code for HBsAg and which can be expressed in yeast which comprises fusing 35 a nucleotide sequence which can code for HBsAg with a 2049 - 31 - regulatory region derived from the yeast arg3 gene.

43. The process of claim 42 wherein the HBsAg sequence is fused in a position relative to the regulatory region such that the HBsAg synthesized by expression of the HBsAg 5 sequence is devoid of extraneous amino acid residues.

44. The process of claim 42 in which the regulatory region is derived by digestion of pYeura3arg3 with a restriction endonuclease.

45. The process of claim 42 in which the regulatory region 10 is derived from digestion of pYEura3arg3 with Hindlll restriction endonuclease to prepare a 3300 bp yeast DNA fragment specifying the arg3 gene.

46. The process of claim 42 which comprises inserting the HBsAg sequence and the regulatory region into a vector. 15

47. The process of claim 42 which comprises treating HBV DNA with BamHI restriction endonuclease to prepare the HBsAg sequence.

48. The process of claim 46 which comprises inserting a BamHI fragment from HBV DNA extracted from Dane particles 20 of adw serotype into the Bglll site of a Hindlll fragment of the regulatory region previously inserted into a vector.

49. The process of claim 46 which comprises inserting a Hhal fragment derived from a fused pair of BamHI fragments of HBV DNA extracted from Dane particles of ayw serotype 25 into the Xbal site of the vector which is a 1480 bp fragment of pMC200 containing the regulatory region and having an EcoRI extension at one extremity and a terminating in a 3' T residue at the other extremity which is blunt-ended, fused to a 5700 bp Xbal-EcoRI fragment of 30 pMC200 after DNA polymerase repair of the Xbal site in the 5700 bp fragment.

50. The process of claim 47 which comprises fusing a BamHI fragment of HBV DNA extracted from Dane particles of adw serotype to the regulatory region. 204952

51. The process of claim 47 which comprises fusing a Hhal fragment derived from a fused pair of BamHI fragments of HBV DNA extracted from Dane particles of ayw serotype to the regulatory region. 5

52. The process of claim 48 wherein the Hindlll fragment is previously inserted into pBR322.

53. The process of claim 48 which further comprises releasing the Hindlll fragment containing the HBsAg insert and the regulatory region by treatment with Hindlll and 10 inserting the Hindlll fragment into a yeast vector.

54. The process of claim 49 which comprises treating the fused 1480 bp-5700 bp fragments with Hindlll to excise a Hindlll fragment carrying the HBsAg sequence and the regulatory region and inserting the Hindlll fragment into a 15 yeast vector.

55. The process of claim 50 which comprises inserting the BamHI fragment into the Bglll site of the regulatory region.

56. The process of claim 51 which comprises fusing the Hhal fragment to the regulatory region at the Xbal site of 20 a 1480 bp fragment of pMC200 containing the regulatory region and having an EcoRI extension at one extremity and terminating in a 31 T residue at the other extremity fused to a 57UU bp Xbal-EcoRi fragment of pMC200 after polymerase repair of the Xbal site in the 5700 bp fragment. 25

57. The process of claim 52 which further comprises releasing the Hindlll fragment containing the HBsAg sequence and the regulatory region by treatment with Hindlll and inserting the Hindlll fragment into a yeast vector. 30

58. The process of claim 54 wherein the yeast vector is YEpl3 which has been previously modified by excision of a Hindlll fragment.

59. The process of claim 57 wherein the yeast vector is YEpl3 which has been previously modified by excision of a 35 Hindlll fragment. - 33 - 2049 5 2.

60. A process for preparing HBsAg which comprises inserting into yeast cells the recombinant DNA molecule of claim 1, culturing the yeast and collecting the HBsAg which is produced. 5

61. A process for preparing HBsAg which comprises inserting into yeast cells the recombinant DNA molecule of claim 2, culturing the yeast and collecting the HBsAg which is produced.

62. A process for preparing HBsAg which comprises 10 inserting into yeast cells the recombinant DNA molecule of claim 11, culturing the yeast and collecting the HBsAg which is produced.

63. A process for preparing HBsAg which comprises inserting into yeast cells the recombinant DNA molecule of 15 claim 13, culturing the yeast and collecting the HBsAg which is produced.

64. The process of claim 60 wherein the yeast is S. cerevisiae strain DC5 or lcl697d.

65. The process of claim 61 wherein the yeast is S_. 20 cerevisiae strain DC5 or lcl697d.

66. The process of claim 62 wherein the yeast is S_. cerevisiae strain DC5 or lcl697d.

67. The process of claim 62 wherein the arginine biosynthesis pathway is derepressed in the yeast. 25

68. The process of claim 63 wherein the arginine biosynthesis pathway is derepressed in the yeast.

69. The process of claim 63 wherein the yeast is S. cerevisiae strain lcl697d.

70. A process for preparing the vector of claim 35 which 30 comprises fusing the regulatory region, which is a 1480 bp fragment of pMC200 having an EcoRI extension at one extremity and terminating in a 3' T residue at the other extremity which is blunt-ended, to a 5700 bp Xbal-EcoRl fragment of pMC200 after polymerase repair of the Xbal site 35 of the 5700 bp fragment.

71. A molecule as claimed in any one of claims. 1 to 17 substantially a,s hereinbefore, described with, reference to any of the accompanying drawings.

72. A micro-organism as claimed in any one of claims 18 to 34 substantially as hereinbefore, described with reference to any of the accompanying drawing?;..

73. A. vector as claimed in any- one of claims. 35 to 37 substantially as hereinbefore described with reference to any of the accompanying drawings.

74. A vaccine as claimed in, any one of claims, 38 to 41 substantially a,s hereinbefore described with, reference to any of the accompanying drawings.

75. A process' as claimed in any one of claims. 42 to 7Q when performed substantially as hereinbefore.' described with or without reference to any of the accompanying drawings. DATED "I Hi*: }°1^- DAY OF \ A. J. PARK & SON PER AGENTS FOR THE APPLICANTS "■y il . \9 JUUW8S | \ / " </div>