AP56A

AP56A - Hepatitis B virus surface antigens and hybrid antigehs containing them.

Info

Publication number: AP56A
Application number: APAP/P/1988/000078A
Authority: AP
Inventors: Teresa Cazebon; Wilde Michael De; Nigel Harford
Original assignee: Smithkline Biologicals S A
Priority date: 1987-01-30
Filing date: 1988-01-25
Publication date: 1989-09-26
Also published as: PT86640B; ZA88488B; CN1031395A; DK43188D0; EP0278940A2; HUT50876A; AU1093088A; NO880395L; EP0278940A3; JPS6463382A; AP8800078A0; NO880395D0; YU17488A; DD285994A5; PT86640A; DD285612A5; FI880428A; NZ223297A; OA08801A; IL85216A0

Abstract

The invention relates to hepatitis b virus antigens and hybrid antigens containing them and to the cloning of genes which code for said antigens in yeast by use of recombinant dna techniques. Said antigens are valuable for the preparation of vaccines, e.G. hepatitis b virus vaccines or malaria vaccines.

Description

BACKGROUND OF THE INVENTION A. Hepatitis B vaccines

Infection with hepatitis B virus (HBV) is a serious, widespread health problem. Infection can be manifested in acute or chronic phases. In the United States, the number of cases of acute hepatitis is estimated to be at least 100,000 per year with a fatality rate of 1 to 2 per cent and the prevalence of chronic carriers of HBV among healthy adults varies between 0.1 and 1% depending on age and social class. In South America, the prevalence of chronic carriers is about l to 3%; in the U.S.S.R. and southern Europe, about 3 to 6%; and in Asia and Africa, more than 10%.

In developed countries, there exists a need for a vaccine for people at high risk of exposure, such as patients and personnel in medical units where blood is handled, military personnel, spouses of chronic carriers, travelers to areas of high HBV endeinicity, newborns of chronic carriers, homosexuals, prostitutes and drug 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

AP ο ο ο o a 6 mortality from chronic active hepatitis and ’.epatocellular care inoma.

Dane particles, which are believed to be hepatitis B virions and which are isolatable from infectad patients, have a diameter of about 42nm. Each consists of an envelope comprising the hepatitis B surface antigen (HBsAg), a capsid (HBcAg). an endogenous polymerase and a DNA genome. The genome is circular and doublestranded with a single strand region consisting of about 200 bases. The single strand region can be CiLled in, in vitro, by the action of the endogenous p- lymerase. The longer strand contains approximately 3,200 bases .

For a long time, 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. However, chimpanzees are also able to be infected in the laboratory with the virus.

Valenzuela et al. (Nature, 298. 347-350. 1982) report synthesis of HBsAg in yeast using an expression vector wherein the HBsAg coding sequence is an 835 base pair (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), 14(1). 21-22. 1981) which reports exit.ssion in yeast of a DNA fragment containing ; 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), which states that a team of U. S. researchers inc I cii.c.j P. Vc'.cr.zuela and W. J. Rutter have announced production in yeast of the protein-coating surrounding hepatitis B virus; and Zuckerman (Nature. 295. 98-99. 1962) which

reports that W. J. Rutter has reported expression of glycosylated HBsAg in yeast cells.

Antigenic components of HBV, such as HBsAg. are reported to have been prepared in bacteria following insertion of a recombinant DNA molecule containing a gene which codes for the antigen. Burrell et al. (Nature, 279. Number 5708, 43-47, 1979) report expression in E. coli strain HB101 of HBV DNA sequences cloned .in plasmid pBR322.

Murray et al., European Patent Application Publication Number 13.828, 1980, disclose preparation of a recombinant vector which can code for HBV antigens, including HBsAg. in E. coli strain HB101. 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 host exemplified in the application is E. coli.

Charnay. et al. (Nature. 286, 893-895. 1980) report construction of a bacteriophage aarrying a fusion of the β-galactosidase gene and the HBsAg structural gene. The bacteriophage directs synthesis of a fusion protein comprising antigenic determinants of both HBsAg and β-galactosidase.

Tiollais et al.. UK Patent Application Number

2,034,323, disclose preparation of a coliphage containing HBV DNA. Fused phage-HBV DNA is transformed into E. coli strain C600.

In UK Patent Application Number 2,070.621. a plas30 mid 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. coli is disclosed.

Rutter et al.» European Patent Application Publication Number 20,251, 1980, disclose recombinant vec35 tors including a recojnbinant vector comprising plasmid .J —

APO 0 0 0 5 6 pBR322 and BamHI fragments of HBV DNA, which can be used to transform E. coli. Another vector, 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, 291, Number 5815. 503-506,

1981) describe construction of plasmids which direct synthesis of HBcAg and a β-lactamase-HBsAg fusion protein,, under the control of the tryptophan operon regulatory region, in E. coli.

Pumpen et al. (Gene, 30, 201-210, 1984) disclose that small amounts of HBsAg monomer proteins and fusion proteins are synthesized in E. coli using antibodies raised against the denatured HBsAg monomer for detection .

Other references disclosing insertion of HBV dna into bacteria include Chacnay, et al. (Prog. Med. Virol., 27, 88-92.(1981); MacKay et al. (Proc. Natl.

Acad. Sci. U.S. 78., Number 7. 4510-4514, 1981); Fritsch et al. (C.R. Acad. Sci., 287. Number 16, 1453,(1978);

U. K. Patent Specification 2,034,323 (Derwent No. 46874C) and Pasek et al. (Nature, 282 , No. 6, 575,

1979).

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_. 72. Number 8. 4549-4553, 1980) report transformation of mouse cells with a plasmid containing the HBV genome and expression of HBsAg; Hirschman et al. (Proc. Natl. Acad. Sci. U.S.. 77, Number 9. 55075511, 1980) report production of HBV-like particle' by

HeLa cells transformed with HBV DNA.

Procedures for preparing HBV vaccine using HBsAg from human blood are reported by Funakoshi et al.

(Prog. Med. Virol., 27. 163-167, 1981) and Maupas et

bad ORIGINAL

al. (Prog. Med. Virol., 27 . 185-201, 1981). The vaccine prepared by Funakoshi et al. contains 40 pg of purified, formalin-treated HBsAg, phosphate, sodium chloride, 20 mg of mannitol; and 0.1% of aluminum hydroxide as adjuvant. In the latter paper, Maupas et al. report that one dose of vaccine was 1 mL of purified, formal in-treated HBsAg containing 2-10 pg/mL of protein (Lowry's method) and 0.1% of i aluminum hydroxide. The protocol used in the study reported by

Maupas et al. called for three injections at one month intervals with a booster after one year: the authocs propose a protocol consisting of two injections of concentrated HBsAg at three month intervals.

Additional references to preparation of HBV vacci15 nes 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 expression of certain other non-HBV 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) contains a report that a type of interferon is being prepared in yeast. In European Patent 11,562 (Derwent No. 38762C) are reported hybrid yeast plasmids containing the ura+ yeast gene in the 2 μ plasmid.

The authentic Hepatitis B Virus surface ’antigen (HBsAg) can be recovered from plasma of infected individuals as a particle of about 22 nra comprised of two proteins known as P24, and its glycosylated derivative GP28. both of which are encoded by the 226 amino acid coding sequence on the HBV genome known as the S pro35 tein coding sequence.. The entire 163 amino acid coding

APO 0 0 0 5 6 sequence which immediately precedes the £ protein coding sequence on the HBV genome is referred to herein as the Pre S coding sequence. The 55 amino acid^ of the Pre S coding sequence which immediately precede the

S protein coding sequence are referred to herein as the Pre S2 coding sequence, and the remaining 108 amino acids of the Pre S coding sequence are referred to herein as the Pre SI coding sequence. The Pre S coding sequence, or any smaller portion thereof, is also referred to herein as the HBsAg precursor protein.

For completeness, it should be noted that the entire Pre S coding sequence for some HBV subtypes (e.g., ayw) is 163 codons, while the entire Pre S coding sequence for other HBV subtypes (e.g., adw₂) is 174 codons. In either case, the Pre S2 region is the 55 codons immediately preceding the S protein coding sequence.

The Pre S2 coding sequence codes for the polyalbumin binding or receptor site found on the surface of

Dane particles and on the surface of some HBsAg particles isolated from sera of HBV-infected patients. Although the Pre S2 region immediately precedes the S protein coding region on the HBV genome, the Pre S2 region is not involved in the assembly of HBsAg parti25 cles. See, e.g., Persing et al. (Proc. Natl. Acad. Sci. U.S.A.. 82., 3440-3444, 1985).

Valenzuela et al. (Nature, 298, 347-350, 1982) disclose that particles resembling HBsAg were found after disruption of yeast cells transformed with a vec30 tor containing the S protein coding sequence, and concluded that HBsAg particles are synthesized in yeast.

Harford et al. (Developments in Biological Stank dardization, 54, 125-139, 1983) disclose that particles resembling HBsAg were found after disruption of yeast cells transformed with a vector containing 18 amino-

acids of the coding sequence of ornithine carbamoyl transferase immediately preceding the N-terminal 42 amino acids of the Pre S2 coding sequence immediately preceding the S protein coding sequence.

Laub et al. (J. Virology, 48.(1), 271-280, 1983) disclose construction of a Simian Virus 40 early replacement vector that has a large portion of HBV genome DNA including the Pre S-S protein coding sequence, and expression of such portion by SV40- transformed CV-1 cells (COS cells) transformed by such vector.

Stibbe et al. (J. Virology, £6(2), 626-628. 1983) disclose that minor HBsAg glycoproteins, named GP33 and GP36, are coded for by the Pre S2-S protein coding sequence. Stibbe et al. (Dev. Biol. Stand., 5 <., 33-43,

1983) disclose that HBsAg particles containing large amounts of GP33 and GP36 did not induce higher antis protein antibody titers than those HBsAg : particles almost devoid of GP33 and GP36.

Heerman et al. (J. Virol., 52 (2). 396-402, 1984) disclose that the entire 389 amino acid coding sequence comprising both the S protein coding sequence and the Pre S coding sequence codes for a polypeptide, P39, which is found in HBV particles and viral surface antigen filaments along with its glycosylated form, GP42, and the other HBV surface antigen associated polypeptides P24. GP27. GP33 and GP36.

Neurath et al. (Science, 224, 392-394, 1984 ) disclose that polypeptides having the sequence of the 26 amino-terminal amino acids of the Pre-S2 coding sequence act as an immunogen, and suggest that antibodies elicited by the immunogen can be utilized for diagnostic tests.

Michel et al. (Proc. Natl. Acad. Sci. U.S.A., 81,

7708-7712, 1984) disclose synthesis of HBsAg carrying

APO 0 0 0 5 6 hamster carrying .

human serum polyalbumin receptors in Chinese ovary (CHO) cells transfected with a plasmid the Pre S2-S protein coding sequence.

Persing et al. (Proc. Natl. Acad. Sci. U.S.A

3440-3444, 1985) disclose that mouse L cells transformed with a Pre S2-S protein coding sequence produce three HBsAg related polypeptides (24.000, 27,000 and

35,000 daltons) all of which may be organized into complex, immunoreactive HBsAg particles of 22 nm diameter 10 which bind to polymerized human serum albumin (HSA); while mouse L cells transformed with the Pre S2-S protein coding sequence bearing a frame-shift mutation near the 3' end of the Pre S2 region produce only the 24,000 and 27,000 dalton polypeptides organized into 22 15 nm diameter immunoreactive HBsAg particles which are unable to bind HSA. Persing et al. conclude that the Pre S2-S protein coding sequence encodes the 35,000 dalton species, that the Pre S2 protein accounts for the HSA binding activity of HBsAg but is not required 20 for assembly and secretion of the HBsAg particles; and that the major polypeptide of HBsAg (i.e., the 24,000 dalton species) is not derived primarily by cleavage of larger precursors (i.e., the 27,000 and 35,000 dalton species) encoded by the Pre S2-S protein coding sequence.

Milich et al. (Science. 228, 1195-1199,(1985) disclose that vaccines that contain HBV particles with both Pre S2 and HBsAg, prepared from Chinese Hamster Ovary (CHO) cells transfected with a plasmid containing the Pre S2-S protein coding sequence, can circumvent nonresponsiveness in certain mice to vaccines which just contain only HBsAg; and that the 26 amino acid residues at the

NH₂-terminus of the 33.000 dalton polypeptide coded for by the Pre S2-S protein coding sequence represent a dominant antibody binding site on the Pre S2 region.

Michel et al., Vaccines 86, Brown et al., Ed., Cold Spring Harbor Laboratory (1986), 359-363, discuss the synthesis in CHO cells of Hepatitis B surface antigen particles containing the Pre S2 region expression product.

Neurath et al. (Nature, 315. 154-156, 1985) disclose that the Pre S region codes for proteins on the

HBV envelope with domains specifically recognized by liver cells; the Pre S2-S protein coding sequence codes for a protein present in HBV particles; synthetic peptides corresponding to the gene encoding Pre-S proteins are immunogenic; and conclude that HBV vaccines should contain Pre S determinants.

Valenzuela et al. (Biotechnology, 3.» 317-320,

1985) disclose the expression of the entire Pre S2-S protein coding sequence in yeast transformed with such coding sequence; and also disclose that such yeast cells do synthesize a particle containing both HBsAg and Pre S2 which is very similar in electronmicroscopy and sedimentation properties to particles containing only HBsAg, and that the Pre-S2 region does not infer the ability to form particles resembling the 22 nm

HBsAg particles. Valenzuela et al. also disclose that it has been hypothesized that HBV gets into the liver by binding polyalbumin to its polyalbumin receptor which in turn binds the polyalbumin receptGr to the liver cell and thus, the virus gets internalized, and that the polyalbumin receptor of the HBV is encoded by the Pre S2 region. Valenzuela concludes that a vaccine containing Pre S2 will elicit antibodies which. in *

addition to inactivating HBV through the normal mechaAP 0 0 0 0 5 6

Takeda Chemical Ind. KK, European Patent Application Publication No. 171,908-A, claims the production of non-glycosylated hepatitis-B virus surface antigen P31 protein in yeast.

Chiron Corporation. European Patent Application

Publication No. 0,174,444 A2, claims a method for preparing HBsAg particles containing P31 protein in yeast.

The following Table I compares known Pre S2 region amino acid sequences with the HBV Pre S2 amino acid sequence of the subject invention :

/ /

/

Table I Comparison of amino acid sequence of pre S2 region from different serotypes of HBV

HBV Pre S2 coding region subtype —55 —50 -40 —30 —20 —10 0 Reference adw MQWNSTAFHQALQDPRVRGLYFPAGGSSSGTVNPAPNIASHISSSSARTGDPVTN The subject

Pre S2 coding region

adw adw	L	T	L L	I	1 n 4
I	I
adw							I		3
adw			K				I		4
adr	T		L		V TT	P	IFS	AP	5
adr	T		L		V TT	P	I S	AP	6
ayw	T	T			VLTT	PL	IFS I	AL	7
adyw H	T	T			V TTT	P	IFS I	AL	8 <©

Amino Acid Abbreviation Key

Asp	0	Aspartic acid	He	I	Isoleucine
Thr	T	Threonine	Leu	L	Leucine
Ser	s	Serine	Tyr	Y	Tyrosine
Glu	E	Glutamic acid	Phe	F	Phenylalanine
Pro	P	Proline	His	H	Histidine
Gly	G	Glycine	Lys	K	Lysine
Ala	A	Alanine	Arg	R	Arginine
Cys	C	Cysteine	Trp	W	Tryptophan
Val	V	Valine	Gin	Q	Glutamine
Met	M	Methionine	Asn	N	Asparagine

AP o 0 0 0 5 6

Reference Key

1. Valenzuela et al.. In Animal Virus Genetics (Field, Jaenisch and Fox eds) p. 57-70, Academic Press.

New York (1980).

2. Chow et al.. Fourth Annual Congress for Recombinant DNA Research, San Diego, USA, 1984.

3. Fujisawa et al.. Nucl. Acids Res. . 11, 46014610 .(1983) .

10	4 .	Lo	et	al., Biochem Biophys. Res. Com.. 2,
	382-388 5 .	(1986 ) . Fu j isawa	et al., cited above.
	6 .	Ono	et	al.. Nucl. Acid Res., 11. 1747-1757
15	(1983) . 7 .	Galibert	et al.. Nature. 281. 646-650 (1979).
	8 .	Pasek et	al.. Nature. 282. 575-579 (1579).

B. Malaria Vaccines

Numerous recent studies suggest that protective immunity in a sensitive host to infection by the sporozoite stage of a particular species of Plasmod ium can be mediated via antibodies produced by such host to the circumsporozoite protein (CS protein) of the particular sporozoite .

The sporozoite proteins of some types of Plasmodium have been cloned and some of those have been expressed by recombinant DNA techniques.

Nussenzweig et al. (U.S. Patent 4,466,917) disclose a sporozoite polypeptide, identified as the P-44 protein, and its cloning and expression in E. co 1i.

Sharma et al. (Science, 228, 879-682, 1 985 ) disclose the expression of a portion of the ciLcumsporo‘zoite (CS) protein of Plasmodium knowlesi in yeast by employing an expression vector containing the 5' regu35 latory region of the yeast alcohol dehydrogenase I gene

in place of the 5' upstream region of the P. knovl.es i CS gene sequence.

New York University, PCT Application Publication Number W084-02922-A, disclose cloning of a portion of the coding region for the P. knowlesi circumsporozoite (CS) protein repeat unit and expression of beta-lactamase and beta-galactosidase fusions thereof in E. coli.

Kemp et al.. PCT Patent Application Publication Number WO84-02917-A, disclose cloning and expression in

E. coli of a coding sequence for the CS protein derived from the blood stage of P. falciparum.

Dame et al. (Science. 225. 593, 1984) report cloning and expression of the CS protein of P. falciparum in E. coli. The protein is described as comprising about 412 amino acids with an approximate molecular weight of 44,000. It comprises 41 tandem repeats of a tetrapeptide. Synthetic 7-, 11- and 15- residue peptides derived from the repeat^: region bound to monoclonal antibodies raised against the CS protein.

Ellis et al. (Nature. 302, 536-538, 1983) report expression of a beta-lactamase - P. knowlesi CS protein fusion in E. coli.

Enea et al. (Proc. Natl. Acad. Sci., USA, 81. 7520-7524, 1984) report an analogous repeat unit struc25 ture within the CS protein of P. cynomolqi. and the cloning and expression of the CS protein in E. coli as well as a disclosure of its coding sequence.

Ballou et al. (Science, 228. 996-999, 1985) disclose that synthetic peptides of the repeating region of the CS protein of Plasmodium falciparum Conjugated to bovine serum albumin (BSA) or to thyroglobulin produced antibody responses in mice and rabbits, and conclude that a vaccine against the sporozoite stage of the malaria parasite can. be developed by using syntheAP 0 0 0 0 5 6 tic peptides of the repeating region of the CS protein conjugated to a carrier protein.

Weber et al. (Molecular and Bacterial Parasitology» 15 , 305-316. 1985) disclose the use of a cloned CS protein gene of a Brazilian strain of P. falciparum as a probe to analyze the structure of 17 other p. falciparum strains by nucleic acid hybridization, and conclude that the CS protein gene is highly conserved and that malaria vaccine development with the CS prptein is unlikely to be complicated by strain variation.

Arnot et al. (Science. 230, 815-818, 1985) disclose the cloning of the CS protein of P. vivax and disclose its entire coding sequence and concxude that the coding sequence of the CS protein of P. vivax is homologous to that of the CS gene of P. knowlesi but not P. falciparum. i

Sharma et al. (Science. 229. 779-782, 1985) disclose the complete nucleotide sequence of t)ie coding region of the CS gene of the Nuri strain of P. knowlesi.

Young et al. (Science, 228, 958-962, 1985) disclose the expression of P. falciparum CS proteins in E. coli for potential use in a human malaria vaccine.

The Walter and Eliza Hall Institute of Medical Research, PCT Patent Application Publication No. W084/02917, 1984, disclose artificially constructed polynucleotide sequences substantially corresponding to all or a portion of P. falciparum mRNA or genomic DNA; the peptide corresponding to such sequence and a method for production thereof; a composition for stimulating immune responses against P. falciparum antipens in a mammal comprising such peptide.

Mazier et al. (Science, 231, 156-159, 1986) disclose that antibodies raised in mice immunized with several recombinant and synthetic peptides of the circumsporozoite protein of P. falciparum were evaluated

for protective activity in a human hepatocyte culture system and were found to exhibit a protective effect against the parasite at three points:

sporozoite attachment to the hepatocyte surface, entry and subse 5 quent intracellular development.

C. Polyvalent vaccines

Valenzuela et al. (Biotechnology, 3., 323-327,

1985) disclose the use of the HBsAg polyalbumin recep10 tor coded for by the Pre S2 coding sequence as a means to prepare polyvalent vaccines. Valenzuela e; al. also disclose preparation of a hybrid HBsAg-Herpes simplex 1 virus glycoprotein D (HSV-lgD) particle by expressing a hybrid HSV-lgD-Pre S2-S protein coding sequence in yeast.

Valenzuela. Hepatitis B subunit vaccines using recombinant DNA Techniques, May 15, 1985 Bio-Expo-5Meeting, Boston Massachussetts, discloses that with a hybrid, such as the HBsAg-HSV-lgD particle described above, there is a problem of immunodominance of the HBsAg over the foreign immunogen presented on its surface .

Chiron Corporation, European Patent Application Publication No. 0.175,261 claims a novel hybrid parti25 cle containing at least a major portion of the Hepatitis B surface antigen fused to one or more oligopeptides, where the hybrid polypeptide is capable of forming a particle in a cellular host with presentation of at least one epitope of at least one oligopeptide.

Proteins can undergo post-translational modifications and some of these can profoundly affect the protein's conformation and function. The presence of oligosaccharide chains on human derived preSl-preS2-S and preS2-S protein (Heerman et al. in J. Virol.,,52., 39635 402, 1984; Stibbe and. Gerlich in Virology. - 123 , 436AP 0 0 0 0 5 6

442, 1982 ; Stibbe and Gerlich in J. Virol., 46 . 626628, 1983) and presumably of myristic acid on the preSl- preS2-S protein (Persing et al., Abstracts of papers presented at the 1986 meeting on Molecular Biology of Hepatitis B viruses, August 28-31, 1986, Cold Spring Harbor Laboratory) might influence particle formation, conformation of the proteins in the particle and the antigenicity and immunogenicity of these particled.

Yeast (Saccharomyces cerevisiae) has the enzymaticcapability for protein glycosylation (Ballou. The Molecular Biology of the Yeast Saccharomyces, Metabolism and Gene Expression; Strathern, Jones and Broach Ed.. Cold Spring Harbor Laboratory (1982). p. 335-360) and fatty acid acylation (Towler and Glasler in Proc. Natl. Acad. Sci.. 8 3 , 2812-2816, 1986 ) similar to those of higher eukaryotic cells.

Viral surface glycoptoteins expressed in yeast have been found to be glycosylated. Jabbar et al.

(Proc. Natl. Acad. Sci^, 82, 2019-2023. 1985) disclose that expression of the influenza viral hemagglutinin gene in yeast resulted in a glycosylated hemagglutinin protein. Wen and Schlesinger (Proc. Natl. Acad. Sci., 83, 3639-3643, 1986) disclose that expression of Sindbis and Vesicular stomatitis virus glycoproteins in yeast gave glycosylated forms of these viral proteins. Fujisawa et al. (Abstracts of papers presented at the 1986 meeting on Molecular Biology of Hepatitis B viruses, August 28-31, 1986, Cold Spring Harbor Laboratory, page 62) disclose that expression of the preS2-S gene in yeast results in the synthesis of two g lycosyla t forms of the preS2-S protein. Kniskern et al. (Abstracts of papers presented at the 1986 meeting on Modern Approaches to New Vaccines, Including Prevention of AIDS. September 9-14. 1986. Cold Spring Harbor Laboi

ratory, page 89) disclose that expression of the preSl-preS2-S gene in yeast results in the synthesis of two preSl-preS2-S proteins of 45 kD and 39 kD. Persing et al. (Abstracts of papers presented at the 1986 mee5 ting on Molecular Biology of Hepatitis B viruses, August 28-31, 1986, Cold Spring Harbor Laboratory, page 19) disclose that labeling of COS7 cells Expressing preSl-preS2-S, preS2-S and S proteins with Hmyristic acid.results in the presence of label in the p.eSl10 preS2-S protein.

Fusion proteins expressed in yeast and reassembled in a particle are purified by the general schene:

Aerosil adsorption and desorption (1) -» CaCl₂ precipitation (2) -» phenylagarose or phenylboronate15 agarose adsorption and desorption (3) -» CsCl gradient centrifugation or DEAE-ion exchange chromatography.

Step 1 is described in EP application publication 0 204 680 and step 3 is described in EP application publication 0 199 698 as far as phenylagarose is con20 cerned. The specific combination of step (1) and (3) is able to perform a drastic clean-up of contaminating material (elimination of 90¾ protein, carbohydrates and 50¾ lipids). This novel combination allows the good functioning of the next chromatographic steps.;

In case that fusion proteins are glycosylated, phenylboronate (PBA) is used as an affinity adsorbent. At pH 9.0 the boronate group makes covalent' complexes with potential cis-diol groups on the glycosylated sidechains. PBA gels have been used occasionally for the purification of soluble glycoproteins but not for the purification of particles containing glycoproteins embedded in the lipid bilayer (See. Cook, et al.. Analytical Biochemistry, 149, 349-353, 1985; Middle. et al.. Biochemical Journal, 209, 771-779, 1983; Cook, et al.. Biochimica et Bio.physica Acta, 828. 205-212, 1985;

S 0 0 0 0 dV

Maestasane, et al.. Journal of Chromatography, 189. 225-231, 1980). Because of the multiple ligand binding sites on the particle. PBA gels with very low PB-ligand density have to be used·, e.g., PBA gels with boronate content < 10pg/ml gel, preferably 5pg boronate/ml gel. The use of phenylboronate gels with (very low

I 1 boronate content allow affinity chromatography of particles containing embedded glycoproteins.

SUMMARY OF THE INVENTION

This invention relates to a recombinant DNA molecule comprising a functional DNA coding sequence fused, in phase, to a portion of the Pre S2 region of a Hepatitis B virus (HBV) Pre S2-S protein coding sequence.

The term coding sequence or coding region as used herein also encompasses any functional derivative thereof. By the terra functional derivative is meant a coding sequence with amrno acid alterations which, where appropriate, don't interfere with particle forma20 tion and/or which retain immunogenicity where such retention is desirable. Such functional derivative can be prepared by conventional site specific mutagenesis such as by the methods disclosed by Botstein et al.

(Science. 229. 1193-1201, 1985. Preferably such DNA molecule also comprises a” regulatory region which is 3 preferably derived from the yeast arq gene.

This invention also relates to a recombinant vector which comprises a recombinant DNA molecule operatively linked to a regulatory region, wherein the DNA molecule contains a functional DNA coding sequence fused, in phase, to a portion of the Pie S2 region of a HBV Pre S2-S protein coding sequence. By the term regulatory region as used herein is meant a DNA

I

- 19 sequences necessary for transcription and translation of a coding sequence.

This invention also relates to a eukaryotic host cell transformed with a. recombinant vector, wherein said vector comprises a DNA molecule operatively linked to a regulatory region, and wherein said dnA molecule contains a functional DNA coding sequence £used, in

J , phase, to a portion of the Pre S2 region of ,'U HBV Pre S2-S protein coding sequence. This invention llso relates to a method of preparing such transformed host cell which comprises transforming a eukaryptic host cell with such recombinant vector.

This invention also relates to a method for preparing a hybrid particle containing HBsAg protein which contains and/or presents a peptide encoded by a functional DNA coding sequence, which comprises culturing a eukaryotic host cell transformed with a recombinant vector in appropriate culture media and isol'ating the particle from a cell lysate or extract of such₍host's culture, wherein said vector comprises a DNA¹ molecule operatively linked to a regulatory region, wherein the DNA molecule contains the functional DNA coding sequence fused, in phase, to a portion of the Pre S2 region of a HBV Pre S2-S protein coding sequence.

This invention also relates to a hybrid immunogeI nic particle comprising a Hepatitis B virus surface antigen (HBsAg) particle which contains and/or presents a peptide encoded by a functional DNA coding sequence.

This invention also relates to a vaccine fcompri30 sing an immunoprotective amount of such hybrid particles .

This invention also relates to micelle comprising ’such hybrid particle and polysorbate and to a vaccine comprising an immunoprotective amount of such micelles.

AP 0 0 0 0 5 6

By the term functional DNA coding sequence is meant a DNA coding sequence which, when fused, in phase, to a portion of the Pre S2 region of ' a HBV Pre S2-S protein coding sequence, is not involved with, and i

does not interfere with, the assembly of the HBsAg particle .

Preferred functional DNA coding sequencer include, but are not limited to. (a) the entire HBV Pre S protein· coding sequence, or an immunogenic derivative thereof, such as, but not limited to. the Pre S2, Pre Si or Pre Sl-Pre S2 protein coding sequence; and (b) other immunogenic coding sequences such as the coding sequence of the circumsporozoite protein of Plasmodium, or any immunogenic derivative thereof, the envelope gene coding sequence of HIV or any immunogenic ^derivative thereof, especially the C_? peptide, peptide 121 or

Dreesman peptide coding sequence or an immunogenic ♦ derivative thereof. ·<Γ

This invention also relates to a HBV Pre SI pro20 tein coding region which codes for the following amino acid sequence:

-163

ATG GGG ACG AAT CTT TCT GTT CCC AAC CCT CTG

Met Gly Thr Asn Leu Ser Val Pro Asn Pro Leu >

f

GGA TTC TTT CCC GAT CAT CAG TTG GAC CCT

Gly Phe Phe Pro Asp His Gin Leu Asp Pro

GCA TTC GGA GCC AAC TCA AAC AAT CCA GAT Ala Phe Gly Ala Asn Ser Asn Asn Pro Asp

	CCA Pro	GCA Ala	GCC Ala	AAC Asn	CAG Gin	GTA Val	GGA Gly	GTG Val	GGA Gly	GCA Ala
	TTC	GGG	CCA	GGG	CTC	ACC.	CCT	CCA	CAC	GGC
5	Phe	Gly	Pro	Gly	Leu	Thr	Pro	Pro	His	Gly
	GGT	ATT	TTG	GGG	TGG	AGC	CCT	CAG	GCT	CAG
	Gly	lie	Leu	Gly	Trp	Ser	Pro	Gin	Ala	Gin
10	GGC	ATA	TTG	ACC	ACA	GTG	TCA	ACA	ATT	CCT
	Gly	lie	Leu	Thr	Thr	Val	Ser	Thr	lie	Pro
	CCT	CCT	GCC	TCC	ACC	AAT	CGG	CAG	TCA	GGA
	Pro	Pro	Ala	Ser	Thr	Asn	Arg	Gin	Ser	Gly
15
	AGG	CAG	CCT	ACT	ccc	ATC	TCT	CCA	CCT	CTA
	Arg	Gin	Pro	Thr	Pro	lie	Ser	Pro	Pro	Leu
	AGA	GAC	AGT	CAT	CCT	CAG	GCC
20	Arg	Asp	Ser	His	Pro	Gin	Ala

or to a HBV Pre S2 protein coding region or to a HBV Pre S2-S protein coding region wherein the Pra S2 portion thereof codes for the following amino acid sequence:

-55 -50

Met-Gln-Trp-Asn-Ser-Thr-Ala-Phe-His-Gln-Ala-Leu-Gln-Asp-40 -30

Pro-Arg-Val-Arg-Gly-Leu-Tyr-Phe-Pro-Ala-Gly-Gly-Ser-Ser30 Ser-20

Gly-Thr-Val-Asn-Pro-Ala-Pro-Asn-Ile-Ala-Ser-His-Ile-SerAP 0 0 0 0 5 6

Ser-Ser-Ser-Ala-Arg-Thr-Gly-Asp-Pro-Val-Thr-Asn; a

or Pre S2-S protein coding region operatively linked to a regulatory region; a transformed host cell :ontaining such vector; a process of preparing such transformed cell which comprises transforming a host cell with such vector; the protein coded for by such coding region; a process for preparing such protein; a vaccine comprising an immunoprotective amount of such protein, the particle coded for by such Pre S2-S protein coding region; a process for preparing such particle, a vac10 cine comprising an immunoprotective amount of such particle. and a micelle comprising such particle and polysorbate, and a vaccine comprising an immunoprotective amount of such micelles.

This invention also relates to a recombinant DNA vector comprising an entire Pre Sl-Pre S2-S protein coding sequence or PreSl-functional DNA coding sequence-PreS2-S protein coding sequence, or immunogenic derivative thereof, operatively linked to a regulatory region; to a yeast host cell· transformed with such vector; to a method of preparing such transformed host which comprises transforming a yeast host cell with such vector; to a method of preparing protein encoded by the Pre Sl-Pre S2-S protein coding sequence, or Pre-Sl-functional DNA coding sequence - Pre S2-S pro25 tein coding sequence. or an immunogenic derivative thereof; to the protein prepared by such method; to a vaccine comprising an immunoprotective amount of such protein; to a particle containing polypeptide encoded by the Pre Sl-Pre S2-S protein coding sequence or Pre

Sl-functional DNA coding sequence - Pre S2-S protein coding sequence or an immunogenic derivative thereof; to a method of preparation thereof, a vaccine comprising an immunoprotective amount of such particles, a micelle comprising such particle and polysorbate, and a vaccine comprising an immunoprotective amount of such Micelles .

This invention also relates to a Pre Sl-Pre S2 protein coding sequence or Pre Sl-Pre S2-S protein coding sequence of which the Pre Sl-Pre S2 portion comprises the following amino acid sequence:

PRE-SI REGION

-163

	ATG	GGG	ACG	AAT	CTT	TCT	GTT	CCC	AAC	CCT	CTG
10	Met	Gly	Thr	Asn	Leu	Ser	Val	Pro	Asn	Pro	Leu
	GGA	TTC	TTT	CCC	GAT	CAT	CAG	TTG	GAC	CCT
	Gly	Phe	Phe	Pro	Asp	His	Gin	Leu	Asp	Pro
15	GCA	TTC	GGA	GCC	AAC	TCA	AAC	AAT	CCA	GAT
	Ala	Phe	Gly	Ala	Asn	Ser	Asn	Asn	Pro	Asp
	TGG	GAC	TTC	AAC	CCC	ATC	AAG	GAC	CAC	TGG
	Trp	Asp	Phe	Asn	Pro	He	Lys	Asp	His	Trp
20	CCA	GCA	GCC	AAC	CAG	GTA	GGA	GTG	GGA	GCA
	Pro	Ala	Ala	Asn	Gin	Val	Gly	Val	Gly	Ala
	TTC	GGG	CCA	GGG	CTC	ACC	CCT	CCA	CAC	GGC
25	Phe	Gly	Pro	Gly	Leu	Thr	Pro	Pro	His	Gly
	GGT	ATT	TTG	GGG	TGG	AGC	CCT	CAG	GCT	CAG
	Gly	lie	Leu	Gly	Trp	Ser	Pro	Gin	Ala	Gin
30	GGC	ATA	TTG	ACC	ACA	GTG	TCA	ACA	ATT	CCT
	Gly	lie	Leu	Thr	Thr	Val	Ser	Thr	lie	Pro
	CCT	CCT	GCC	TCC	ACC	AAT	CGG	CAG	TCA	GGA
	Pro	Pro	Ala	Ser	Thr	Asn	Arg	Gin	Ser	Gly

AP 0 0 0 0 5 fi

	AGG Arg	CAG Gin	CCT Pro	ACT Thr	CCC Pro	ATC lie	TCT Ser	CCA Pro	CCT Pro	CTA Leu
	AGA	GAC	AGT	CAT	CCT	CAG	GCC
5	Arg	Asp	Ser	His	Pro	Gin	Ala
	PRE-	-S2 REGION
	-55
	ATG	CAG	TGG	AAT	TCC	ACT	GCC	TTC	CAC	CAA
10	Met	Gin	Trp	Asn	Ser	Thr	Ala	Phe	His	Gin
	GCT	CTG	CAG	GAT	CCC	AGA	GTC	AGG	GGT	CTG
	Ala	Leu	Gin	Asp	Pro	Arg	Val	Arg	Gly	Leu
15	TAT	TTT	CCT	GCT	GGT	GGC	TCC	AGT	TCA	GGA
	Tyr	Phe	Pro	Ala	Gly	Gly	Ser	Ser	Ser	Gly
	ACA	GTA	AAC	CCT	GCT	CCG	AAT	ATT	GCC	TCT
	Thr	Val	Asn	Pro	Ala	Pro	Asn	lie	Ala	Ser
20
	CAC	ATA	TCG	TCA	AGC	TCC	GCG	AGG	ACT	GGG
	His	He	Ser	Ser	Ser	Ser	Ala	Arg	Thr	Gly
	GAC	CCT	GTG	ACG	AAC
25	Asp	Pro	Val	Thr	Asn

This invention also relates to a recombinant vector comprising such novel Pre Sl-Pre S2 or Pre Sl-Pre S2-S protein coding sequence operativeLy linked to an expression control sequence; a host cell transformed with such vector; a method of preparing such transformed host which comprises transforming a host cell with such vector; to a method of preparing protein encoded by such Pre Sl-Pre S2 or Pre Sl-Pre S2-S protein coding

protein prepared by such method; to a vaccine comprising an immunoprotective amount of such protein; to a particle containing polypeptide encoded by such Pre Sl-Pre S2-S protein coding sequence or an immunogenic derivative thereof; to a method of preparation thereof, a vaccine comprising an immunoprotective amount of such particles, and a micelle comprising such particle and polysorbate.

The following represents the key to the amino acid 10 abbreviations used in this application;

Asp	Aspartic ‘acid	He	Isoleucine
Thr	Threonine	Leu	Leucine
Ser	Serine	Tyr	Tyrosine
Glu	Glutamic acid	Phe	Phenylalanine
Pro	Proline	His	Histidine
Gly	Glycine	Lys	Lys ^;.ne
Ala	Alanine	Arg	Arginine
Cys	Cysteine	Trp	Tryptophan
Val	Valine	Gin	Glutamine
Met	Methionine	Asn	As parag i ne

BRIEF DESCRIPTION OF

THEFIGURES

Figure 1 is a map of pRIT10601.

Figure 2 is a map of pRIT10616.

Figure 3 is a restriction restriction restriction endonuclease endonuclease endonuclease cleavage cleavage cleavage

AP000056 map of pMC200.

Figure 4 is the nucleotide sequence of a portion 30 of the 3300 bp] Hindlll yeast DNA insert in pMC200 which portion contains HiacII, Bglll and EcoRI sites.

Figure 5 is a flow sheet illustrating preparation of PRIT10671 and pRIT10673.

Figure 6 is a flow sheet illustrating preparation of pRIT10749.

Figure 7 is a flow sheet illustrating preparation of pRIT10761 and pRIT10764.

Figure 8 is a flow sheet illustrating preparation of PRIT10167 (Example 1.), pRIT10158 and pRIT10162 (Example 3); pRIT10158, pRIT10677. and pRIT10909 (Example 4); PRIT10911 (Example 5); PRIT10679. pRIT10903,

PRIT10914 (Example 6); PRIT12211. pRIT12209 and pRIT12230 (Example 7); and pRIT12288 and pRIT12322 (Example 8).

Figure 9 is a restriction endonuclease cleavage map of pRIT10172.

Figure 10 is a restriction endonuclease cleavage map of pRIT1229O.

Figure 11 is a restriction endonuclease cleavage map of pRIT12322 which illustrates which portions thereof were derived from pRIT1223O and pRIT12288.

Figure 12 is a flow sheet illustrating preparation of pRIT12571; pRIT12573; pRIT12572 and pRIT12574.

Figure 13 is a restriction endonuclease map of PRIT12309. ?

Figure 14 is a flow sheet illustrating preparation of pRIT12314 and pRIT12544.

Figure 15 is a flow sheet illustrating preparation of pRIT12658.

Figure 16 is a restriction endonuclease cleavage map of pRIT12662.

Figure 17 is a flow sheet illustrating preparation of pRIT12660.

Figure 18 is a flow sheet illustrating preparation of PRIT12845 (a, b, c, d, e, f).

Figure IA is a flow sheet illustrating the preparation Of pRIT12662. . ·.

Figure 2A shows a schematic restrictibn map and sequencing strategy of clone XMPfl.

Figure 3A shows th'.- nucleotide sequence of the circumsporozoite protein gene from P. falciparum.

Figure 4A shows the PreSl-PreS2 DNA coding sequence comprised by pRIT12792.

Figure IB is a schematic restriction map of the

HIV B virus envelope region.

Figure 2B illustrates the construction of plasmid PRIT12893.

Figure 3B illustrates the construction of plasmid pRIT12897. ;

Figure 4B illustrates the construction of plasmid

PRIT12899.

Figure 5B illustrates the construction o; plasmid pRITX.

DETAILED DESCRIPTION OF THE INVENTION EXPRESSION OF HBsAg IN YEAST

By regulatory region as used herein is meant a DNA sequence which contains sequences necessary or desirable for transcription and translation of a coding sequence. Such regulatory regions are generally placed 5' and adjacent to the coding sequence which is to be expressed. Preferably a further region of yeast DNA containing a signal for the termination of trar.scrip25 tion is placed immediately 3' to the coding region to be expressed so as to effect termination of the transcript .

By HBsAg as used herein is meant an antigen containing proteins encoded by the S protein coding sequence of the HBV genome, wherein such HBsAg is structurally similar to authentic HBsAg or has substantially the same S protein coding sequence encoded antigenic determinants as authentic HBsAg, that is, is capable of stimulating an immune response which speciAP000056 fically recognize and react with authentic HBsAg or is specifically recognized by anti-HBsAg antibodie,;.

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, 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, the HBsAg coding sequence of HBV DNA of certain Dane particles of the adw serotype can be isolated on a single BamHl fragment; the HBsAg coding sequence of HBV DNA of certain Dane particles of the ayw serotype can be isolated on a HhaI fragment. HBV DNA of Dane particles of the same serotype may also exhibit different patterns of restriction sites.

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 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 remaiii at about 7.0 to 11.0 and the temperature is kept below about 60°C. Preferably 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 ligase is the preferred ligase. '

The various fragments and final constructions may be joined together in accordance with conventional

res29 triction mapped, as well as sequenced. To tha: extent, one can select the sequence of interest, such as the HBsAg sequence, by restriction of the gene, employing further manipulation as necessary such as resection with Bal31. in vitro mutagenesis, primer repair, or the like, to provide a fragment of a desired size, including the desired sequence, and having the appropriate termini. Linkers and adapters can be used for joining sequences, as well as replacing lost sequences.’> where a restriction site employed was internal to the region of interest. The various fragments which are isolated, may be purified by electrophoresis, electroeluted, ligated to other sequences, cloned, reisolated'and further manipulated.

The use of regulatory regions for controlling transcription of the structural gene of inteiast, such as the HBsAg sequence, may allow for growing the host cells to high density with no or low levels of expression of the structural gene, such as the HBsAg sequence, and then inducing expression by changing the environmental conditions, e.g., nutrient, temperature, etc .

For example, with the.GAL4 regulatory region, the yeast cells can be grown in rich media with a glyce25 rol-lactic acid combination to high density, e.g., mid or late log phase, followed by switching the carbon source to galactose. As another example, +or PHO5 regulation, one could grow the cells at a high phosphate concentration of about 7 mM ^KH2^PO4' and then recover and resuspend them in medium lacking phosphate to effect decepression and expression. For temperature sensitivity, one could grow the cells at 25° to 37°C and then change the temperature as appropriate by about 5° to 20°C. The host cells would have the regulatory system associated with the regulatory region employed.

Fusing of the HBsAg sequence to the regulatory region can be accomplished by use of intermediate vectors. Alternatively, the HBsAg sequence can oe inserted directly into a vector which 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 (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.

In one embodiment of this invention the HBsAg coding sequence together with 42 codons of the HBsAg precursor (preS-S) protein were fused in phase after the 18th amino acid of the coding sequence of, the yeast 3

OCT protein which is transcribed from the arq promoter. This construct gives a hybrid particle containing HBsAg protein containing an additional 60 K^terminal amino acids to the 226 amino acids of the S-protein proper. ^!

An exemplary regulatory region is derivedjfrom the 3 yeast arq gene which codes for ornithine carbamoyl3 transferase (OCT). The arq regulatory region is subject to both specific and general control mechanisms. It has been cloned in E. co 1 i on plasmid

3 pYeura arg as reported by Crabeel et al., Proc.

Natl. Acad. Sci. U.S.A., Volume 78. 6026 (1981). Preferred hosts are S. cerevisiae strains in which the arginine biosynthetic pathway is derepressed? such as strain lcl697d. Use of such strains results in increa3 sed expression from the arq promoter as compared to strain DC5. Examples of other useful regulatory

regions include those derived from yeast genes of the glycolytic pathway such as those coding for enolase, aldolase, phosphoglycerate kinase and glyce:aldehyde3-phosphate dehydrogenase; the yeast alcohol dehydroge5 nase gene; and, in general, genes involved in catabolism such as the arginase gene.

A preferred vector for cloning the fused DNA fragment into yeast is the plasmid YEpl3. which is capable 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 simultaneously. Transformation with plasmid vectors containing autono15 mous replication sequences (replicons) functional in yeast will normally result in extra-chromosomal maintenance of the DNA molecule of the invention is a plasmid. Such replicons are well known in the art. Transformation with plasmid vectors not containing replicons functional in yeast can result in incorporation of the DNA molecule into chromosomal DNA.

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 or aluminum phosphate, is desirable. The HBsAg so produced can also be combined with other immunogens to prepare combination vaccines. The HBV or combination vaccines can be administe30 red, for example, by the subcutaneous, intravenous or intramuscular route. The DNA fragment of the invention and the HBsAg produced thereby, can also Le used as c probe for detection of HBV in biological samples by DBA hydridization techniques and various immunoassays.

AP00005«

EXPRESSION OF A DNA MOLECULE CONTAINING A FUNCTIONAL DNA CODING SEQUENCE FUSED, IN PHASE, TO A PORTION OF

THE PRE S2 REGION OF A HBV PRE S2-S PROTEIN CODING

SEQUENCE

This invention also relates to a recombinant DNA molecule comprising a functional DNA coding sequence fused, in phase, to a portion of the Pre S2 region of a HBV Pre S2-S protein coding sequence. The term coding sequence or coding region as used herein also encom10 passes any functional derivative thereof. Ey the term functional derivative is meant a coding sequence involving amino acid alterations which, where desired, don't interfere with particle formation and/or which retain immunogenicity. Such functional derivatives can be prepared by conventional site specific mutagenesis. Site specific mutagenesis techniques are taught by Botstein et al.. Science. 229. 1193-1201 (1985). Such fusion may occur at the N-terminus of the Pre S2 region or at some point within the Pre S2 region. Th’tre can be four types of fusions, i.e., at the 5'-terminus of the entire Pre S2 region, at some point within the Pre S2 region so that Pre S2 DNA is flanking both sides of the functional DNA coding sequence, at the S'-terminal of a truncated portion of the Pre S2 region, or at the

5'-terminus of the S-protein coding region so that the fusion contains no Pre S2 DNA. Various Pre S2-S protein coding sequences are known and can be prepared or isolated by known techniques. [See. references cited herein, supra 1 . In one embodiment of this invention, the ARG3 regulatory region together with 18 amino acids of the yeast OCT protein were fused, in ph»se. to a truncated Pre S2-S protein coding sequence ’such that the fused sequence contained 42 codons of the Pre S2-S protein coding sequence together with the complete S protein coding sequence. Preferred functional DNA

coding sequences include the circumsporozoite protein coding sequence of Plasmodium, or any immunogenic derivative thereof, the envelope gene coding se ;uence of HIV or any immunogenic derivative thereof, especially 5 the C_? peptide. peptide 121 or Dreesman peptide coding sequence or any immunogenic derivative thereof, and the Pre S2, Pre SI or the entire Pre Sl-Pre S2-S protein coding sequence or any immunogenic derivative thereof. For example, a preferred functional PNA 10 coding sequence is the first 13 Nterminal codons of the Pre S2 coding sequence which are then ligated to the C-terminal 42 codons of a truncated Pre S2 region of a Pre S2-S protein coding sequence. By the term immunogenic derivative is meant a sequence which is a deri15 vative or modified version of a naturally occurring sequence which retains or enhances the protective immunogenic activity of the natural sequence. Such immunogenic derivatives can be prepared by conventicnal techniques. The term Pre S2, Pre SI or the entire Pre 20 Sl-Pre S2-S protein coding sequence as used herein includes any immunogenic derivative thereof.

By the term the circumsporozoite protein of Plasmodium is meant the immunodominant surface antigen on the sporozoite of Plasmodium or any immunogenic deriva25 tive thereof. Useful circumsporozoit<

APO 0 0 0 5 6

proteins	(CS)
der ived	f r om
, and P.	mala-
and P.	ma la -
Lasmod ium	f al-
Science,	2 2 5 .

riae infect humans.

The CS protein coding sequence of ciparum is disclosed by Dame et al., ___

593-599 ( 1984 ). The CS protein coding sequence of. r_ vivax is disclosed by Arnot et al.. Science, 2 30. 815

818 (1985). The CS protein coding sequence of· P. know35 bad original les i is disclosed by Sharma et al., Science, 229. 779 782 ( 1985). Enea et al., Proc. Natl. Acad. Sci. us,A.

81, 7520-7524 (1984). disclose the CS protein codino sequence of P. cynomolqi. The CS protein coding sequences of other species of Plasmodium are known (sec references cited above) and/or can be derived by conventional techniques.

As used herein, the terms CSP or CS protein” will include both the natural, full length circumsporo10 zoite protein of Plasmodium as well as any immunogenic derivative thereof. By the term immunogenic derivative of the circumsporozoite protein of Plasmod ium is meant (a) any derivative of the circumsporozoite protein of Plasmod ium which retains protective im.runogen ic activity or (b) any protein derived from a modified coding sequence of the circumsporozoite protein of Plasmodium which retains protective immunogenic activity. Such immunogenic derivatives can be prepared by conventional techniques. Some immunogenic synthetic derivatives of the CS protein coding sequence of

E a 1c i pa rurn are disclosed in Ballou et al.. Science, 228. 996-999 ( 1985). See also. Mazier et al.. Sc ience,

231, 156-159 (1986).

The CS coding sequence for various species of

Plasmodium is known and can be prepared or isolated by known techniques. [See, e.g., Colman et al., WO84 02922-A (P. knowlesi CS protein) and Dame et al.. Science, 225. 593 (1984) (P. fale iparum CS protein)]. A DNA molecule containing the CS coding sequence, or any other functional DNA coding sequence, fused in phase to a portion of the Pre S2 region of a HBV t :.-: S2-S protein coding sequence can be construct id by con ventional techniques, such as by ligating the CS coding sequence, or any other functional DNA coding sequence.

BAD ORIGINAL in phase to any codon within the 55 amino acid Pre S2 coding region of the Pre S2-S protein coding sequence.

Preferably, enough of the Pre S2 coding sequence remains after ligation^- of the CS coding sequence, or any other functional DNA coding sequence, to insure optimal presentation of the CS protein, of ] any other functional DNA coding sequence protein, on the HBsAg particle surface, i.e.. enough Pre S2 region should remain so that the Pre S2 region encoded polypeptide acts as a bridge between the functional DNA protein coding sequence product and the HBsAg particle surface. It has been reported that the 26 amino-terminal amino acids of the Pre S2 coding sequence represent a dominant antibody binding site on the Pre S2 region.

[See. e.g., Milich et al.. Science. 228. 1195-1199 (1985)]. Thus, if it is desired to prepare a hybrid particle which contains and/or presents both Pre S2 epitopes and the CS, or any other functional coding sequence, epitopes, it is preferable to insert the CS coding sequence, or any other functional DNA coding sequence, at a point within the Pre S2 coding region which enables preparation of a hybrid particle which contains and/or presents both Pre S2 epitopes and CS, or any other functional DNA coding sequence, epitopes.

The recombinant vector of this invention comprises the recombinant DNA molecule of the subject invention (i.e., a functional DNA coding sequence fused, in phase, to a portion of the Pre S2 region of a HBV Pre S2-S protein coding sequence) operatively linked to a regulatory region. Such vector may additionally also comprise a replicon functional in yeast. By the term replicon is meant that minimum region of DNA which functions to maintain stably, and extrachromosoma 1ly, such recombinant vector.in a host yeast organism trans35 formed with such vector.' Such replicons are well known

9S0000d¥

in the art. By the terra regulatory region is meant any DNA region which regulates transcription and translation of the coding sequence desired to be expressed.

i

Such regulatory regions are well known in the art.

Such a vector can be prepared by conventional techniques such as by inserting the DNA molecul^/ of this invention in phase into a vector already containing a replicon and regulatory region in such a way that the

I

DNA molecule is operatively linked to such regulatory region. Preferably the DNA molecule is ligated to a vector capable of expression in the yeast genus Saccharomyces .

This invention also relates to a yeast host cell transformed with such recombinant vector and to a method of preparing such transformed host which comprises transforming a yeast host cell with the recombinant vector of this invention. Such transformation is carried out by conventional γ techniques such j'as those already described herein. Preferred yeast cells include those belonging to the genus Saccharomyces. particularly those belonging to the species S^ccharomyces cerevisiae and Saccharomyces car lsberqensi-s.

This invention also relates to a method,of preparing a hybrid particle containing HBsAg proteyi which contains and/or presents the functional DNA coding sequence product wherein such method comprises culturing the transformed yeast host cells of this invention in appropriate culture media, and isolating the particle from a cell lysate or extract of such host's cul30 ture. By the term presented or contains and/or presents is meant that the functional DNA coding sequence protein is contained within the hybrid particle contai* ning HBsAg protein in such a way as to enable i'-an immune response to the functional DNA coding sequence protein

tial, the presentation of the functional DNA coding sequence protein does not interfere with the immunogenic activity of the HBsAg epitopes, thus yielding a bivalent vaccine. Most preferably the functional DNA coding sequence protein is presented on the HBsAg particle in such a way that the maximum number of either the functional DNA coding sequence protein's epitopes or both the functional DNA coding sequence protein and HBsAg epitopes are properly exposed. By appropriate culture media is meant that media which will enable the transformed host to survive and which will also enable such host to express the hybrid functional DNA coding sequence-Pre S2-S protein coding sequence protein product of the vector it is transformed with in recoverable quantity. It will be appreciated by one of skill in the art that the appropriate culture media to use will depend upon the host cell employed. The isolation of the hybrid particle of this invention from a cell lysate or extract of such host's culture is car20 ried out by conventional protein isolation techniques.

This invention also relates to a vaccine containing the hybrid particles of this invention. Such vaccine will contain an immunoprotective quantity of the hybrid particles of this invention and is prepared by conventional techniques. By immunoprotective” is meant that enough of the hybrid particles of this invention are administered to elicit sufficient protective antibody response against the agent desired to be protected against without serious side effects. Prefe30 rably, the vaccine of this invention comprises the micelle of this invention, i.e., a micelle comprising the particle of this invention and polysorbate. The preferred amount of polysorbate comprised by such micelle is 5-50 yg polysorbate per 100 yg protein.

The micelle may be prepared by the method disclosed in European Patent Application Publication No. 0 199 698.

In the vaccine of the invention, an aqueous solution of the hybrid particles of the invention, preferably buffered at physiological pH, can be used directly. Alternatively, the particle, can be admixed or adsorbed with any of the various known adjuvants. Such adjuvants include. among others, aluminum hydroxide.

Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press. Baltimore, Maryland, U.S.A.. 1978.

The amount of the hybrid particle of the present invention present in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and whether or not the vaccine is adjuvanted. Generally, it is expected that each dose will comprise 1-1000 pg of protein, preferably 1-200 pg . An optimal amount for a particular vaccine can be ascertained by standard stud.es involving observation of antibody titres and other responses in subjects. Following an initial vaccination, subjects will preferably receive a boost in about 4 weeks, followed by repeated boosts every six mourns for as long as a risk of infection exists.

The immune response to the hybrid particle of this invention may be enhanced by the use of adjuvant, such as, but not limited to, aluminum hydroxide (alum).

To exemplify the hybrid particle of this invention, a 64 codon DNA sequence coding for the repetitive epitope of the circumsporozoite protein (CS) of Plasmodium falciparum and a-n eight codon CS coding sequence

bad origin».39 [see Dame et al.. Science. 225. 593-599 (1984)] were each inserted in phase to a portion of the Pre S2 coding sequence in a yeast expression vector comprising a replicon, the Pre S2-S protein coding sequence and an appropriate regulatory region. Both expression vectors. i.e., one containing the 64 codon CS sequence and one containing the 8 codon CS sequence, ware each introduced into yeast host cells, and extracts of such transformed cells were analyzed for the presence of

HBsAg epitopes and CS epitopes on the same molecule.

The results obtained from immunoblot analyses using a pool of five monoclonal antibodies specific for CS and a monoclonal antibody raised against purified yeast-derived HBsAg showed that a 37,000 kilodalton (Kd) hybrid protein was obtained from lysates of yeast transformed with the vector containing the 64 codon CS coding sequence fused in phase with the Pre S2-S protein coding sequence, and a 30,000 Kd hybrid protein was obtained from lysates of yeast transf ormefd. iwith the vector containing the 8 codon CS coding sequence fused in phase with the Pre S2-S protein coding sequence. Assembly of these proteins into HBsAg par’ticle-l ike structures was shown by CsCl and sucrose grauient analysis. high pressure liquid chromatography (HPLC) and electron microscopy. Presentation of the CS e'pitopes on the exterior of such particles was shown by the accessibility of the CS epitope to CS specific antibodies in an ELISA assay. Presentation of HBsAg epitopes on the exterior of such particles was shown by radioimmune assay (AUSRIA II, Abbott) or ELISA assay (EnzygnostHBsAg, Behringwerke).

NOVEL PRE S2 AND PRE S2-S PROTEIN CODING SEQUENCES

The novel HBV Pre S2 and Pre S2-S protein coding sequences of this invention are from an HBV adw subtype which was isolated from plasmid pRIT10616. The term

AP000056 coding sequence or coding region as used herein also encompasses any functional derivative thereof. By the term functional derivative is meant a coding sequence with amino acid alterations which, where appropriate, don't interfere with particle formation and/or which retain immunogenicity where such retention is desirable. Such functional derivatives can be prepared by conventional site specific mutagenesis such as by the method of Botstein et al.. Science, 229. 11931201 (1985). Such Pre S2 and Pre S2-S protein coding sequences can be recovered by conventional recombinant DNA procedures from plasmid pRlT10616 which was deposited (within E. coli K12 strain C600) in accordance with the Budapest Treaty at the American Type Culture Collection. Rockville, Maryland, on June 2, 1582, under accession number ATCC 38131. The Pre S2 region of such novel protein coding sequences codes for the following amino acid sequence:

-55 -50

Met-Gln-Trp-Asn-Ser-Thr-Ala-Phe-His-Gin-Ala-Leu-Gln-Asp-40 -33

Pro-Arg-Val-Arg-Gly-Leu-Tyr-Phe-Pro-Ala-Gly-Gly-Ser-Ser-20

Ser-Gly-Thr-Val-Asn-Pro-Ala-Pro-Asn-Ile-Ala-Ser-His-Ile-10

Ser-Ser-Ser-Ser-Ala-Arg-Thr-Gly-Asp-Pro-VaI-Thr-Asn.

Such region may also be obtained by conventional synthetic oligonucleotide synthesis. A preferred functional derivative of the Pre S2-S coding sequence of this invention is one wherein the alanine (GCT) amino acid at -45 is modified via site specific mutagenesis to a threonine (ACT). Such functional derivative results in two times greater level of expiession by Saccharomyces cerevesiae transformed therewith as comBAD ORIGINAL-

pared to the unmodified coding sequence of this invention.

A recombinant vector of this invention comprises the Pre S2 or Pre-S2-S protein coding sequence of the subject invention operatively linked to a regulatory region. Such vector may also additionally contain a replicon depending on preference and/or the host to be employed. By the terra replicon is meant that minimum region of DNA which functions to maintain stably, and extrachromosomally, such recombinant vector in a host eukaryotic or prokaryotic organism transformed with such vector. Such replicons are well-known in the art. By the term regulatory region is meant any DNA sequence which contains a promoter region and other sequences necessary for transcription of a coding sequence. Such regulatory regions are well-known in the art. Such a vector can be prepared by conventional techniques such as by inserting the Pre S2 or Pre S2-S protein coding sequence of this invention in phase into a vector already containing a replicon and regulatory region in such a way that the DNA molecule is operatively linked to such regulatory region. Preferably the DNA molecule is ligated to a vector capable of expression in yeast such as Saccharomyces.

This invention also relates to a host cell transformed with such recombinant vector and to a method of preparing such transformed host. Such transformation is carried out by conventional techniques. Host cells can be prokaryotic or eukaryotic. Preferred host cells are eukaryotic especially yeast cells and include those belonging to the genus Saccharomyces. particularly those belonging to the species Saccharomyces cerevisiae and Saccharomyces carlsberqensis .

This invention also relates to a method of prepa35 ring a protein encoded by the Pre S2 or Pre S2-S proAP000056 tein coding sequence of this invention wherein such method comprises culturing the transformed host cells of this invention in appropriate culture media, and isolating the protein from the host cell culture fluid or from a cell lysate or extract of such host's culture depending upon the host cell's ability to secrete such protein. By appropriate culture media is meant that media which will enable the transformed hos£_; of this

1’ invention to survive and which will also enable such 10 host to express the Pre S2 or Pre S2-S protein coding sequence of this invention in recoverable quantity. It will be appreciated by one of skill in the art that the appropriate culture media to use will depend upon the host cell employed. The isolation of the Pre S2-S pro15 tein of this invention from a culture lysate or the culture fluid of such host is carried out by conventional protein isolation techniques. The protein prepared by the method of this invention may be glycosylated depending upon the host cell's ability to do so. If a deglycosylated protein is desired, the protein of this invention should be prepared by utilizing a host cell incapable of effecting such glycosylation or by employing conventional site specific mutagenesis on the protein's coding sequence prior to carrying out the process of this invention, such as by the method of Botstein et al.. Science. 229. 1193-1201 (1985).

This invention also relates to a vaccine containing an immunoprotective amount of the Pre S2 or Pre S2-S protein of this invention. Such vaccine nay be prepared by conventional techniques.

This invention also relates to a method of preparing a hybrid particle containing HBsAg protein which ‘ comprises protein encoded by the PreS2-S protein coding sequence of this invention wherein such method compri35 ses culturing the transformed eukaryotic hoeV cells of

this invention in appropriate culture media, and isolating the particle from the host cell culture fluid or from a cell lysate or extract of such host's culture depending upon the transformed host cells ability to secrete such particles. By appropriate culture media is meant that media which will enable the transformed host of this invention to survive and which¹ will also enable such host to express the Pre S2-S protein coding sequence of this invention in particle form and in recoverable quantity. It will be appreciated by one of skill in the art that the appropriate culture media to use will depend upon the host cell employed. The isolation of the hybrid particle of this invention from a culture lysate or the culture fluid of such host is carried out by conventional isolation techniques.

This invention also relates to a vaccine containing the hybrid particles of this invention. Such vaccine will contain an immunoprotective quantity of the hybrid particles of this invention and are prepared by conventional techniques. Preferably, the vaccine of this invention comprises the micelle of this invention, i.e., a micelle comprising the particle of this invention and polysorbate. The preferred amount of polysorbate comprised by such micelle is 5-50 yg polysorbate per 100 yg protein. The micelle may be prepared by the method disclosed in European Patent Application Publication No. 0 199 698.

In the vaccine of the invention, an aqueous solution of the Pre S2-S protein or hybrid particle of the invention, preferably buffered at physiological pH. can be used directly. Alternatively, the protein or particle can be admixed or adsorbed with any of the various known adjuvants. Such adjuvants include, amdng others, aluminum hydroxide.

APO 00 0 5 6

Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller University Park Press, Baltimore, et al. U.S.A. , 5 cribed.

Maryland ,

1978. Encapsulation within liposomes is desfor example, by Fullecton,

u.s.

Patent

4.235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, U.S. Patent 4.372,945 and by Armor et al., U.S. Patent 4,474,757.

The amount of the Pre S2-S protein or hybrid par10 tide of the present invention contained in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and whether or not the vaccine is adjuvanted. Generally, it is expected that each dose will comprise 1-1000 yg of protein/ preferably 1-200 ,yg. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Following an initial vaccination, subjects will preferably receive a boost in about 4 weeks, followed by repeated boosts every six months for as long as a risk,of infection exists.

The immune response to the Pre S2-S protein or hybrid particle of this invention is enhanced by the use of adjuvant such as, but not limited to, alum.

NOVEL PRE SI PROTEIN CODING SEQUENCE

The novel HBV Pre SI protein coding sequence of this invention is from an HBV adw subtype which was isolated from plasmid pRIT10616. The term coding sequence or coding region as used herein also encompasses any functional derivative thereof. By the term functional derivative is meant a coding sequence

don't interfere with particle formation and/or which retain immunogenicity. Such functional derivatives can be prepared by conventional site specific mutagenesis such as by the method of Botstein et al.. Science, 229.

1193-1201 (1985). Such Pre SI protein coding sequence can be recovered by conventional recombinant DNA procedures from plasmid pRIT10616 which was deposited (within E. coli K12 strain C600) in accordance with the Budapest Treaty at the American Type Culture Collec10 tion. Rockville, Maryland, on June 2 sion number ATCC 38131. The Pre

1982 under accesS1 region codes for the following amino acid sequence: -163

	ATG	GGG	ACG	AAT	CTT	TCT	GTT	CCC	AAC	CCT
15	Met	Gly	Thr	Asn	Leu	Ser	Val	Pro	Asn	Pro
	GGA	TTC	TTT	ccc	GAT	CAT	CAG	TTG	GAC	CCT
	Gly	Phe	Phe	Pro	Asp	His	Gin	Leu	Asp	Pro
20	GCA	TTC	GGA	GCC	AAC	TCA	AAC	AAT	CCA	GAT
	Ala	Phe	Gly	Ala	Asn	Ser	Asn	Asn	Pro	Asp
	TGG	GAC	TTC	AAC	CCC	ATC	AAG	GAC	CAC	TGG
25	Ttp	Asp	Phe	Asn	Pro	lie	Lys	Asp	His	Ttp
	CCA	GCA	GCC	AAC	CAG	GTA	GGA	GTG	GGA	GCA
	Pro	Ala	Ala	Asn	Gin	Val	Gly	Val	Gly	Λ 1 a
	TTC	GGG	CCA	GGG	CTC	ACC	CCT	CCA	CAC	GGC
30	Phe	Gly	Pro	Gly	Leu	Thr	Pro	Pro	His	Gly
	GGT	ATT	TTG	GGG	TGG	AGC	CCT	CAG	GCT	CAG
	Gly	lie	Leu	Gly	Trp	Ser	Pro	Gin	Ala	Gin

AP000056

GGC	ATA	TTG	ACC	ACA	GTG	TCA	ACA	ATT	CCT
Gly	lie	Leu	Thr	Thr	Val	Ser	Thr	He	Pro
CCT	CCT	GCC	TCC	ACC	AAT	CGG	CAG	TCA	GGA
Pro	Pro	Ala	Ser	Thr	Asn	Arg	Gin	Ser	Gly
AGG	CAG	CCT	ACT	CCC	ATC	TCT	CCA	CCT	CTA
Arg	Gin	Pro	Thr	Pro	lie	Sec	Pro	Pro	Leu
AGA	GAC	AGT	CAT	CCT	CAG	GCC
Arg	Asp	Ser	His	Pro	Gin	Ala
	Such region may also	be	obtained

synthetic oligonucleotide synthesis.

A recombinant vector of this invention comprises 15 the Pre SI protein coding sequence of the subject invention operatively linked to a regulatory region and may additionally comprise a replicon depending on preference and/or the host to be employed. By the term replicon is meant that minimum region of DNA which and extrachromosoma 1ly. host eukaryotic or prokawith such vector. Such art. By the term the functions to maintain stably, such recombinant vector in a ryotic organism transformed replicons are well-known in regulatory region is meant a DNA sequence which con25 tains a promoter region and other sequences necessary for transcription of a coding sequence. Such regulatory regions are well-known in the art. Such a vector can be prepared by conventional techniques such as by inserting the Pre SI protein coding sequence of this .30 invention in phase into a vector already containing a replicon and regulatory region in such a way that : ΙΩΝΑ molecule is operatively linked to such regulatory region. Preferably the DNA molecule is ligated to a vector capable of expression in yeast such as Saccha ro35 myces.

This invention also relates to a host cell transformed with such recombinant vector and to a method of preparing such transformed host. Such transformation is carried out by conventional techniques. Host cells can be prokaryotic or eukaryotic. Preferred host cells are eukaryotic especially yeast cells and include those belonging to the genus Saccharomyces. particularly those belonging to the species Saccharomyces cerevis iae and Saccharomyces carlsberqensis.

This invention also relates to a method of preparing protein encoded by the Pre SI protein coding sequence of this invention wherein such method comprises culturing the transformed host cells of this invention in appropriate culture media, and isolating the protein from the host cell culture fluid or from a cell lysate or extract of such host's culture depehding upon the transformed host cell's ability to secrete such protein. By appropriat'e culture media is meant that media which will enable the transformed host of this invention to survive and which will also enable such host to express the Pre SI protein coding sequence of this invention in recoverable quantity. It will be appreciated by one of skill in the art that the appropriate culture media to use will depend upon the host cell employed. The isolation of the Pre SI protein of this invention from a culture lysate or the culture fluid of such host is carried out by conventional isolation techniques. The protein prepared by the method of this invention may be glycosylated depending upon the host cell's ability to do so. If a deglycosylated protein is desired, the protein of this'¹ invention should be prepared by utilizing a host cell incapable of effecting such glycosylation or by employing conventional site specific mutagenesis on the protein's coding sequence prior to carrying out the process of

APO00056 this invention. Site specific mutagenesis is carried out by such as by the method of Botstein et al.. Science, 229, 1193-1201 (1985).

This invention also relates to a vaccine containing the Pre SI protein of this invention. Such vaccine will contain an immunoprotective quantity of the Pre SI protein of this invention and is prepared by conventional techniques.

r

In the vaccine of the invention, an aqueous solution of the Pre SI protein of the invention, preferably buffered at physiological pH, can be used directly. Alternatively, the Pre SI protein, can be admixed or adsorbed with any of the various known adjuvants. Such adjuvants include, among others, aluminum hydroxide.

Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al.. University Park Press. Baltimore, ^: Maryland, U.S.A., 1978. Encapsulation within liposomes is described, for example, by Fullerton, U.S. Patent

4,235,877. Conjugation of proteins to macramolecules is disclosed. for example. by Likhite, U.S. Patent 4,372,945 and by Armor et al., U.S. Patent 4,474,757.

The amount of the Pre SI protein of the present invention present in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and whether or not the vaccine is adjuvanted. Generally, it is expected that each dose will comprise 1-1000 pg of protein, preferably 1-200 pg. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Following an initial vaccination, subjects will preferably receive a boost in about 4 weeks.

followed by repeated boosts every six months for as long as a risk of infection exists. The immune response to the PreSl protein of this invention -is enhanced by the use of adjuvant such as, but not limited to.

alum.

NOVEL PRES! - PRES2 - S PROTEIN CODING SEQUENCE

This invention also relates to a HBV PreSl-PreS2-s protein coding sequence of which the PreSl-PreS2 portion. comprises the following amino acid sequence:

PRE-SI REGION -163

ATG	GGG	ACG	AAT	CTT	TCT	GTT	CCC	AAC	CCT	CTG
Met	Gly	Thr	Asn	Leu	Ser	Val	Pro	Asn	Pro	Leu
GGA	TTC	TTT	CCC	GAT	CAT	CAG	TTG	GAC	CCT
Gly	Phe	Phe	Pro	Asp	His	Gin	Leu	Asp	Pro
GCA	TTC	GGA	GCC	AAC	TCA	AAC	AAT	CCA	GAT
Ala	Phe	Gly	Ala	Asn	Ser	Asn	Asn	Pro	Asp
TGG	GAC	TTC	AAC	CCC	ATC	AAG	GAC	CAC	TGG
Trp	Asp	Phe	Asn	Pro	He	Lys	Asp	His	Trp
CCA	GCA	GCC	AAC	CAG	GTA	GGA	GTG	GGA	GCA
Pro	Ala	Ala	Asn	Gin	Val	Gly	Val	Gly	Ala
TTC	GGG	CCA	GGG	CTC	ACC	CCT	CCA	CAC	GGC
Phe	Gly	Pro	Gly	Leu	Thr	Pro	Pro	His	Gly
GGT	ATT	TTG	GGG	TGG	AGC	CCT	CAG	GCT	CAG
Gly	He	Leu	Gly	Trp	Ser	Pro	Gin	Ala	Gin
GGC	ATA	TTG	ACC	ACA	GTG	TCA	ACA	ATT	CCT
Gly	lie	Leu	Thr	Thr	Val	Ser	Thr	He	Pro

AP 0 0 0 0 5 6

CCT	CCT	GCC	TCC	ACC	AAT	CGG	CAG	TCA	GGA
Pro	Pro	Ala	Ser	Thr	Asn	Arg	Gin	Ser	Gly
AGG	CAG	CCT	ACT	CCC	ATC	TCT	CCA	CCT	CTA
Arg	Gin	Pro	Thr	Pro	lie	Ser	Pro	Pro	Leu
AGA	GAC	AGT	CAT	CCT	CAG	GCC
Arg	Asp	Ser	His	Pro	Gin	Ala

PRE-S2 REGION

-55	GCC Ala	TTC Phe	CAC His	CAA Gin
ATG Met	CAG Gin	TGG Trp	AAT Asn	TCC Ser	ACT Thr
GCT	CTG	CAG	GAT	CCC	AGA	GTC	AGG	GGT	CTG
Ala	Leu	Gin	As p	Pro	Arg	Val	Arg	Gly	Leu
TAT	TTT	CCT	GCT	GGT	GGC	TCC	,AGT	TCA	GGA
Tyr	Phe	Pro	Ala	Gly	Gly	Ser	Ser	Ser	Gly
ACA	GTA	AAC	CCT	GCT	CCG	AAT	ATT	GCC	TCT
Thr	Val	Asn	Pro	Ala	Pro	Asn	He	Ala	Ser
CAC	ATA	TCG	TCA	AGC	TCC	GCG	AGG	ACT	GGG
His	lie	Ser	Ser	Ser	Ser	Ala	Arg	Thr	Gly
GAC	CCT	GTG	ACG	AAC
Asp	Pro	Val	Thr	Asn

The novel HBV Pre Sl-Pre S2-S protein coding sequence of this invention is from an HBV aiw subtype which was isolated from plasmid pR!T10616 which, as stated above, is available from the American Type Culture Collection. Rockville. Maryland, under accession number ATCC 38131. The term coding sequence or

BAD ORIGINAL

coding region as used herein also encompasses any functional derivative thereof. By the term functional derivative is meant a ceding sequence with amino acid alterations which, where appropriate, don't interfere with particle formation and/or which retain immunogenicity where such retention is desirable. Such functional derivatives can be prepared by conventional site specific mutagenesis such as by the method of Botstein et al.. Science. 229. 1193-1201 (1985).

A recombinant vector of this invention comprises the Pre-Sl-Pre S2-S protein coding sequence of the subject invention operatively linked to a regulatory region. Such vector may also additionally contain a replicon depending upon preference and/or the host to be employed. By the term replicon is meant that minimum region of DNA which functions to maintain stably, and extrachromosomally. such recombinant vector in a host eukaryotic or .prokaryotic organism, transformed with such vector. Such replicons are well-known in the art. By the terra regulatory region is meant any DNA sequence which contains a promoter region and other sequences necessary for transcription of a coding sequence which regulates transcription of a structural gene's coding sequence. Such regulatory regions are well-known in the art. Such a vector can be prepared by conventional techniques such as by inserting the Pre SI- Pre S2-S-protein coding sequence of this| invention in phase into a vector already containing a replicon and regulatory region in such a way that the UNA mole30 cule is operatively linked to such regulatory region. Preferably the DNA molecule is ligated to a vector capable of expression in yeast such as Sacchacomyces.

This invention also relates to a host cell transformed with such recombinant vector and to a method of

APO00056

is carried out by conventional techniques. Host cells can be prokaryotic or eukaryotic. Preferred host cells are eukaryotic especially yeast cells and include those belonging to the genus Saccharomyces, particularly those belonging to the species Saccharomyces cerevisiae and Saccharomyces carlsberqensis .

This invention also relates to a method of preparing a protein encoded by the Pre Sl-Pre S2-S protein coding sequence of this invention wherein such method comprises culturing the transformed host cells of this invention in appropriate culture media, and isolating the protein from the host cell culture fluid or from a cell lysate or extract of such host's culture depending upon the host cell's ability to secrete such protein.

By appropriate culture media is meant that media which will enable the transformed host of this invention to survive and which will also enable such host to express the Pre-Si Pre S2-S protein coding sequence product of this invention in recoverable quantity. It will be appreciated by one of skill in the art that the appropriate culture media to use will depend upon the host cell employed. The isolation of the Pre Sl-Pre S2-S protein of this invention from a culture lysate or the culture fluid of such host is carried out by conventional protein isolation techniques. The protein prepared by the method of this invention may be glycosylated depending upon the host cell's ability to do so. If a deglycosylated protein is desired, the protein of this invention should be prepared by utilizing a host cell incapable of effecting such glycosylation or by employing conventional site specific mutagenesis on the protein's coding sequence prior to carrying out the process of this invention, such as by tri^ method of Botstein et al.. Science. 229. 1193-1201 (1986). The Pre Sl-Pre S2-S coding 'Sequence of this invention will be expressed as a glycosylated protein by a yeast or mammalian host cell transformed therewith. The Pre Sl-Pre S2-S coding sequence of this invention will be expressed as a N-myristylated protein by a yeast host cell transformed therewith.

This invention also relates to a vaccine containing an immunoprotective amount of the Pre Si- Pre S2-S protein of this invention. Such vaccine may be prepared by conventional techniques.

This invention also relates to a method of preparing a hybrid particle containing HBsAg protein which comprises protein encoded by the Pre Sl-PreS2-S protein coding sequence of this invention wherein such method comprises culturing the transformed eukaryotic host cells of this invention in appropriate culture media, and isolating the particle from the host cell culture fluid or from a cell lysate or extract of such host's culture depending upon the ^transformed host cells ability to secrete such protein. By appropriate, culture media is meant that media which will enable the transformed host of this invention to survive and which will also enable such host to express the Pre Sl-Pre S2-S protein coding sequence of this invention in particle form and in recoverable quantity. It will be apprecia25 ted by one of skill in the art that the appropriate culture media to use will depend upon the host cell employed. The isolation of the particle of this invention from a culture lysate or the culture fluid of such host is carried out by conventional isolation techni30 ques.

This invention also relates to a vaccine containing, the hybrid particles of this invention. Such vaccine will contain an immunoprotective quantity of the hybrid particles of this ’invention and are prepared by conventional techniques.’ Preferably. the Vaccine of

S 0 u o 0 dV

thia invention comprises the micelle of this invention, i.e., a micelle comprising the particle of this invention and polysorbate. The preferred amount of polysorbate comprised by such, micelle is 5-50 pg polysorbate per 100 pg protein. The micelle may be prepared by the method disclosed in European Patent Application

Publication No. 0 199 698.

In the vaccine of the invention, an aqueous solution of the Pre Sl-Pre S2-S protein or the hybrid particle of the invention, preferably buffered at physiological pH. can be used directly. Alternatively, the protein or particle can be admixed or adsorbed with any of the various known adjuvants. Such adjuvants include, among others, aluminum hydroxide.

Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al.. University Park Press. Baltimore. Maryland, U.S.A., 1978. Encapsulation within liposomes is described, for example, by Fullerton. U.S. Patent

4,235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, U.S. Patent 4,372,945 and by Armor et al., U.S. Patent 4,474,757.

The amount of the Pre Sl-Pre S2-S protein or hybrid particle of the present invention present in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and whether or not the vaccine is adjuvanted. Generally, it is expected that each dose will comprise 1-1000 vg of protein, preferably 1-200 vg. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Following an initial vaccination, subjects will preferably

receive a boost in about 4 weeks, followed by repeated boosts every six months for as long as a risk of infection exists.

The immune response to the Pre Sl-Pre S2-S protein or hybrid particle of this invention is enhanced by the use of adjuvant such as. but not limited to, alum.

EXPRESSION OF PRES1-PRES2-S PROTEIN IN YEAST

This invention also relates to a recombinant DNA i

vector comprising an entire PreSl-PreS2-S protein coding sequence operatively linked to an expression control sequence. The term coding sequence or coding region as used herein also encompasses any functional derivative thereof. By the term functional derivative is meant a coding sequence with amino acid alterations which, where appropriate, don't interfere with particle formation and/or which retain immunogenicity where such retention ,is desirable. Such functional derivatives can be prepared by conventional site specific mutagenesis such as by the method of Botstein et al.. Science. 2 29. 1193-1201 ( 1985). Such vector may additionally comprise a replicon depending upon preference and/or the host to be employed. Preferably, the PreSl-PreS2-S protein coding sequence employed is the one of this invention.

By the terra replicon is meant that minimum region of DNA which functions to maintain (stably, and extrachromosomally, such recombinant vector in a host yeast organism transformed with the vector of this invention. Such replicons are well-known in the art. By the term regulatory region is meant any DNA sequence which contains a promoter region and other sequences necessary for transcription of a coding sequence which regulates transcription of a structural gene's coding sequence. Such regulatory regions are

9SOoOOdV well-known in the art. Such a vector can be prepared by conventional techniques such as by inserting a PreSl- PreS2-S protein coding sequence in phase into a vector already containing a replicon and regulatory region in such a way that the DNA molecule is operatively linked to such regulatory region. Preferably the DNA molecule is ligated to a vector capable of expression in the yeast genus Saccharomyces. Preferably the Pre SI- Pre S2-S protein coding sequence contained by the vector of this invention is the Pre SI- Pre S2-S protein coding sequence of this invention described hereinabove. Such vectors are also useful for the insertion therein of functional DNA coding sequences to enable expression of the resulting Pre Sl-functional DNA coding sequence-Pre S2-S protein coding sequence comprised by such vector. Therefore, this invention also relates to a recombinant DNA molecule comprising a functional DNA coding sequence fused in phase into the Pre SI region of the Pre Sl-Pre S2-S protein coding sequence. in such a way that the functional DNA sequence either forms an insertion in the Pre Si region or replaces part of the Pre SI region and possibly a portion of the Pre S2 region, and to a vector comprising such recombinant DNA molecule. See, for example. Example 21A. infra. By the term functional DNA coding sequence is meant a DNA coding sequence which, when fused, in phase, into the Pre SI region of the Pre SlPre S2-S protein coding sequence, is not involved with, and does not interfere with, the assembly of the HBsAg particle. Preferred functional DNA coding sequences include but are not limited to, the circumsporozoite coding sequence of Plasmodium or any immunogenic derivative thereof, the envelope gene coding sequence of HIV or any immunogenic derivative thereof, especially the C_? peptide, peptide 121 or Dreesman peptide

coding sequence as hereinafter exemplified, or sequences coding for peptides of interest from other viruses especially those for coat proteins or for surface proteins from other parasites.

This invention also .relates to a yeast host cell transformed with such recombinant vector and to a method of preparing such transformed host. Such transformation is carried out by conventional techniques. Preferred yeast cells and include those belonging to the genus Saccharomyces. particularly those belonging to the species Saccharomyces cerevisiae.

This invention also relates to a method of preparing a protein encoded by a Pre Sl-Pre S2-S protein coding sequence or Pre Sl-functional DNA coding sequence - Pre S2-S protein coding sequence wherein such method comprises culturing the transformed host cells of this invention in appropriate cultute media, and isolating the protein from the host cell culture fluid or from a cell lysate or extract of such host's culture depending upon .the host cell's ability to secrete such protein. By appropriate culture media is meant that media which will enable the transformed host of this invention to survive and which will also enable such host to express a Pre Sl-Pre S2-S protein coding sequence or Pre Sl-functional DNA coding sequence - Pre S2-S protein coding sequence product in recoverable quantity. It will be appreciated by one of skill in the art that the appropriate culture media to use will depend upon the host cell employed. The iso30 lation of the Pre Sl-Pre S2-S protein or Pre Sl-functional DNA coding sequence - Pre S2-S protein from a cell lysate or the culture fluid of such host culture is carried out by conventional protein isolation techniques.The protein prepared by the method of this invention may be glycosylated depending upon the host

AP000056 cell's ability to do so. If a deglycosylated protein is desired, the protein of this invention should be prepared by utilizing a host cell incapable of effecting such glycosylation or by employing conventional site specific mutagenesis on the protein's coding sequence prior to carrying out the process of this invention, such as by the method of Botstein et al.. Science, 229. 1193-1201 (1985).

This invention also relates to a vaccine¹ containing an immunoprotective amount of Pre-Sl-Pre S2-S protein or Pre Sl-functional DNA coding sequence - Pre S2-S protein produced by the method of this invention. Such vaccine may be prepared by conventional techniques.

This invention also relates to a method of preparing a hybrid particle which comprises protein encoded by a PreSl-PreS2-S protein coding sequence or Pre Slfunctional DNA coding sequence - Pre S2-S protein coding sequence, wherein such method comprises culturing the transformed yeast host cells of this invention in appropriate culture media, and isolating the particle from the host cell culture fluid or from a cell lysate or extract of such host's culture depending upon the transformed host cells ability to secrete such particles. By appropriate culture media is meant that media which will enable the transformed host to survive and which will also enable such host to express the Pre Sl-Pre S2-S protein coding sequence or Pre Sl·-functional DNA coding sequence - Pre S2-S protein coding sequence in particle form and in recoverable quantity.

It will be be appreciated by one of skill in the art that the appropriate culture media to use will depend upon the host cell employed. The isolation of the particle of this invention from a culture lysate or the culture fluid of such host is carried out by conventional isolation techniques.

This invention also relates to a vaccine containing the particles of this invention. Such vaccine will contain an immunoprotective quantity of the particles of this invention and are prepared by conventional techniques. Preferably, the vaccine of this invention comprises the micelle of this invention, i.e., a micelle comprising the particle of this invention and polysorbate. The preferred amount of polysorbate comprised by such micelle is 5-50 pg polysorbate per

100 pg protein. The micelle may be prepared by the method disclosed in European Patent Application Publication No. 0 199 69Θ.

In the vaccine of the invention, an aqueous solution of the Pre Sl-Pre S2-S protein or Pre SI functio15 nal DNA coding sequence - Pre S2-S protein or the particle of the invention, preferably buffered at physiological pH, can be used directly. Alternatively, the protein particle can be admixed or adsorbed with any of the various known adjuvants. Such adjuvants include, among others, aluminum hydroxide.

Vaccine preparation is generally described in New

Trends and Developments in Vaccines, edited by Voller et al.. University Park Press, Baltimore. . Maryland, U.S.A.. 1978. Encapsulation within liposomes is des25 cribed, for example, by Fullerton, U.S. Patent 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, U.S. Patent 4,372,945 and by Armor et al., U.S. Patent 4,474,757.

The amount of the Pre Sl-Pre S2-S protein' or Pre

Sl-functional DNA coding sequence - Pre S2-S protein or particle of the present invention present in each vaccine dose is selected as an amount which induces an ‘ immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon * which specific immunogen is

APO 0 0 0 5 6 employed and whether or not the vaccine is adjuvanted. Generally, it is expected that each dose will comprise 1-1000 pg of protein. preferably 1-200 pg. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Following an initial vaccination, subjects will preferably receive a boost in about 4 weeks, followed by repeated boosts every six months for as long as a risk -of infection exists.

The immune response to the Pre Sl-Pre S2-S protein or Pre Sl-functional DNA coding sequence - Pre S2-S protein or particle of this invention is enhanced by the use of adjuvant such as, but not limited to, alum.

EXAMPLES

In the following examples of the invention, which are illustrative and not limiting

- All percentages are by weight and all temperatures are in degrees Celsius (Centigrade).

- The enzymes used in DNA manipulations were obtained from Bethesda Research Laboratories, New England Biolabs, and/or Boehringer, and were employed according to the suppliers' directions.

- Yeast growth media : selective medium : YNB (yeast nitrogen base without amino acids (Difco Labs) 0.675 % (w/v) containing 2 % (w/v) glucose).

- YEPD medium 1 % (w/v) yeast extract. 2 % w/v peptone and 2 % (w/v) glucose.

- DNA recombinant methode are described in Molecular Cloning, T. Maniatis et al.. Cold Spring Harbor Lab. (1982).

- The following abbreviations may be employed:

PBS : Phosphate Buffered Saline {per liter): (pH 7.4) 8.0 gram NaCl

0.2 gram KC1

1.15 gram Na.HPO.

Z 4

0.2 gram KH₂PO₄0.1 gram CaCl₂

0.1 gram MgCl₂ · 6H₂0

BSA: Bovine Serum Albumin (A , Sigma)

3 7 8

X-gal : 5-bromo-4-chloroindoyl-fl-D-galactopyranoside, commercially available from Sigma Chemical Co., St. Louis, Missouri.

L-BROTH (per liter):

gram Tryptone 5 gram NaCl 5 gram Yeast extract 1 ml 0.1M MgS04

Add 5 ml of aseptic solution of thiamine (1 mg/ml) after autoclaving. For solid media, add 15 g agar per litre.

APO 0 0 0 56

Example 1. Construction of plasmid PRIT10167.

The starting material was plasmid p6y containing a

2.1 kb Hindi 11 fragment of yeast DNA encoding the TDH3 gene cloned on pBR322. pBR322 is described by Bolivar et al. (Gene, 2, 95-113, 1977). The p6y plasmid was constructed and characterized by Musti et al. (Gene, 25, 133, 1983) and received from Dr. M. Rosenberg of the National Institute of Health. The HindiII fragment was recloned onto a pBR322 derivative in which the EcoRI site had been destroyed to give plasmid pRIT10164 (a non essential manouver). The following manipulations were carried out so as to create a BamHI site located at the G residue of the ATG codon of the TDH3 coding sequence and to obtain a Hindi II-BamHI DNA fragment with TDH3 promoter activity in which the TDH3 sequences are intact and unchanged by the manipulation. DNA of pRITLO164, prepared as described above, was purified by two cycles of CsCl - ethidium bromide density gradient centrifugation essentially as described by Kahn et al. (Methods in Enzymology, 68. 268 ,

1979). 150 pg of pRIT10164 DNA were digested to completion with 75 units of XbaI endonuclease, extracted with phenol and ether, precipitated with ethanol and the DNA resuspended in 0.01 M tromethamine-HCl buffer at a concentration of 1 pg per pi. Samples of 20 pg of this Xba I treated DNA were digested with Bal31 nuclease to resect the 61 base pairs of DNA between the ATG codon and the Xba I site. Digestions with Ba 131 were carried out at 30° for 1 to 3 minutes in buffer containing 600 mM NaCl, 12 mM CaC12, 12 rrtM KgC12, 1 mM EDTA, 20 mM tromethamirie-HCl, pH3. 1 using one unit of Bal31 nuclease per 20 pg DNA in a final reaction volume of 200 pi.

The enzyme reactions were stopped by addition of ethylenebis (oxyethyleriemtrilo) tetraacetic acid (EGTA)

I to give 20 mM final concentration and the samples were extracted with equal volumes of phenol, ether and precipitated with ethanol. Each DNA sample was resuspended in 20 pi 10 mM tromethamine-HCl buffer pH 7.5.

The extent of Bal31 digestion was measured by digesting about 2.5 pg of each DNA sample with Hpa I endonuclease and comparing the size of the Hpal-Xbal fragments to the 335 bp Hpal-Xbal fragment from pRIT10164. A 2 minute digestion with Bal31 was found to remove about 41 to 88 nucleotides from the Xbal-Hpal fragment of pRIT10164.

This result indicates that a similar number of base pairs had been removed from the other Xbal extremity towards the ATG codon. 5 pg of the DNA recove15 red from the 2 minute Bal31 digest was digested with BamHI endonuclease, extracted with phenol and recovered by ethanol precipitation. This DNA was treated with 5 units of T4 polymerase in the presence of deoxynucleotide triphosphates to fill in and render flush ended the BamHI and any Ba 131 single stranded extremities, extracted with phenol and recovered by ethahol precipitation. 2.3 pg of this DNA was treated with 5 units of T4 DNA ligase and half of the ligation mixture used to transform competent cells of E. co 1i K12 strain

MM294 prepared according to the method of Cohen et al. (Proc. Natl. Acad. Sci. 69. 2110, 1972). One ml of the transformed E. coli population was diluted into 350 ml L-broth with 200 pg/ml ampicillin and total plasmid DNA was purified from the resulting culture.

80 pg of this plasmid DNA containing a population of plasmid molecules with Ba 131 induced deletions of about 40 to 80 base pairs was digested sequentially • with 75 units of Hindi 11 endonuclease and 96 units of BamHI endonuclease to release DNA fragments from those 35 plasmids in which a BamHI site had been recreated after

APO00056

I the treatments described above, that is, where the Bal31 digestion had terminated at a G residue. This DNA digest was run on a preparative 1% agarose gel and the desired Hindi II-BamHI fragments corresponding to sizes from 1100 to 1000 bp excised from the gel in two slices, one corresponding to DNA of about 1070 bp and the other of about 1030 bp. The DNA was recovered from the two agarose gel slices by cycles of freezing and thawing followed by high speed centrifugation to pellet the agarose. The supernatant liquid was filtered through a Millipore GV Millex filter and the DNA recovered by two cycles of ethanol precipitation and resuspended in 20 yl of 0.01 M tromethamine-HCl buffer pH 7.5.

Analysis by agarose gel electrophoresis and comparison to a Hind 111 plus Xbal digest of pRIT10154 DNA and to the fragments from a Hind 111 plus EcoRI digest of phage X DNA showed that two distinct populations of Hindi! I-BamHI fragments were obtained, one.'with an estimated size of about 1070 bp and one with an estimated size of about 1030 bp compared to the 1120 bp parental Hindi I I-Xbal fragment from pRlT101;64. About 100 ng of the 1030 bp Hindi II-BamHI fragment population was ligated with 200 ng of plasmid pUC9 which had been digested with HindiII and BamHI endonucleases and treated with alkaline phosphatase (See, Shine, U.S. Patent No. 4,264,731) and the ligation mixture was used to transform competent cells of E. coli strain JM103 with selection being made for ampicillin resistance. Transformation of E. coli strain JM103 was effected according to the method of Cohen et al., cited aoove.

Both the pUC9 vector plasmid and the recipient E. coli strain JM103 have been described by Vieira and Messing in Gene, .19., 259. 1982 and were obtained from

J. Messing (Univ. of Minnesota). The pUC9 vector is

commercially available from Amersham (Buckinghamshire, United Kingdom) and Pharmacia (Uppsala, Sweden). e . coli strain JM1O3 is available from J. Messing (Univ. of Minnesota). Another E. coli strain with the charac5 teristics of E. coli strain JM103, i.e., JM101. can be obtained from the American Type Culture Collection, Rockville, Maryland, under accession number ATCC 33876, and can also be employed as host. About 400 ampicillin resistant colonies per ml were obtained and of 98 colo10 nies tested on medium containing X-gal. 95 were white indicating the successful insertion of a foreign DNA fragment between the Hindlll and BamHI sites on the vector .

Plasmids from individual transformant colonies were prepared by a small scale procedure (Birnboim and Doly, Nucl. Acid Res., T_. 1513, 1979) after amplification of the plasmids by addition of spectinomycin (150 pg/ml) to the growing cultures.

The recombinant plasmids were analyzed oni 7.5% acrylamide gels after do'uble digestion with ¹ Aval I and BamHI endonucleases and compared to the 450 bp Aval IXbal fragment comprising the promoter-,N-terminus coding region of pRIT10164 and to the fragments of a Hpa 11 digest of pBR322 DNA. An AvaI I -BamHI fragment corresponding to deletions of from 20 to 90 base pairs was present in 35 of 36 plasmids examined when compared to pRIT10164. Three plasmids representing deletions of about 80 base pairs (pRITlOI66) 50 base pairs (pRIT10167-Figure 8) and 45 base pairs (pR!T10165) were chosen for further study. Plasmid DNA was prepared by CsCl-ethidium bromide density centrifugation and 25 vg of each digested with EcoRI

32.

The EcoRI extremiAP000056 ties were labelled witri γ- P-ATP by exchange kination and the nucleotide sequence of the HindiII-EcoRI 35 fragments from each plasmid determined by t'he chemical

I modification methods of Maxara and Gilbert (Methods in

Enzyroology. 65., 499. 1980).

sequencing from the EcoRI site portion of each recombinant plasmid

Labelling of and in the pUC9 vector is a convenient means of determining the sequence around the adjacent BamHI site marking the end of the deletion into the TDH3 DNA frag- ment. This sequencing analysis showed that in plasmid pRIT10166, the BamHI site is recreated at a G residue located in the 5' non translated region 25 base pairs upstream of the ATG codon; in pRlT10l65 the BamHI site is located at the second base of the third codon; and in pRIT10167 the BamHI site is located at the G residue of the ATG codon.

The Hindi 11-BamHI fragment of TDH3 DNA thus constructed in pRlT10167 contains all the sequences necessary for TDH3 promoter activity in an unaltered form.

Example 2. Construction'of vector pRIT10172.

The 1050 bp Hindi I Ι-BamHI TDH3 DNA insert from prepared as described in Example 1, was shuttle vector described by (Gene, 8., 121, 1979) between the Hind 111

PRIT1O167 recloned onto the YEpl3 Broach et al site in the 2 micron DNA portion of the vector and the

BamHI site to give the recombinant plasmid pRIT12159.

DNA of pRIT12159 was then digested with Xba'l and BamHI endonucleases and the resulting 1650 bp fragment was ligated in place of a similar XbaI-BamHI fragment con. . 3 taming the arg promoter on plasmid pRIT10774. Plasmid pRIT10774 is described by Cabezon et al. (Proc. Natl. Acad. Sci. U.S., '81. 6594-6598, 1986)', and comprises the YEpl3 replicon from which the natural vector's BamHI and Xhol sites have been deleted by in vitro manipulation together with a 1470 bp Hindi 11. . 3 ;

BamHI insert comprising the arg promoter region and a 1150 bp BamHI-Hindl11 fragment comprising the arg³ transcription termination region. Replacement of the arq promoter insert on pRIT10774 by the TDH3 promoter insert gives plasmid pRIT10172 which therefore contains a single BamHI site located at the ATG codon of the TDH3 promoter insert and preceeding a functional signal for transcription termination on the 1150 bp BamHI-HindllI arq DNA fragment. Foreign DNA can therefore be cloned at this site. Furthermore the two DNA . inserts are bounded by Hindi11 sites enabling remo10 val of the expression unit module (cassette) as a 2200 bp Hindi11 fragment for insertion on other vectors. Figure 9 is a restriction endonuclease cleavage map of PRIT10172.

Example 3. Construction of pRIT12290.

A 1050 bp Hindi II-EcoRI fragment from pRIT10167 (Figure 8), prepared as described in Example 1, and containing the HindiII-BamHI TDH3 promoter fragment together with the BamHI-Smal-EcoRI part of the pUC9 polylinker was recloned between the Hindi 11 and EcoRI sites of a pBR322 derivative plasmid previously deleted fro the BamHI site by filling in with T4 DNA polymerase and religation. The resulting recombinant plasmid was identified as pRIT12176.

Plasmid DNA of pRIT 10158 (Figure 8), prepared as described in Example 4 (b), below, was digested with

EcoRI endonuclease and treated with T4 DNA polymerase to fill in the EcoRI single strand extensions. This preparation was further digested with Clal endonu30 clease. A further sample of pRIT10158 DNA was digested with Clal and Hael11 endonucleases and a 1150 bp

ClalHael11 fragment carrying the transcription termina. 3 tion region of the arq gene was recovered by preparative agarose gel electrophoresis and electroelution.

This fragment was ligated with the EcoRI. T4 DNA polyAPO00056 a plasmid, identified as pRIT10162, ³ transcripon pRIT10158 raerase, Clal treated pRIT10158 DNA. and the ligation mixture was used to transform competent cells of e. coli strain MM294 prepared as described by Cohen et al., above, to ampicillin resistance. From the trans5 formed colonies, was isolated in which the Clal-Hae111 arc tion termination fragment was inserted between the Clal and filled in EcoRI sites and in which the EcoRI site has been recreated. Figure 8 is a flow 10 sheet which illustrates the preparation of pRlT10162. Plasmid DNA of pRIT10162 was digested with EcoRI and Pstl endonucleases, mixed with plasmid DNA of pRIT12176. prepared as described above, (also digested with EcoRI and Pstl endonucleases) and the mixture was 15 ligated and used to transform cells of E. coli K12 strain MM294 to ampicillin resistance by the methods of Cohen et al., cited above. A plasmid. pRIT12208, was recovered from a colony from this transformation in which the large 4630 bp EcoRI-Pstl fragment from 20 pRIT12176 had been ligated to the small 1930 bp fragment from pRIT10162, and in which the

EcoRIPstl 3 arg transcription termination region is now juxtaposed to the TDH3 promoter. The arg³ and >TDH3 DNA regions can be excised from pRIT12208 as a single 2200 25 bp fragment by digestion with Hind 111 endonuclease. To obtain the other orientation of this fragment with respect to vector sequences the 2200 bp Hindi 11 fragment from pRIT12208 was recloned into the Hindi 11 site of a pBR322 derivative plasmid in which the natural EcoRI 30 and BamHI sites had separately been deleted by filling in with T4 DNA polymerase and religation. The resulting. plasmid, with the 2200 bp Hi rid I 11 fragment inser• ted in the opposite orientation with respect to vector sequences compared to pRIT12208, is identified as 35 pRIT12290. Figure 10 * is a restriction endonuclease cleavage map of pRIT12290. In both the pRIT12208 and PRIT1229O vectors, the TDH3 promoter and arq³ transcription termination fragments are separated by unique BamHI. Smal and EcoRI restriction sites useful for the cloning of DNA fragments, carrying coding sequences, and the promoter and transcription termination regions can be excised together on a 2200 bp Hind 111 fragment as a module or cassette for transfer to other vectors.

The BamHI site is especially useful as it is loca10 ted at the ATG codon of the TDH3 gene and can be employed for fusion of other genes to the TDH3 promoter with a view to expressing them in yeast as exemplified below. Such fusions can be recovered from pRIT122O8 or pRIT12290 derivatives in the form of cassettes or modu15 les for insertion on yeast shuttle vectors by digestion with Hindi 11 endonuclease or by the use of other restriction endonucleases cutting at flanking restriction sites.

Example 4. Construction of Plasmids ipRlT10677, PRIT10909 and pRIT10158. ' a) PRIT10677

A 1372 bp BamHI fragment of cloned HBV DNA was excised from pRIT10616 (Harford et al., Dev. Biol.

Standard, 54., 125-130, 1983) and mixed and ligated with

BamHI endonuclease digested pBR327 DNA. This BamHI fragment contains part of the HBsAg precursor region, the HBsAg coding sequence and 3¹ non coding sequences. From the ligation mixture a plasmid, pR!T10677, was

AP 0 0 0 0 5 6

25 selected	which has	the	HBV BamHI	fragment inserted at
the	BamHI	site of	the	vector in	the orientation	such
that	the	Xbal site	in	the HBV DNA is proximal to	the
' Sail	site	in the	pBR327 vector.	Figure 8 is a	flow

sheet which illustrates the preparation of pRIT10677.

b) PRIT10909

The 3300 bp Hindi 11 fragment from plasmid pMC200 (available from the American Type Culture Collection, Rockville, Maryland, U.S.A.. without restriction under accession number ATCC 39131), described by Crabeel M. et al (EMBO J., 2., 205-212, 1983), was recloned onto a pBR322 derivative plasmid on which the natural EcoRl· site had been deleted by filling in with T4 DNA polymerase and religation, A resulting recombinant plasmid, pRIT10158 (Figure 8). was selected in which the Hindi I I . . 3 arq gene insert has the 3' region of the arq gene proximal to the Clal site on the modified pBR3 22 vector. From pRIT10158, a 1150 bp Cla1-HaeI11 fragment was recovered which carries the transcription termination region of the arq gene. This 1150 tp fragment w; s ligated with Clal and Hpa1 endonucleases digested D'JA of plasmid pRIT10677, (prepared as described in Part a), above, to fuse the arq³ termination region at the Hpal site downstream of the HBsAg coding region. The resulting plasmid is pRIT10909. Figure 0 is a flow sheet which illustrates tne preparation of

PRIT10909. cleavage map	Figure 3 is of pMC200.	a	restriction	endonuclease
Example 5. PRIT10912.	Construction	of	plasmids	PRIT10911 and

The BamHI site on pRITL0167 (Example 1) and the natural BamHI site located in the precursor region of the HBsAg gene from a HBV virus of adw serotype cloned on pRIT10616 (Harford et al.. Dev. Biol. Standard, 54, 125-130, 1983 ) are in the same tra ns 1 ationa1 reading frame enabling the construction of a fusion of the TDH3 promoter fragment which ^; supplies the ATG codon to 42 codons of the HBsAg precursor sequence followed by 226 codons of the HBsAg coding sequence proper. The source

of the HBV DNA fragment for this fusion was a plasmid pRIT10909 (see. Example 4, part b. above) containing a 935 bp BamHIHpaI insert encoding part of the HBsAg precursor region, the full HBsAg coding sequence and 128 5 bp of 3' non translated DNA to which had been fused, downstream of the Hpal site, an 1150 bp Haelll-Clalll DNA fragment from pRIT10158 transcription termination region, bed in Example 4, part B, above.

BamHI fragment was excised from pR!T10909 and inserted between the EcoRI and BamHI sites of pRIT10167, prepared as described in Example A, so as to create plasmid pRIT10911. The 3135 bp Hindi 11 fragment from pRIT10911 containing the yeast DNA transcriptional signals and 15 the HBsAg coding sequences was then inserted on the YEpl3 shuttle vector to give plas- mid pF.IT10912.

containing the arq pRITL0158 is descriThe 2085 bp EcoRIFigure 8 i3 of pRIT10911 a flow sheet illustrating the preparation

Example 6. Construction of plasmids pRITiG9O3 and PRIT10914

The aim of the constructions described below was to remove excess nucleotides from the 5' end of a DNA fragment encoding at least the N-terminal portion of the mature HBsAg coding sequence in such a fashion as to create a 6 base pair restriction site at the first base of the second codon. The resulting fragment can then be fused to promoter fragments with a 6 base pair restriction site at the initiation codon using Mung

Bean or SI nucleases to trim away the single strand extensions and create blunt ends for ligation as shown by Rosenberg et al., Methods in Enzymology, 101C. 123 (1983).

A 1225 bp FnuDII fragment of HBV DNA from pRIT10616 (See, Harford et al. (1983) Devel. Biol.

AP000056

72‘ Standard, 54.. 125-130) and encoding the HBsAg gene was excised from this plasmid and ligated with synthetic octomer EcoRI linkers, and the fragment clonei. into the

EcoRi site of vector pACYC184 to give pRIT10679 (Figure

8). Vector pACYC184 was received from S. Cohen and is described by Chang A.C.Y. and Cohen S. (1978), j.

Bacteriol.. 134 . 1141- 1156. pACYC184 is also available from the American Type Culture Collection,

Rockville, Maryland, U.S.A., under accession number

ATCC 37033. From this EcoRi (FnuDII) fragment, a 125 bp EcoRI-Xbal fragment was obtained which contains the

Cterminal region of the HBsAg precursor, the HBsAg ATG codon and 90 bp of HBsAg coding sequence. This 125 bp

EcoRi-Xbal fragment was introduced between the EcoRi and Xba I sites of pRIT10158 (see, Exampl· 4 (b), above). The resulting plasmid, pRIT10903, therefore contains a sin- gle EcoRi site located at the junction 3 between arg DNA and HBV DNA and has a single Xho I 3 site located in the arq promoter region. Figure 8 is a flow sheet which illustrates preparation of PRIT10903 .

150 yg of pRIT10903 plasmid DNA, prepared by CsCl-ethidium bromide density gradient centrifugation, was digested with 80 units of EcoRi endonuclease, extracted with phenol, ethanol precipitated and resuspended at 1 yg per yl in 10 mM tromethamine-HCl buffer (pH 7.5). The DNA was divided into three 50 yg samples and incubated for 50, 75 and 90 seconds with Bal31 nuclease at 30°. Each reaction mixture contained the same reaction buffer cited in Exa.aple 1, 50 yg of EcoRi digested pRIT10903 DNA, end 2.5 units of Bal31 nuclease in a final volume of 500 yl. The reactions were stopped by addition of EGTA' to 20 mM final concentration, chilling on ice, extraction with an equal volume of phenol and precipitation with etha- 73 nol. The Bal31 treated DNA samples were each taken up in 50 yl 10 mM tromethamine-HCl buffer and 2.5 yg digested with BstEII endonuclease and compared on a 7.5% acrylamide gel to an EcoRI plus BstEII endonu5 clease digest of pRIT10903 which releases a 285 bp fragment. Treatment with Ba 131 nuclease for 75 seconds at 30° was found to reduce the size of this EcoRlBstEII fragment by 10 to 60 base pairs giving a series of discrete bands estimated to represent removal of 10,30,45 and 60 base pairs of DNA. Resection of 29 base pairs from the original FnuDII site would remove all the HBsAg precursor DNA and the HBsAg ATG codon.

yg of pRIT10903 DNA which had been treated with Ba 131 for 75 seconds was digested with 8 units of

Xhol nuclease, extracted with phenol and ethanol precipitated. This DNA was then incubated with 5 units of T4 DNA polymerase in the presence of deoxynucleotide triphosphates to fill in and render flush ended the Xho1 and Ba 131 extremities, treated with phenol and ethanol precipitated. The DNA was then incubated with 5 units of T4 DNA ligase for 16 hours at 16° and the mixture used to transform competent cells of E. co 1i K12 strain MM294 to ampicillin resistance according to the method of Cohen et al., cited above. About 2000 ampicillin resistant colonies per ml were· recovered after plating. Plasmids were prepared from 47 individual colonies by a small scale procedure (See, Bimbo im et al, cited above), and 23 of 47 were found to retain a Xhol site indicative of successful filling in of the original Xhol site and ligation to a 3' end terminating in a G residue. These 23 plasmids were furtner digested with Xhol and XbaI endonucleases to determine the size of the small restriction fragment in comparison with the original EcoRI-Xbal fragment from pRIT10903.

AP000056

Several plasmids were identified with deletions estimated to range from 25 to 40 base pairs. One plasmid, pRIT10914. which was estimated to contain a XhoIXbal fragment of 95.to 100 base pairs was selected for further study. Figure 8 is a flow sheet which illustrates preparation of pRIT10914. DNA of this plasmid was purified by CsClethidium bromide density gradient centrifugation and 25 pg of such DNA was digested with 140 units of Xbal endonuclease.

The Xbal terminii were exchange labelled with 3 2 γ- P-ATP, and the labelled DNA was digested with 15 units of Hind 111 endonuclease. The small 765 bp Hindi II-Xbal fragment was isolated by acrylamide gel electrophoresis and electroelution followed by ethanol precipitation. The nucleotide sequence at and around the Xhol site was determined by sequencing this fragment from the labelled Xbal terminus using the chemical modification methods of Maxam and Gilbert. cited above. The sequence data showed that the Xhol site was located at the first base of the second codon of the HBsAg coding sequence.

Xhol

CTCGAGAAC

HBsAg nucleotides

This fragment is therefore suitable for fusion to the BamHI extension of the TDH3 promoter fragment on pRIT10167 (Example 1) after removal of single stranded ends .

Example 7. Construction of plasmids pRIT122ll.

PR1T12209. pRIT1223O. and pRIT12265

Plasmid pRIT10911, prepared as described in Example 5, was used as a scaffold for further manipulations. To introduce .desirable restriction sites, the

plasmid was digested with BamHI and Xba I endonucleases, and the excised fragment replaced by a 2275 BamHI-Xbal fragment from plasmid pRIT10158 (Example 4). This manipulation results in plasmid pRIT12211 in which the

Hind111-BamHI TDH3 promoter fragment is next to a

BamHI-Xbal fragment containing a Xhol site followed by an Xbal-Hindl11 fragment comprising the C-terminal region of the HBsAg coding sequence and 128 bp of 3' 3 non coding DNA fused to the 1150 bp arg transcnp10 tion termination region. Figure 8 is a flow sheet which illustrates preparation of pRIT12211. Plasmid pRIT12211 was digested with Xhol and Xba I endonucleases thereby removing a 1600 bp fragment which was replaced by the 94 bp XhoIXbal fragment of modified HBsAg DNA from pRIT10914, prepared as described in Example 5. This 94 bp fragment was purified by acrylamide gel electrophoresis from a Xhol plus XbaI digest of pRIT10914 DNA followed by electroelution of the appropriate gel slice and recovery of the DNA by etjhanol precipitation.

The plasmid, pRIT122O9, resulting from the introduction of the 94 bp fragment (See. Figure 8), was purified by CsCl-ethidium bromide density centrifugation, and 50 yg of pRIT12209 DNA was digested with 90 units of BamHI endonuclease and 60 units of Xhol endonuclease to remove the 990 bp of extraneous DNA between the TDH3 promoter and the HBsAg coding region, and then was phenol extracted and ethanol precipitated. 16 yg of this DNA was incubated for 30 minutes at 30° with 20 units of Mung Bean nuclease (PL Biochemicals) in a volume of 125 yl. The buffer used Cor the incubation was 30 mM Sodium acetate (pH 4.6 ), 250 mid NaCl, 1 mil ZnCl₂ and 5% glycerol. The reaction was stopped by the addition of sodium dodecyl sulphate to give 0.2% final concentration and extraction with an equal volume

AP 0 0 0 0 5 6

I

- 76 of phenol followed by ethanol precipitation. An aliquot of 0.5 yg of this Mung Bean nuclease treated sample was incubated with 2 units of T4 DNA .igase for 16 hours at 16° and then digested with 2 units of Ba mil I endonuclease to destroy any vector molecules which had escaped the above treatments. This mixture was used to transform competent cells of E. coli K12 strain MM294 with selection for ampicillin resistance according to the method of Cohen et al.. cited above. About 2000 ampicillin resistant colonies per ml of transformation mixture were recovered and plasmids were prejared by a small scale procedure (according to the method of Birnboim et ai.. cited above) from amplified cultures of 24 of these transformants.

of 24 plasmids gave two bands of DNA with sizes to 2700 bp (pUC9 vector) and 3000 bp after digestion with Hind 111 endonuclease. The 3000 bp band is the size expected for the ¹ fusion of the HBsAg coding sequence and arg³ termination region to the TDH3 promoter fragment.

Four candidate plasmids were taken for further study. DNA of each plasmid was digested with Xba I endonuclease and labelled by exchange kination with

....

y- P-ATP followed by digestion with Hind 111 endonuclease and isolation of the labelled 1190 bp Hindi II-Xbal fragment by elect roelution aod ethanol precipitation following acrylamide gel electrophoresis. The sequence of each fragment was determined by the chemical modification methods of Maxam anc Gilbert, cited above. Two plasmids were found to hav= the correct sequence ATGGAGAAC for perfect fusion of the TDH3 promoter region to the HBsAg gene. One of these plasmids. pRIT1223O, was used in further manipulations. Figure 8 is a flow sheet which illustrates preparation Of PRIT1223O. DNA of' this plasmid (pRIT12230) was

digested with Hind111 endonuclease and the 3000 bp fragment containing the HBsAg coding sequence fused to the TDH3 promoter was ligated onto shuttle vector YEpl3. The resulting plasmid, pRIT12265, was transfor5 med into yeast strain DCS (MATa. l_eu2-3, Ieu2-ll2. his3 , canl-11) (obtained from J. Broach (State University of N.Y. Stony Brook) and available without restriction from the American Type Culture Collection under accession number ATCC 20630) using the transfor10 mation procedure of Ito et al., J. Bact, 153. 163 ( 1983 ) .

Example 8. Construction of plasmids pRIT122B8, PRIT12322

The construction on plasmid pRIT1223O (Example 7) contains 128 bp non coding HBV DNA located 3' downstream of the stop codon of the HBsAg structural gene.

To remove the 128 bp of DNA. about 25 pg of pRIT12230, prepared as described in Example 7 above, is digested with AccI and EcoRI endonucleases. pRIT12230 contains a single AccI site located in the S-gene coding sequence 7 nucleotides before the TAA stop codon. The digested DNA was electrophoresed on a 1% agarose gel and the larger 4200 bp vector fragment was recovered from the gel by electroelution and ethanol precipitation.

Artificial DNA linker molecules composed of a 12mer strand and a 14-mer strand with the following sequences are synthesized for insertion between the

AccI and EcoRI sites of pRIT12330:

APO00056

5' ATACATTTAACG 3¹12 mer

3' TGTAAATTGCTTAA 5' 14 mer

This will restore the correct C-terminal HBsAg codons, the TAA stop codon and provide an EcoRI site not present elsewhere in the TDH3 promoter or HBsAg coding sequences for addition of transcription termination fragments.

100 pmoles of the synthetic 12 mer and loo pmoles of the synthetic 14 mer single strands are mixed together in a final volume of 10-20 yl, heated at 70° for 15 minutes, and allowed to return slowly to room temperature over a period of 3 hr permitting the annealing of the two strands along their complementary sequences. To this annealed mixture is added about 0.15 pmole (400 ng) of the 4200 bp Acc I -EcoRI fragment of PRIT1223O, lOx concentrated ligation buffer and 2 units of T4 DNA ligase.

This mixture is incubated for 4 hours at 16° before the addition of a further 2 units of T4 DNA ligase and' then incubated overnight on ice. The ligated mixture is used to transform competent cells of strain MM294 to ampicillin resistance according to the method of Cohen et al. (Proc. Natl. Acad. Sci., U.S.A., 69. 2110, 1972). Plasmid’s are prepared from 12 or more individual colonies by the rapid procedure of Birnboim and Doly (Nucl. Acids Res., ]_. 1513, 1979 ) and examined by restriction endonuclease analysis. Plasmids with insertion of the Acc I to EcoRI linker will show a single band of DNA of 4200 bp on digestion with either AccI or EcoRI enzyme. The correctness of the linker insertion on such a plasmid can be verified by DNA sequencing from either the EcoRI site or AccI site by the chemical modification method of Maxam and Gilbert (Methods in Enzymology, 63, 499, 1980). ‘

To provide a transcription termination fragment, a plasmid with the AccI-EcoRI linker insert, obtained as described above, is digested with EcoRI enlonuclease and treated with alkaline phosphatase (Shine J., u.s. Patent 4,264,731). The 2650 bp Hind 111 fragment of PRIT1O162, obtained as described in Example 3, was recloned in the Hindi II site of the pBR327 vector p las 5 mid to form plasmid pRIT12288. pBR327 is described in Soberon et al. (Gene. £, 287-305, 1980), and can be prepared as described in Example 10, part b, infra, from plasmid pRIT123O9 which can be recovered from the American Type Culture Collection under accession number

ATCC 67187. Figure 8 is a flow sheet which discloses construction of pRIT12288.

The orientation of the Hindi 11 fragment on 3

PRIT12288 is such as to juxtapose the arq transcription termination region with the EcoRI and Clal res15 triction sites on the pBR327 moiety. From pRIT12288, a

1180 bp EcoRI fragment is obtained which carries the 3 . . . .

arq transcription termination region. This fragment is ligated to the EcoRI digested, alkaline phosphatase treated pRIT1223O derivative described above, and plas20 mids issuing from the ligation mixture are ext,mined for insertion and the orientation of the 1180 bp EcoRI fragment. The orientation of the insert can be obtained by digestion with Hindi 11 endonuclease which will liberate fragments of 2880 and 2720 bp if the insert orientation is as desired.

A plasmid, pRIT12322, was constructed in which the

AccI-EcoRI linker replaces the 128 bp of unwanted HBV DNA and on which the 1180 bp EcoRI fragment from pRIT12288 was present in the desired orientation, down30 stream of the HBsAg coding sequence. A 2900 bp Hind 111 fragment can be excised from pRIT12322 which carries the expression module for HBsAg synthesis including the . 3

TDH3 promoter, the HBsAg coding sequence and the arq transcription termination region. Figure 8 is a flow sheet which discloses .construction of pRIT12322. See,

AP000056 also. Figure 11, which is a restriction endonuclease map of pRIT12232 and shows which portions thereof come from pRIT12288 and pRIT1223O.

Example 9. Construction of plasmid pRITl2329

As described in Example 8 above, pRIT12322 contains a 2900 bp Hindi 11 fragment with all the necessarysignals for expression of HBsAg from the TDH3 promoter. This module was ligated onto a shuttle vector for introduction into yeast cells.

DNA of shuttle vector YEpl3 was digested with Hind 111 endonuclease and alkaline phosphatase and used as recipient for the introduction of the Hi no 111 fragment from pRIT12322, prepared as described in Example 8. Plasmid pRIT12329 was isolated which consists of the YEpl3 replicon together with the 2900 bp Hind 111 module fragment described in Example 8.

Example 10. Construction of plasmid vectors for the expression of the hybrid antigen HBsAg-CS in yeast

A. Construction of Plasmids pRIT12573 and pR!T12574

Plasmid WR201 was obtained from the Walter Reed Army Institute of Research and results from insertion of a 2.3 kilobase (kb) EcoRI fragment from XmPfl (Dame et al.. Science, 225, 593-599, 1984) encoding the complete circumsporozoite protein gene of Plasmodium falciparum into vector pUC8. Figure 2A shows a schematic restriction map and sequencing strategy of clone

XmPf1.		The	positions of restriction enzyme	cleavage
sites	s hown	in the figure were	determined	from	t h e
sequence	and	confirmed by digestion: A, Avail;	Ac ,
Acc I;	B,	Bstnl; D, Dral; Dd, Ddel;	F, FokI; N.	Ndel;	R,
Rsal;	s.	Stul	; T, Tthlll; Tq, TaqI;	X. XhoII.	Arrows
indicate	the	origin,· direction	and extent	of	the

sequences determined. The CS protein coding region is shown as a heavy line. Figure 3A shows the nucleotide sequence of the CS protein gene from P. falciparum. The nucleotide sequence of the CS protein gene in

XmPfl is shown. Vector pUCB is described by Vieira et al. (Gene. .19., 259. 1982). pUC8 is commercially available from Amersham and Pharmacia. A 1215 base pair (bp) Stul-Rsal fragment of plasmid WR201 containing the CS protein coding sequence minus the first 52 bp (Figure 12) was purified by electroelution from a 7.5% polyacrylamide electrophoresis gel. This fragment was digested with Sau3A to generate smaller fragments which included, among others, a 192 bp Sau3A fragment coding for 16 tetrapeptide repeats of the CS protein and three identical 24 bp Sau3A fragments coding for 2 tetrapeptide repeat of the CS protein.

Plasmid pRIT10911 (prepared as described in Example 5) is a pUC9 derivative carrying a 3136 bp Hind I 11 cassette containing a 1050 bp Hind 111-BamHI yeast gly20 ceraldehyde-3P-dehydrogenase (TDH3) promoter fragment (which supplies the ATG) fused at the BamHI site to a

935 bp BamHI-HpaI fragment of HBV DNA encoding the Cterminal 42 amino acids (aa) of the pre-S2 region, 226 aa of the S gene (serotype adw) and 128 bp of 3' non25 coding DNA. The S gene is flanked by a 1150 bp yeast 3

DNA fragment carrying the arq transcription terminator. pUC9 is described by Vieira et al. (Gene, 19, 259-268, 1982). The BamHI site of pRlT10911, located at the ATG initiation codon, is in phase with the Sau3A sites of the repetitive epitope of CS of Plasmod ium falciparum. DNA of plasmid pRIT10911 was digested with BamHI endonuclease, treated with alkaline phosphatase, extracted with phenol and recovered by ethanol precipitation. 0.2 yg of this DNA was mixed with 0.2 yg of the Sau3A digest of the Stul-Rsal CS gene fragment

S 0 0 0 0 dW prepared as described above, treated with T4 DNA ligase, and the ligation mixture was used to transform competent cells of E . coli K12 strain MM2 94 prepared according to the method of Cohen et al. (Proc. Natl. Acad. Sci., U.S.A.. .69., 2110, 1972). 32 plasmids from individual transformant colonies were prepared by a small scale procedure (Birnboim et al.. Nucleic Acids Research, 7, 1513, 1979) after amplification of the plasmid by addition of spectinomycine (300 yg per ml) to the growing cultures. The recombinant plasmids were analysed on 7.5% acrylamide electrophoresis gel. after double digestion with BamHI and Xba I endonucleases, and were compared to the 219 bp BamHI -Xba I fragment of pRIT10911 (comprising the first 42 amino acid coding sequence of the Pre S2 region precursor and the beginning of HBsAg region) and to the fragments of a HaeI I ί digest of ψχ174 DNA.

Among the 32 plasmids, one recombinartt showed a BamHIXbal fragment of 411 bp which corresponds to the insertion of the 192 bp fragment Sau3A in the correct orientation and was designated pRIT12572. Another recombinant showed a BamHI-XbaI fragment of 243 bp corresponding to the insertion of the 24 bp Sau3A fragment in the correct orientation and was designated pRIT1257l. Figure 12 is a flow sheet illustrating preparation of pRIT12571 and pRIT12572.

The Hind 111 fragments of pRIT12572 and pRIT1257 L, containing the TDH3 promoter, the CS-HBsAg hybrid coding sequence and the arg3 termination region were recloned onto YEpl3, to give, respectively, plasmids pRIT12574 and pRIT12573 (Figure 12). ΎΕρ13 is descri bed by Broach et al. (Gene, 8, 121-133, 1979). Figure is a flow sheet illustrating preparation of PRIT12573 and pRIT12574.

Plasmids pRIT10912, pRIT12574 and pRIT12573 were introduced into S. cerevis iae yeast strain DC-5 (a, leu2-3, leu2-112, his3 , canl-ll). and into S. cerevis iae yeast strain 1OS44C (pep4-3 leu2-3. leu2-112) obtained from M. Crabeel located at the Ceria Institute, Anderlecht, Belgium, (see. also Cabezon, T. et al., Proc. Natl. Acad. Sci. U.S.A., 81. 6594-6598,

1984) by transformation of lithium acetate treated cells (Ito et al., J. Bacteriol., 153, 163-168, 1983) and selection for leucine independence. Yeast strain DC5 was deposited in the American Type Culture Collection, Rockville, Maryland, U.S.A., in accordance with the regulations of the Budapest Treaty on June 2, 1982, under accession number ATCC 20630. Yeast strain 10S44C was deposited in the American Type Culture Collection, Rockville, Maryland, U.S.A., in accordance with the regulations of the Budapest Treaty on August 18. 1986, under accession number ATCC 20819. The expected hybrid protein for pRIT12573 is predicted to contain 277 amino acids, and for pRIT12574 is predicted to contain 333 amino acids .

It is also possible to add to or enlarge the C3 epitope region on the vector of the invention. For example, on plasmids pRIT12572 and pRIT12571, additio25 nal Sau3A fragments of CS protein DNA, e.g., 192 bp or 24 bp Sau3A fragments, can be ligated in to the BamHI site as follows:

Plasmid DNA of pRIT12572 or pR!T12571 is digested with BamHI endonuclease and treated with alkaline phos30 phatase. The 1215 bp Stul-Rsal fragment of plasmid WR201, obtained as described above, is digested with Sau3A endonuclease to liberate the 192 bp and 24 L>p fragments bearing the tetrapeptide repeats. These fragments are mixed with the BamHI and alkaline phos 35 phatase treated pRIt!2572 or pRIT1257l vectors, treated

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APO00056 with T4 DNA ligase and the mixtures transformed into E. coli K12 cells according to conventional procedures with selection for ampicillin resistance. Plasmids from transformant colonies are screened for the size of the BamHI-XbaI fragments by endonuclease digestion and compared to similarly digested DNA of pRItl2572 or PRIT12571, especially the 411 bp and 243 bp BamHI-Xba I fragments from pRIT12572 and pRItl2571 which carry the fusion between the CS repeats and part of the pre S2-S coding sequence. Increase in the size of these fragments by 192 bp or 24 bp is indicative of the insertion of a tetrapeptide repeat fragment at the Bamt.l site of pRIT12572 and pRIT12571. The presence of a BamHI site on such plasmids is indicative of insertion of the 192 bp or 24 bp Sau3A fragments in the correct orientation for fusion at the ATG codon. This process described above may be repeated if desirable to introduce further 192 bp or 24 bp Sau3A fragments encoding the CS tetrapeptide repeats and elongated the CS tetrapeptide region on the hybrid particle. As stated before, the Hindi 11 fragments from such derivatives of p'RlT12572 or pRIT12571 carrying the expression module or cassettes may be excised and inserted on YEpl3 or other suitable yeast cloning vector for introduction into yeast cells by conventional techniques.

(B) Construction of pRIT12309, pRIT12314 and an expression shuttle vector PRIT12377 containing the complete 2 micron DNA sequence from yeast

The plasmid pRIT12309 consists of the complete

6318 bp 2 siicron DNA sequence from yeas· cloned into the EcoRI site of the plasmid vector pBR327. The 6318 bp 2 micron DNA sequence was obtained from plasmid pCV19 from J. Broach, Princeton University, NJ. Plasmid pRIT123O9 was deposited on August 18, 1986 in the

American Type Culture Collection, Rockville, Maryland, U.S.A., in E. coli K12 strain MM924 in accordance with the regulations of the Budapest Treaty under accession number ATCC 67187. Figure 13 is a restriction endonu5 clease map of pRIT123O9. Insertion on pBR327 of the 2 micron DNA sequence through one of the two EcoRI sites present on this molecule interrupts the 2 micron A coding region and renders the resulting hybrid molecule pRIT123O9 ineffective for A gene function, also refer10 red to as FLP function. For a description of 2 micron structure and function see Broach J., The Molecular Biology of the yeast Saccharomyces: Life cycle and

Inheritance, pp. 445-470, Strathern J.N., Jones E.W. and Broach J.R. (Eds). Cold Spring Harbor Laboratory,

New York (1981) .

pBR327 is described by Soberon et al. (Gene, 9, 287-305, 1980) and was obtained from F. Bolivar (Department of Molecular Biology, National University of Mexico, Mexico 20DF). It will be appreciated by one skilled in the art that pBR327 can be recovered from plasmid pRIT12309 above by preparing pRIT12309 plasmid DNA recovered from the E. coli strain deposited in the American Type Culture Collection under the 'accession ATCC 67187 by any of a number of conventional methods, digesting this DNA with EcoRI endonuclease, ligating the digest and transforming competent cells of E. coj_i K12 to ampicillin or tetracycline resistance with the ligation mixture. A substantial number of the trans formant colonies will contain plasmids in which only the pBR327 moiety of pRIT123O9 is present devoid of either of the two 2 micron DNA fragments.

The 2850 bp Clal-Sail fragment containing the 3

TDH3-arq expression cassette and flanking pBR322 sequences from pRIT12290 (described in Example 3, above) was recloned onto the pRIT123O9 vector between

APO 0 0 0 5 6

BAD ORIGINAL the Cla I and Sail sites. The resulting plasmid, pRIT12314, was digested with Sail endonuclease and ligated with the 2218 bpSall-XhoI fragment from YEpl3 containing the yeast LEUZ gene. Figure 14 is a flow sheet illustrating preparation of pRIT12314. YEpl3 is available from the American Type Culture Collection under accession number ATCC 37115. The ligation mixture was used to transform competent cells of strain JA221 prepared according to the method of Cohen et al (above) to both leucine independence and ampicillin res is tance.

Strain JA221 is publicly available, and was obtained from M. Rosenberg. Smith Kline & French Laboratories, Inc., Philadelphia and may be obtained from the American Type Culture Collection under accession number ATCC 33875.

A plasmid, pRIT12544, was obtained from a colony from this transformation in which the 2218 bp Sa1I -XhoI

LEU2 gene fragment was inserted at the Sail site of pRIT12314. The orientation of the insertion is such so as to recreate the Sail Site on the side closest to the 3

TDH3-arg expression cassette. Plasmid pRIT12544 is an E. co 1i - yeast shuttle vector with single BamHI and SmaI sites located between the TDH3 promoter and arq^ transcription termination regions which are suitable for insertion of DNA coding sequences to effect their expression from the TDH3 promoter. Figure 14 is a flow sheet illustrating preparation of pRITl2544.

(C) Construction of Plasmid PRIT12658

The 3328 bp Hindi 11 fragment of pRITl2574, a YEpl3 derivative prepared as described in Example 10, Part A, was recloned onto a pBR322 Δ BamHI plasmid (where the BamHI site has been destroyed by filling in with T4 DNA polymerase) to give ,pRIT12608 where the BamHI site bad original $ located at the ATG initiation codon of the CS - HBsAg coding sequence is unique (Figure 15). The 2550 bp

BamHI-Sa1I fragment from pRIT126O8 containing the 3

CSHBsAg coding sequence, the arg transcription termination region and flanking pBR322 sequence was recloned between the BamHI and Sail sites of pRITl2S!4 (Figure 15), described above, to give plasmid pRIT12658 (Figure 15). This exchange placed the CS-HBsAg coding sequences under the control of the TDH3 promoter on a complete 2 micron yeast shuttle vector. Figure 15 is a flow sheet illustrating preparation of pRIT12658.

Example 11. Synthesis of hybrid protein in yeast

Yeast (Saccharomyces cerevisiae) strains DCS or 10S44C were transformed according to the method of Ito et al. (J. Bacteriol., 153, 163-168, 1983) with each of the plasmids pRIT10912 (Example 5), pRIT12573 (Example

10); pRIT12574 (Example 10) or pRIT12658 (Example 10), or plasmids pRIT12329, (Example 9) and pRIT10172 (Example 2), and were separately grown in liquid medium Lac king leucine (YNB or YNB + 80 yg/ml histidine) and harvested in mid-log phase. Cells from 1 or 2 ml culture were collected by centrifugation and were resuspended in 50 yl of 0.125 M tris-HCl (pH6.8) containing 20% glycerol, 4% SDS, 6 M urea and 10% 2-mercaptoethanol. After incubation for 5 minutes (min.) at 100°, the sample was divided in halves and both were electrophoresed through a 12.5% separating gel, 5% stacking gel according to the method of Laemmli (Nature, 227, 680, 1971).

HBsAg and CS protein sequences were identified by protein immunoblotting technique (See, Burnette, Anal. Biochem., 112, 195-203, 1981; Gershoni and Palade,

Anal. Biochem., 131, 1-15, 1983; Towbin and Gordon, J. Immunol. Methods, 72:,. 313-340, 1984). After electro9fi000OdV

BAD ORIGINAL blotting of the proteins onto a nitrocellulose filter (Schleicher and Schiill, 0.45 μ) (Towbin et al., Proc. Natl. Acad. Sci., U.S.A., 76., 4350-4354 , 1979 ) the filter was preincubated for. one hour at 37°c with 3% gelatin in PBS. Subsequently the filter was washed 5 times for 5 min. each time with PBS containing 0.1% Tween 20 (polyoxyethylene sorbitan monolaurate) . and one-half of the sheet was treated for one hour at room temperature with a mixture of five monoclonal antibodies against CS protein (Dame et al.. Science. 225. 593-599, 1984 ) in

PBS, 1% gelatin. The other half of the sheet was treated with a monoclonal antibody against HBsAg. HepYTAS12, in PBS, 1% gelatin. HepYTAS12 is an antibody raised against purified yeast derived HBsAg which is directed against a reduction and denaturing resistent epitope. The filter sheets were washed 5 times for 5 min. each time with PBS, 0.1% Tween 20, and a second biotinylated sheep anti-mouse antibody (Amersham) in PBS, 1% gelatin was added for one hour at room temperature. The sheets were washed again with PBS. 0.1% Tween 20, 5 times for 5 min. each time, and were incubated for 30 min. at room temperature with streptavidin-biotinylated horseradish peroxidase (HRP) complex (Amersham). The sheets were washed again 3 times for 5 min. each time with PBS, 0.1% Tween 20, and were finally incubated with 30 μΐ ^H2°2 ^{and HRP Color}Development Reagent containing 4-chlorol-naphtol (BioRad), 30 mg in 10 ml methanol, in 50 ml PBS. The molecular weight of the antigens was estimated from prestained protein markers, ovalbumin, alphachymotrypsinogen, beta-lactoglobin, lysozyme, cytochrome C (BRL).

Both yeast strains DC5 and 10S44C. transformed with pRIT12573, showed production of an antigen reacting with both anti-CS and anti-HBsAg antibodies, and of an estimated molecular weight of 30,000 kilodaltons

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(kd). Both yeast strains DC5 and 10S44C, transformed with either pRIT12574 or pRIT12658 showed production of an antigen capable of reacting with both anti-CS and anti-HBsAg antibodies, and of an estimated molecular weight of 37.000 kd. Some lower molecular weight antigen was also detected, indicative of some degradation of the hybrid protein. Control samples of cells containing plasmids without any CS or HBsAg sequences (PRIT10172) did not show any reaction. Control samples of cells containing plasmids with only HBsAg sequences (pRIT12329 or pRIT10912) showed reaction only with the anti-HBsAg antibodies.

These results show that a hybrid protein of the expected molecular weight containing both CS and HBsAg sequences is synthesized in yeast transformed with a plasmid containing the CS coding sequence fused in phase to the PreS2-HBsAg coding sequence.

Furthermore, while the band intensity in the antiHBsAg reaction was about the same for pRIT12573 and pRIT12574, the intensity of the anti-CS reaction was much higher for pRIT12574 than for pRIT12573. This indicates that for the same quantity of HBsAg sequences there are more sequences of the repetitive epitope of the CS protein present in pRIT12574 than in pR!TL2573.

Example 12. Synthesis of hybrid particles presenting the CSP epitope

Yeast (Saccharomyces cerevis iae) strains DC5 or 1OS44C containing either pRIT10172 (Example 2), pRIT12329 (Example 9), pRIT12573 (Example 10) or pRIT12574 (Example 10), were grown in selective mediur (200 ml YNB or YNB + 80 yg/ml histidine) to end of log phase. Cells were collected by centrifugation and resuspended in 10 ml of 50 mM sodium phosphate buffer (pH 7.4) containing 0.5% Tween 20 (polyoxyethylene sor-

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bitan monolaurate), 1 mM PMSF (phenylmethylsulfony1 fluoride) and 2.5% isopropanol. Cells were disrupted by passage twice through a French Press at 20.000 psi

Q (1.38 x 10 Pa). The suspension was centrifuged for 30 min. at 30.000 xg. The total protein concentration of the supernatant liquid (crude cell extract), was measured by the method of Lowry et al. (J. Biol. Chem., 193. 265-275, 1951) with bovine serum albumin as the standard. The presence of HBsAg was assayed using a radioimmune assay kit (AUSRIA II) commercially available from Abbott Laboratories, North Chicago, Illinois or using an ELISA kit (Enzygnost-HBsAg micro) commercially available from Behringwerke, Marburg, W. Germany. The availability of CS sequences in the HBsAg particles to an immunoreaction was tested in the abovementioned ELISA assay. Appropriate dilutions of samples to be tested (in PBS containing 0.2% BSA) were allowed to bind (overnight at room temperature) to the solid phase anti-HBsAg (Enzygnost kit). The bound HBsAg particles were incubated for one hour at 37°C with an 1/10,000 dilution (in PBS containing 0.1% BSA) of a mixture of five monoclonal antibodies directed against the CS protein· (Dame et al.. Science, 225, 593-599, 1984) as the first antibody, and then incubated with an 1/500 dilution (in PBS containing 0.1% BSA) of biotinylated sheep anti-mouse Ig (Amersham) as the second antibody for one hour at 37°c. Detection was by incubation with an 1/1000 dilution (in PBS containing 0.1% BSA) of streptavidinbiotinylated horseradish peroxidase complex (Amersham) and chromogen from the Enzygnost kit for 30 min. at 37°C. Between incubaticrs the trays were washed four times with washing solution from the Enzygnost kit. Color development was measured at 510 nm in a Titertek Multiskan (Dynatech).

bad

ORIGINAL

Measurement of protein concentration and AUSRIA activity (Table II) in crude cell extracts showed that PRIT12573 directed HBsAg expression at the same level as pRIT12329. However, pRIT12574 showed about a ten5 fold lower AUSRIA activity. Immunoblot analysis of these extracts showed about an equal amount of HBsAg related protein synthesized in those three cases.

Table II - AUSRIA activity in crude cell extracts

Plasmid	Gene	Protein (mg/ml)	HBsAg(RIA) (ng/ml)	HBsAg ng/mg protein
15 pRIT10172			5.6	0	0
PRIT12329	S		3 . 3	5700	1730
PRIT12573	CS( 24	bp)	PreS2-S 3.6	4000	1110
PRIT12574	CS(192	bp)	PreS2-S 2.8	450	160

AP 0 0 0 0 5 6

The ELISA assay showed that a structure expressing both HBsAg and CS epitopes is present in pRIT12573 and pRIT12574 extracts (Table III) and that there are more CS epitopes per HBsAg epitopes that react in pRIT12574 extracts than in pRIT12573 extracts.

To examine the nature of these RIA and ELISA positive structures, one ml of the crude cell extracts was mixed with 1.5 M CsCl in 50 mM sodium phosphate (pH 7.4) and centrifuged for 40 hours at 40,000xg in a

Beckman SW50.1 rotor. Collected fractions were assayed for the presence of HBsAg and CS antibody binding activity with the RIA and ELISA methods described above. HBsAg and CS antigenic activity was found to coequiliBAD ORIGINAL brate in both pRIT12573 and pRIT12574 gradients. Their buoyant density was found to be slightly higher (rho = 3

1.20 g/cm ) than the one for pRIT12329 (rho = 1.18 g/cm³).

Immunoblot analysis of the gradient fractions confirmed the RIA/ELISA results. HBsAg and/or CS sequences were found only in those fractions that did react in tte RIA/ELISA assay.

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Table -II. HBsAg and CS epitopes present in antigen bound to immobilized anti-HBsAq antibody

5	Crude cell extract from:	Dilut ion	ELISA (HBsAg) ELISA (CS)
OD_,_Λ nm 510	°^D510
	1OS44C (pRIT10172)	lOOx	0.000	0.065
	10S44C (PRIT12329)	lOOx	O. 580	0.054
10		200x	0.341	0.041
		4OOx	0.188	0.056
	1OS44C (pRIT12573)	lOOx	1.298	1.596
		2OOx	1.080	1.299
15		400x	0.702	0.898
		800X	0.336	0.560
		1600x	0.155	0.323
		32OOX	0.075	0.189
20	1OS44C (pRIT12574)	100X	0.662	1.678
		200x	0.370	1.506
		4OOx	0.183	1. 130
		800X	0.079	0.798
		1600X	0.044	0.449
25		32OOX	0.061	0.205
	These results	indicate	that the	hybrid protein

produced by expression of pRIT12573 and pRlT12574 does

AP 0 0 0 0 5 6 assemble into particles like the 22 nm HBsAg particles upon synthesis in yeast. The particles produced by expression of pRIT12573 and pRIT12574 do expose the CS sequences on their exterior as shown by their capacity to react with anti-CS antibodies. In the case of pRIT12574, those CS sequences partially obstruct the

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accessibility of the a determinant of HBsAg (the one mainly responsible for AUSRIA activity) in the RIA test.

Example 13.

A. Purification of hybrid particles

The crude cell extract of yeast strain 10S44C containing pRIT12574, prepared as described in Example 10, was clarified by polyethylene glycol (PEG) 400 precipitation and concentrated by ultrafiltration in an AMICON DC equipped with a cartridge having a cut-off of 100.000 daltons.

Further purification of the hybrid particles was done by methods known in the art, for example, CsCl centrifugation, hydroxyapatite chromatography and gel filtration.

The final purified preparation from cell extract of 10S44C containing pRIT12574 showed one main peak in the exclusion volume of a HPLC TSK-3000 column (LKB) which contained both HBsAg and CS antigenicity. SDSpolyacrylamide gel electrophoresis (Laemmli, Nature, 227, 680. 1971) followed by silver al.. Anal. Biochem., 118, 197-203, major band of estimated molecular weight (MW) 37,000 kd. Electron microscopy after negative staining with uranyl acetate showed particles of about the same size and shape as yeast derived HBsAg.

staining (Wray et 1981) showed one

B. Purification of hybrid particles

Crude yeast extracts are prepared by Dyno Mill disruption of the washed yeast cells in the presence of an equal weight of 50 mM sodium phosphate extraction buffer (pH 8.1) supplemented with 4 mM Titriplex III, 1 % Tween 20. 4 mM PMSF and 10 % isopropanol.

0AD

Cell debris are removed by centrifugation at ll.OOOxg for 90 min. Five hundred ml centrifuged crude extract are adjusted to pH 7.2 with IN acetic acid and stirred overnight at 4°C in the presence of 10 gr col5 loidal Silica (Aerosil 380, Degussa). Subsequently, the suspension is centrifuged at 3,500 g for 1 hour and the pellet is washed three times with 150 ml of 0.15 M NaCl supplemented with 2 mM PMSF and 5 mM EDTA during 15 min. Finally the Aerosil pellet is eluted with 250 ml 10 mM pyrophosphate buffer (pH 9.5) containing 2 1 Tween 20 at 37°C for 3 hours. To the desorbate is added dropwise a 1 M CaCl^ solution to a final concentration of 30 mM CaCl₂ and the pH is adjusted to 7. The solution is left overnight at 4°C and centrifu15 ged at 6,500g during 15 minutes.The supernatant is dialysed against 2 x 51 10 mM Tris/HCl (pH 7.1) at 4°C and subsequently diluted four times with dialysis buffer.

After addition of 30 mM CaCl₂ and 1 M NaCl the diluted antigen solution, adjusted to pH 7.1, is passed over a 150 ml column of Phenyl Sepharose FF, equilibrated in the same CaCl^/NaCl containing Tris buffer, at a flow rate of 30 cm/hr. Subsequently the column is washed with equilibration buffer until

280 nm decreases to zero, and elut_ed at the same flow rate with 6 M urea in 10 mM Tris/HCl pH9.

Alternatively, the dialysed and four times diluted antigen solution is supplemented with 20 1 ^^NH4^2^SO4 <^{pH 7}) ^and Passed over a 150 n.l column of Phenyl Sepharose FF, equilibrated in 10 mM Tris/HCl (pH 7.1) 20 % (NH₄)₂SO₄. at a flow rate of 30 cm/hr. After washing the column with equilibration buffer, the column is eluted at the same flow rate with TO mM Tris/HCl pH 7.1.

The antigen positive fractions are pooled and finally centrifuged in.one or two successive CsCl den-

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BAD ORIGINAL jpf sity gradients at 4°C and 245,000g for 65 hrs. The purified hybrid Cs-HBsAg particles have a buoyant den3 sity of 1.23 gr/cm .

Example 14.

Hybrid

Immunogenicity of hybrid particles particles, purified from S. c erevis iae strain 10S44C containing pRIT12574, prepared by the method of Example 13, were inoculated into laboratory animals, as such or with aluminum hydroxide as adjuvant (Young et al.. Science 228. 958-962, 1985).

C57B1/6 mice (6-8 weeks), guinea pigs, and rabbits were immunized subcutaneously and intraperitoneally on days 0, 21 and 49. Animals were bled on days 7. 28, 56 and 84. Blood was allowed to clot at 4°C overnight and sera were separated and stored at -70°C until used.

Rabbits, 2 groups of 2 animals each, received 10 yg of the hybrid CS-HBsAg protein in 1.0 ml doses, with or without aluminum hydroxide, at each immunization. As a control. 2 rabbits were immunized with 10 yg Heptavax B® (Merck Sharpe & Dohme) following the same inoculation scheme. Mice and guinea pigs, in groups of five animals, received 1 ug and 5 _Pg hybrid protein respectively, in 0.5 ml doses at each immunization. Nonimmunized animals served as controls.

For determination of antibody responses, sera from mice were pooled for each group while guinea pig and rabbit sera were assayed individually. Anti-CS antibody titers were measured by ELISA assay using the recombinant CS protein R32tet32 as antigen (See, Young et al.. Science. 228 . 958-962, 1985 ). Anti-HBsAg titers were determined by using the AU3AB kit (Abbott Laboratories) and the WHO reference.

Neither guinea pigs nor mice developed appreciable antibody responses to HBsAg despite repeated boosting.

In contrast, both animal species developed anti-CS

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antibodies and these responses were augmented by boosting. An absorbance of 1.0 in the ELISA assay was obtained with serum dilutions of 1600 fold (mice) and 9000 fold (guinea pigs). Immunogenicity of the hybrid particles was not enhanced by adsorption to aluminum hydroxide .

In contrast rabbits developed antibodies to both CS and HBsAg. Reciprocal titers at an absorbance of 1.0 in the anti-CS ELISA assay were between 800 and

3200. Anti-HBsAg titers obtained with the hybrid particles adsorbed to aluminum hydroxide were between 4000 and 20,000 mUI/ml while the rabbits immunized with Heptavax gave titers of 10^ to 10⁶ mUI/ml. Adsorption of the hybrid particles to aluminum hydroxide enhanced the immune response.

Sera from mice, guinea pigs and rabbits all reacted strongly in the circumsporozoite precipitin reaction as well as inhibited sporozoite invasion of hepatoma cells in vitro (Young et al.. Science. 2^:_3, 95820 962, 1985). (see Table IV). Both reactions are indicative of a protective immune response.

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Table IV. Circumsporozoite precipitin (CSP) reacti-

vity, and	percent	inhibi tion	of sporozoi fe
invasion (%	ISI) of	HepG2-A 16	hepatoma cells

Anima1(s)	CSP Reactivity	% ISI
1 week post 2nd boost	(4 +	2+ 0)

Pooled	mouse sera	14	9	2	91¾
Rabbit	#36	22	3	0	96¾
Guinea	pig #1107	24	1	0	100¾
Degree	of CSP reactivity	is	shown	in parentheses:

- No CSP reactivity detectable

2+ - granular precipitate on surface of sporozoite 4+ - threadlike filament from end of sporozoite

Example 15. Vaccine Preparation

An illustrative vaccine of the subject invention is prepared as follows: To a buffered, aqueous solution of 3¾ aluminum hydroxide (10 mM sodium phosphate, 150 mM NaCl. pH 6.8; sterilized by filtration), the particle of the invention in similar buffer is added with stirring to a final concentration of .,00 pg/ml of polypeptide and 0.5 mg/ml of aluminum (Al³⁺). The pH is maintained at 6.8. The mixture is left overnight at about 0°C. Thimersol is added to a final concentration of 0.005¾. The pH is checked and adjusted, if necessary, to 6.8.

While the above fully describes the invention and all preferred embodiments thereof, it is to be appreciated that the invention is not limited to the embodi ments particularly described but rather includes all modifications thereof coming within the scope of the following claims.

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EXAMPLES OF Pre S2-S PROTEIN AND PreSl-Pre S2-S

PROTEIN PRODUCTION BY YEAST

It has been discovered that the insertion of the 5 Pre S2-S protein coding region of this invention in a yeast expression vector into a yeast results in the synthesis of particles resembling the authentic 22 nm HBsAg particles exposing glycosylated Pre S2 protein on their surface. It has not been previously correctly reported that yeast, transformed with any portion of the HBV genome, express a glycosylated product.

The following examples relate to the expression in yeast of the glycosylated form of a protein containing the Pre S2 region in addition to the S protein of Hepa15 titis B surface antigen. The protein can be extracted and purified from yeast cells in the form of particles for use as a human vaccine. The Pre Sl-Pre S2-S protein expressed in yeast cells is also glycosylated and may also be extracted and purified therefrom i.-. the form of particles for use in a vaccine against Hepatitis B virus .

With respect to the finding of glycosylation, the following should be noted:

1. Examples 25. 26 and 27 concern concanavalin A binding, tunicamycin effect and sensitivity to Endo II, respectively. Each prove that the pre S2-S protein species of 33Kd is glycosylated. Thus, glycosylation has been proven by three independent methods.

2. The specificity of these reagents are, in general terms :

a) concanavalin A binds preferentially to mannose residues .

b) tunicamycin inhibits all types of glycosylation that are N-glygosidically bound to asparagine.

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100

c) endoglycosidase H cleaves asparagine-linked high mannose oligosacchardies leaving one N-i.cetylglucosamine residue attached to the asparagine.

3. From the combined results of Examples 25, 26, 27 and the above, it is deduced that there is a N-linked oligosaccharide chain present on the 33Kc form of the pre S2-S protein (tunicamycin inhibition, sensitivity to Endo H) which is of the high-mannose type (sensitivity to Endo H). The shift in molecular weight observed (about 3000) upon tunicamycin treat ment and Endo H digestion, indicates that the oligosaccharide structure is composed of about 10 sugar residues. This is about the size of a core glycosylation in yeast. Thus, probably only one of the three potential glycosylation sites in the Pre S2-S protein is glycosylated.

4. The purification schemes describeD in Examples 28 and 29 both contain an affinity chromatography step based on sugar residues of a glycoprotein.

5. Phenylboranate has a specificity for 1, i.-c is-d io 1 groups (2 adjacent OH groups sterically positioned in the same direction)·.

6. Lentil Sepharose is glucose/mannose specific.

Example 16. Construction of pRIT12662 - an E. coli vector containing the PreS2-S expression cassette (Figure 16).

Plasmid pRIT12290, obtained as described in Example 3, above, was modified by the insertion of a BamHI-EcoRl synthetic adaptor fragment encoding the Nterminal amino acids of the pre S2 region between the TDH3 promoter and ARG3 terminator fragments. This was done as follows:

Plasmid pRIT12290 was digested with BamHI and EcoRl enzymes and dephosphorylated with alkaline phosBAD ORIGINAL

101 phatase adaptor

200 pmoles of the phosphorylated synthetic

BamHI

Gin Trp 5' GATC CAG TGG

EcoRI

GTC ACCTTAA were ligated with 0.1 pmoles of the BamHI-EcoRI dephosphorylated plasmid pRIT12290 using 1 unit (U) of T4 ligase and used to transform the E. coli strain MM294 to ampicillin resistance. One transformant harboring the desired plasmid with the synthetic adaptor inserted between the BamHI and EcoRI sites was identified and retained. The plasmid is identified as pRIT12621.

Starting with plasmid pRIT12621, the next s ;eps were the elimination of the four extra base pairs (the core of the BamHI site) in order to put the ATG codon in phase with the second codon of the Pre S2 region, and the insertion of an EcoRI fragment containing the rest of the pre S2 coding region and the complete S coding region into the EcoRI site of pRIT12621 to complete the entire pre S2-S coding region.

At this stage the DNA manipulations including subsequent linearization, deletion and recircularization of the plasmid pRIT12621, were realized without amplification by transformation of intermediate plasmids: the procedure was as follows:

pmoles (120 pg) of pRIT12621 DNA were linearized with 120 U of BamHI. After elimination of the restriction enzyme by phenol extraction, the plasmid was ethanol precipitated and resuspended in the appropriate buffer to digest the BamHI single strand extensions with 280 units (U) of mung bean nuclease. The nuclease was eliminated by phenol extraction followed by ethanol precipitation. The so treated plasmid was

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- 102 resuspended in the ligation buffer and recircularized with 10 units of T4 ligase.

The preparation containing the ligated piasmid was phenolized, ethanol precipitated and resuspended into appropriate buffer and digested with EcoRI enzyme. After elimination of EcoRI endonuclease by phenolization and ethanol precipitation, 0.05 pmoles (0.2 pg) of DNA were resuspended in the ligation buffer in order to be ligated with 0.4 pmoles (0.2 pg) of a 838 base pair EcoRI fragment isolated from pRITl2581 that contains the entire C-terminal coding region of the pre S2-S protein. A plasmid essentially similar to

PRIT12581 can be constructed as follows. The vector pUC9 (available commercially from Amersham and Pharma15 cia) is digested with EcoRI endonuclease and treated with alkaline phosphatase (Shine. U.S. patent 4,264,731). Plasmid pRIT10616 (ATCC 39131), is digested with EcoRI and AccI restriction endonucleases and the 826 EcoRI-AccI fragment containing part of the pre

S2-S coding region is recovered by preparative acrylamide gel electrophoresis and electroelution. About 0.4 pmoles (0.2 pg) of this fragment is mixed with about 10 pmoles of the following 12 bp synthetic adaptor previously annealed as

He

Tyr Stop

5' ATACATTTAACG 3’

AccI EcoRI * TGTAAATTGCTTAA 5' described in Example 8, above, and the mixture is treated with T4 DNA ligase and digested with EcoRI restric* tion endonuclease. To the so treated mixture is added about 0.2 pg of the EcoRI digested alkaline phospha35 tase treated pUC9 vector, prepared as described above.

103 and the mixture is treated with T4 DNA ligase and used to transform competent cells of E. coli K12 to ampicillin resistance by conventional procedures. Colonies from the transformant are examined for the presence of a plasmid consisting of the 2650 bp pUC9 vector together with a 838 bp EcoRI fragment comprised cf the 826 bp EcoRI-AccI pre S2-S fragment together with the 12 bp AccI-EcoRI synthetic linker. The correctness of the construction so identified can be verified by DNA sequencing using the chemical modification method of Maxam and Gilbert (Methods in Enzymology, 65 , 499,

1980), preferably from a unique flanking restriction site in the pUC9 polylinker.

The ligation mixture containing plasmid DNA of pRIT12621, treated as described above, and the 8 38 bp EcoRI fragment of pRIT12581 was used to transform the E. coli strain MM294 according to the method of Cohen et al., cited above. One transformant having a plasmid with the EcoRI fragment in correct orientation was retained and the plasmid is identified as pRIT12662 (See, Figure 1A and Figure 16). Figure 1A is a flow sheet illustrating the preparation of pRlT12662. Figure 16 is a restriction endonuclease map of PRIT12662. The pre S2 region from pRIT12662 was sequenced by the chemical modification procedure of Maxam and Gilbert (Methods in Enzymology, 65., 499,

1980) to check that the coding reading frame at the Nterminus was as follows:

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Met Gin Trp Asn Ser

AAAC AAAC AAA ATG CAG TGG AAT TCC pTDH3 pre S2-S coding region

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104

It will be appreciated by one skilled in the a r t that the vector pRIT12662 may also be used for the introduction of further functional DNA sequences into the preS2 region by appropriate in vitro manipulation of vector DNA. The preS2 coding sequence contains restriction sites for EcoRI. Pstl and BamHI endonucleases. Any of these sites may be used for introduction of functional DNA sequences encoding peptides of interest to create in phase fusions with the preS2 region. Such fusions will be of the type preS2 - introduced sequence - preS2-S. These restriction sites may also be manipulated in vitro to create other vectors and provide other sites or reading frames for insertion of functional DNA sequences, such as by the method of Botstein et al. (Science, 229. 1193-1201, 1985).

Example 17. Construction of pRIT12377 and of pRlT12660 a yeast plasmid expressing the preS2-S protein

DNA of plasmid pRIT123O9, obtained as described in Example 10(B), was digested with Sa 11 endonuclease and ligated with a 2218 bp Xhol -Sal I fragment of DNA containing the yeast LEU2 gene obtained by digestion of YEpl3 and purification of the fragment by acrylamide gel electrophoresis. The ligated mixture was used to transform cells of Έ. coli K12 strain JA221, prepared according to the procedure of Cohen et al., cited above, with selection being made Cor both a.npicillin resistance and leucine independence. strain JA221 was obtained from M. Rosenberg, Smith Kline and French Laboratories, Philadelphia. Pennsylvania, U.S.A., and is available from the Amecican Type Cu.ture ;o 11 eci c n under accession number 33875. A plasmid. pRIT12377, was identified from one transformant colony which contains the 2218 bp LEU2 Xhol-SalI fragment inserted at the Sail site of pRIT123O9.

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105

The orientation of the fragment on the vector i .such as to recreate the Sail site on the AvaI side of the pBR327 part of the vector. Plasmid pRIT12377 is an E. co 1 i - yeast shuttle vector having the necessary functions for selection, replication and maintenance in both organisms.

The 3050 base pair Hindi 11 fragment from pRIT12662 (Example 16), containing the pre S2-S expression cassette was purified by acrylamide gel electrophoresis, treated with T4 polymerase and ligated to pRIT12377 previously opened by BamHI enzyme and T4 polymerase repaired. This preparation was used to transform strain E. co 1 i MM294 to ampicillin resistance according to the method of Cohen et al.. cited above. An E. co 1i transformant harboring the plasmid pRITl266O was retained. Figure 17 is a flow sheet illustrating preparation of pRIT1266O.

This plasmid, pRIT12660. was used to transform two strains of S. cerevisiae. i.e., strain DCS cir° and strain 1OS44C cir°, according to the method describe'] by Hinnen et al. (Proc. Natl. Acad. Sci., U.S.A., 2157-2161. 1978).

Both strain DC5 cir° and 10S44C cir° were deposi ted in the American Type Culture Collection, Rockville,

Maryland, U.S.A., in accordance with the Budapest. Treaty on August 18, 1986 under accession numbers ATCC

20820 and ATCC 20818. respectively.

APO00056

Example 18. The preS2 region in plasmid pRlT12662.

The preS2 region in plasmid pRIT12662 was sequenced using the chemical modification method of Maxam anw Gilbert, Proc. Natl. Acad. Sci., 7 4 , 560- 564, 1977 ) a:, it was found that there were four base pair changes leading to three amino acid changes when compared to another virus of adw2 serotype (Valenzuela et al., 198U

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106

- Table I page 11). An alanine residue instead of a threonine residue at position -45, a phenylalanine instead of a leucine at position -34, and a serine residue instead of an isoleucine residue at position

-11. The last change affects a residue that had been found invariable in all known serotypes until now (Lo et al.. Biochem. Biophys. Res. Com.. 2, 382-388, 1986).

Example 19. Synthesis of particles carrying a receptor 10 for polymerized human serum albumin (pHSA)

Yeast (S . cerevis iae) strains DC5 cir° or 10S44C cit° containing either pRIT1266O or pRIT12363 (Example 16) were grown in selective medium [ 200 ml YNB + 80 yg/ml histidine (DC5 cir°) or YNB (1OS44C cir°)J into log phase. (pRIT12363 is identical to pRIT1266O except for the hepatitis derived gene; pRIT12363 contains the S gene only). Cells were collected by centrifugation and resuspended in 50 mM sodium phosphate pH 8.0 containing 0.5% Tween 20 (polyethylene sorbitan mnnolau20 rate), 1 mM PMSF (phenylmethylsu1fony1 fluoride) and 2.5% isopropanol. Cells were disrupted by passagin'/ twice through a French Press at 20,000 psi (1.30 x g

Pa). The suspension was centrifuged for 30 min. at 30,000 X g. Total protein concentration in the supernatant liguid (crude cell extract) was measured by the method of Lowry et al.,(J. Biol. Chem., 193. 2 65275, 1951), with bovine serum albumin as standard. The presence of HBsAg related material was assayed using a radioimmune assay kit (AUSRIA II) commercially avail30 able from Abbott Laboratories. The results (Table V) indicate that pRIT12660 directs the synthesis of material antigenica1ly related to HBsAg particles. The expression level, based on AUSRIA activity, is about the same in yeast strains DC5 cir° and 1OS44C cir°.

5

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In a radioimmune assay to detect a receptor for polymerized human serum albumin (pHSA) (Pontisso et al., J. Virol Methods, 6., 151-159 , 1983 ), crude cell extracts from pRIT1266O containing cells gave a posi5 tive reaction while crude cell extracts fror, pRlTl2363 containing cells did not. No reaction above background was observed with polymerized bovine serum albumin.

CsCl equilibrium centrifugation of crude cell extracts from pRIT12660 containing cells showed the

AUSRIA positive material to band around rho = 1.20 g/cm³ as do serum derived or yeast derived HBsAg particles. Material recovered from this portion of the gradient gives a positive result in the pHSA test.

The aforementioned results show that in pRIT1266O containing yeast cells a HBsAg related protein is expressed which is assembled into a structure like that of 22 nm particles from serum and exposing a receptor for pHSA on its surface.

Table VA. AUSRIA activity in crude cell < :^rtracts

Strain Plasmid Protein HBsAg related HBsAg related

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25	mg/ml	antigenicity ng/ml	ant igenicity nq/mq protein
DC5 cir°	PRIT12660 13.4	7	522
DC5 cir°	PRIT12660 12.6	7	555
10S44C cir°	PRIT12660 10.8	6	556
30 10S44C cir°	PRIT1266O 9.5	6	631

^BAQ original • 108

Example 20. Four S-related protein species are found to be expressed in yeast transformed with pRlT12660.

To analyze the HBsAg related protein monomers expressed in pRIT1266O containing cells, cells were grown in selective medium, either in Erlenmeyer flasks or in fermenters, into log phase and collected by centrifugation.

To the packed cells (0.1 gram), first 20 pi 40 mM PMSF in isopropanol, and then 200 pi sample buffer for SDS-ροlyacrylamide gel electrophoresis (0.125 M tris-HCl, pH 6.8 containing 20% glycerol. 4% SDS, 6 M urea and 10% 2-mercapto-ethanol), were added Samples were vortexed and aliquots of 2 to 20 pi were further diluted in sample buffer to a final volume of 40 pi, incubated for 5 minutes at 100° and electrophoresed through a 12.5% separating. 5% stacking gel according to the method of Laemmli (Nature, 227, 680, 1971).

After electroblotting (Towbin et al., Ploc. Natl. Acad. Sci., U.S.A., 7 6 , 350-4354, 1979 ) of the proteins onto a nitrocellulose filter (0.45 pm, BioRad), the filter was preincubated for one hour at 37°C with 3¾ gelatin in PBS. The filter was incubated with a monoclonal antibody directed against a denaturation and reduction resistant epitope of human derived H3sAg (monoclonal 6 obtained from H. Thomas. Royal Free Hospital, London. England) and antibody binding was detected by successive incubation with biotinylated sheep anti-mouse Ig (RPN 1021, Araersham, dilution 1/250 in PBS containing 1% gelatin), steptavidi ι-biotinyla ted horseradish peroxidase complex (RPN 1051, Amersham, dilution 1/400 in PBS containing 1% gelatin) and finally 30 pi ^H2°2 ^an<^ ^HR£> Color Development reagent containing 4-chloro-l-naphtol (BioRad), 30 mg in 10 ml methanol, in 50 ml PBS. Between incubations the filter was washed with PBS containing 0.1% Tween 20.

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109

The immunoblot showed 4 protein bands related to the S protein (p23) of estimated molecular weights of about 33Kd« 30Kd, 28Kd and 25Kd which are detected by the monoclonal antibody. The majority of S related material is found in the 33K protein band and was found to migrate slightly faster than the gp33 protein described by Stibbe and Gerlich (Virology, 123 , 436-442,

1982). The smallest protein band detected migrates slower than the S protein (p23) synthesized in yeast.

Example 21. The four S-related protein species (of 33Kd, 30Kd, 28Kd and 25Kd) are assembled in particles.

Crude cell extracts of pRIT12660 (Example 16) containing yeast cells were prepared as described in Exam15 pie 19 or by grinding yeast cells in a Dynomill in the presence of an equal weight of extraction buffer (200 mM tris, 3 M NaCl, 10 mM EDTA, 10 mM ethylene glycol bis (beta-aminoethyl ether)' N.N.N',N'-tetraacetic acid (EGTA), 4 mM PMSF, 10% isopropanol) and subsequent cen20 trifugation for 45 minutes at 30,000 X g.

The pRIT12660 directed particles were partially purified from the crude cell extract by ultrafiltration, CsCl equilibrium centrifugation and hydroxyapatite chromatography which are methods known in the art. The four protein bands of S-related material detected by Western blot analysis copurify during these purification steps. An immuno-blot analysis (as described in Example 20) of the AUSRIA positive CsCl fractions after equilibrium centrifugation showed:hat the four S-related bands were present in the same relative proportions in each fraction.

The material is thus distributed in homogenous fashion throughout the peak fractions of the gradient.

AP000056

110

Example 22. The 33Kd and 30Kd proteins carry a receptor for polymerised human serum albumin.

Human serum albumin (Sigma. A 8763) was polymerised by glutaraldehyde treatment (Merck. A-12179) and purified according to Pontisso et al (J. Virol, methods. 6 151-159, 1973). Reactivity of the four Srelated proteins towards pHSA was tested in ' a Western blot procedure. Partially purified preparations of pRIT12660 directed particles (Example 21) were separa10 ted by SDS polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane as described in Example 20. The nitrocellulose filter was successively incubated with pHSA (15 yg/ml in PBS containing 1% gelatine), rabbit anti-serum to human albumin (Beh15 ringwerke, ORCB 04/05, 1/125 dilution in PBS containing

1% gelatin), biotinylated donkey anti-rabbit Ig (Amersham RPN 1004, 1/250 dilution in PBS containing 1¾ gelatin), streptavidin-biotinylated horseradish peroxidase (Amecsham RPN 1051 1/400 dilution in PBS contai20 ning 1% gelatin) and finally with ^H2°2 ^{and 4}~ chloro-l-naphtol as described in Example 20.

The two largest S-related protein bands (the 33Kd and 30Kd) reacted with pHSA, the two smallest ones (28Kd and 25Kd) gave no reaction.

Example 23. The 33Kd - and 3OKd proteins contain a sequence specific for the preS2 region of the preS2-S protein.

A peptide corresponding in 23 of 26 positions to 30 the 26 N-terminal amino acids of the preS2-S protein was synthesized (NH^-Met-Glu-Trp-Asn-Ser-Thr-AlaPhe-His-Gln-Ala-Leu-Gin-Asp-Pro-Arg-Val-Arg-Gly-Leu-Tyr• Leu-Pro-Ala-Gly-Cys-COOH) and coupled via t'nb C-terminal to keyaole Limpet hemocyanin (Peninsula Laborato35 ties). Rabbits were immunized subcutaneously with 100

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Ill yg of the conjugate (which corresponds to 20 yg peptide) in Freund's complete adjuvant, boosted at days 8 and 15 with another 100 yg conjugate in Freund's complete adjuvant and at days 28, 69 and 149 with 100 yg conjugate in PBS. Development of antibody titer was followed by taking blood samples at regular intervals and testing serum dilutions by incubation on solid-phase fixed synthetic peptide. Bound antibodies were detected by iodinated protein A (New England Nuc10 lear). For immunoblot applications, sera with antipeptide titers greater or equal to 1/5120 were selected. The serum was used as such after a 100 fold dilution or after partial purification of IgG. IgG was partially purified by precipitation with Na₂SC>₄ (120 mg per ml of serum), resuspension in PBS and reprecipitation with 14% Na₂SO₄- The resuspended IgG solution was desalted by passage on a PD10¹ column (Pharmacia).

Detection of reactivity of these antibodies towards the S-related proteins in immunoblots¹ was by biotinylated donkey anti-rabbit Ig, streptavidin-biotinylated horseradish peroxidase and ^H2°2 ^{and 4}chloro-l-naphtol as described in Example 22.

Proteins from partially purified preparations of PRIT12660 directed particles (Example 21) were separa25 ted by SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose as described in Example 20. Also run on the gel was S protein (p23) from particles purified from yeast harboring pRIT12363. The immunoblot showed that the two largest S-related pro30 tein bands (the 33Kd and 30Kd) react with the anti-peptide antibodies while the two smallest ones (28Kd and 25Kd) give no reaction. The S protein (p23) gives no reaction with the anti-peptide antibodies.

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112

Example 24. Human anti_THepatit is B antibodies from naturally infected persons recognize epitope(s) on the 33Kd and 30Kd preS2-S proteins not present jn the s protein.

Reactivity of pooled gamma globulin from antihepatitis B positive human subjects (Belgian Red Cross, 100-150 IU/ml Lot 85AO8) towards the four S-related proteins was tested by the immunoblot procedure described in Example 20. A partially purified preparation (Example 21) of pRIT12660 directed particles was dissociated by SDS and the proteins separated by iOS polyacrylamide gel electrophoresis. After transfer of the proteins to nitrocellulose, the membrane was incubated with a ten-fold dilution of anti-Hepatitis B ga.rma glo15 bulin in PBS containing 1% gelatine. Antibody binding was detected by successive incubation with biotinylated sheep anti-human Ig (Amersham RPN1003, 1/250 dilution in PBS containing 1% gelatin), steptavidin-biotinylated horseradish peroxidase and finally with ^H2°2 and

4-chloro-l-naphtol as described in Example 20.

The two largest S-related protein bands (the 33Kd and 30Kd band) react with the human antibodies, the two smallest ones (the 28Kd and the 25Kd band) give no reaction. The S protein (p23) derived from particles from yeast cells containing pRIT12363 does not react with these human antibodies. These results show that gamma globulin obtained from infected persons specifically recognizes epitopes present on denatured and reduced preS2-S proteins which are absent on denatured and reduced S protein. It is known that the epitopes present on S protein derived from human serum and yeast are predominantly conformational.

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Example 10A. The 33Kd protein species carries a rnannosyl containing oligosaccharide structure.

PreS2-S protein from partially purified prepara tions of particles from pRIT1266O containing 10S44C cir ° cells was electrophoretica1ly separated and transferred to nitrocellulose as described in Example 20. Also run on the gel were S protein from particles purified from yeast containing pRIT12363 and HLsAg particles from infected human serum obtained from Dr. W.H.

Gerlich, Department of Medical Microbiology, University of Gottingen, Federal Republic of Germany.

One half of the nitrocellulose filter was incubated in 50 yg/ml concanavalin A (Calbiochem A grade) in 10 mM tris pH 7,5, 150 mM NaCl 1 mM CaCl₂ 1 mM

MnCl₂ 0.05% Tween 20 and subsequently with 30 yg/ml horseradish peroxidase (Sigma type VI P 8375) in 10 mf-l tris pH 7.5 150 mM NaCl 0.05% Tween 20 and finally ^H2°2 ^and 4-chloro-l-naphtol as described in Example 20 .

Between incubations the filter was washed with mM tris pH 7.5 150 mM NaCl 0.05% Tween 20 (Hawkes,

Anal Biochem, 123. 143-146, 1982; Clegg, Anal.

Biochem.,127. 389-394. 1982). The other half of the nitrocellulose filter was incubated with monoclonal antibody 6 and treated as described in Example 20.

The 33K protein band reacts with the concanavalin

A-peroxidase reagents while none of the 30Kd, 28Kd,

25Kd protein bands or the S protein derived from pRIT12363 containing cell reacts. In the presence of

0.1M methyl alpha-D-mannopyranoside the binding of concanavalin A to the 33K protein is abolished.

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114

Example 26. An oligosaccharide structure is N-glycosidically bound to asparagine pRIT12660 and pRIT12377 (Example 17) containing cells were grown in selective medium (YNB + 80 ug/ml histidine (DC5 cir°) or YNB (10S44C Cir°)l to OD

2 0nm = 0.2. Tunicamycin (Calbiochera) was added to a final concentration of 10 yg/ml and the cultures incubated 35 for 30 mm at 30°C. Then 20 yCi S-methionine (1000 Ci/mmol) (New England Nuclear) per ml was added and the cultures were incubated another 15 minutes. 2 ml of labeled cell culture, either treated or untreated with tunicamycin, were centrifuged in a microfuge and the packed cells were resuspended in 40 yl 1% SDS and disrupted by agitation with a Vortex mixer for 2 min.

in the presence of 0.3 gram glass beads (diameter 0.45- 0.50 mm).

Immune precipitation was essentially according to Julius et al. , (Cell, 36. -309-318, 1984). Che lysate was heated for 3 minutes (min.) at 100°, diluted with

0.5 ml PBS containing 1% Triton X100, 0.5% sodium deoxycholate 0.1% SDS and heated again for 1 min. at 100° .

yl of a 10% suspension of prewashed Immunoprecipitin (Formalin-fixed Staph A cells, Bethesda

Research Laboratories) was added to the boiled extract and incubated at 0° for 30 min. The mixturi was clarified by centrifugation for 5 min. in a microfuge. To the supernatant liquid. 2.5 yl monoclonal antibody 6 specific for S protein (see Example 20) was aided, and the mixture was incubated for 1 hour at 0°: Another portion (25 yl) of Immuno-pr ec i p i t i n was then added and incubated for 30 min. at 0°. The immune complexes were collected by centrifugation (5 min. in a micro-

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115 fuge) and washed with PBS containing 2M urea. PBS containing 1% 2-mercaptoeth^nol and finally PBS. The final washed pellets were resuspended in sample buffer for electrophoresis as in Example 20.

. . 5

Samples containing 10 cpra were electrophoresed through a 12.5% SDS polyacrylamide gel (see Example 20). After electrophoresis, gels were fixed in 40% methanol 10% acetic acid, treated with Amplify (Amersham) for fluorography, dried on filter paper under vacuum and subjected to fluorography using Kodak X-Omat S film at -70°.

This showed that pRIT12660 containing oils synthesize a 33Kd protein in the absence of tunicamycin and a 30Kd protein in the presence of tunicamycin.

Both bands are absent in the corresponding samples of pRIT12377 containing cells.

This results shows that the 33Kd preS2-S protein expressed in yeast strains ,DC5 cir° carries an oligosaccharide portion N-glycosydically bound to aspara20 gine. There is most probably only one oligosaccharide chain attached to the 33Kd form of the preS2^-S protein which is not much larger than a core glycosylation.

AP 0 0 0 0 5 6

Crude cells or

Example 27. The oligosaccharide structure is of the 25 high-mannose type.

cell extracts from pRIT1266O containing partially purified preparations of preS2-S containing particles from these extracts (Example 21) were treated with Endo-N-acetylglucosaminidase (Endo 30 H. ; EC.3.2.1.96) from Streptomyces piicatus obtained from New England Nuclear.

Partially purified particles (50 yl, 450 yg/ml protein) were boiled for 3 minutes at 100° in the presence of 0.1% sodium dodecyl sulphate, diluted ten-fold with 100 mM sodiura-ci.trate pH5 and to 240 yl of this

116 mixture 2.4 pi Endo H (74 pg/ml) was added. Samples with and without added Endo H were incubated for 4 hrs at 37°. Analysis of the Endo H treated and untreated samples by SDS-polyacrylamide gel electrophoresis and immunoblotting with detection by the monoclonal antibody 6 directed against the S protein as described in Example 20. showed the disappearance of the 33Kd band and the appearance of a 31Kd band upon Endo H treatment. The 31Kd band still reacts with the concanavalin A-peroxidase reagents as described above. The position of the other bands (30Kd, 28Kd and 25Kd) did not shift upon Endo H treatment. Longer incubation times or higher Endo H concentrations gave identical results. The result shows that the oligosaccharide structure linked to the 33Kd protein is of the highmannose type and has about the size of a core glycosylation. <

Example 28. Purification of preS2-S protein containing particles.

Crude yeast extract containing preS2-S particles is prepared by grinding the washed yeast cells in a Dynomill in the presence of an equal weight of extraction buffer, either 50 mM sodium phosphate pH 8.1, 4 mM EDTA 1.0% Tween 20, 4 mM PMSF and 10% isopzopanol or

200 mM tris-HCl pH 9.0, 3.0 M NaCl, 10 mM EDTA, 10 mM

EGTA (ethylene glycol bis (beta)aminoethyl ether Ν,Ν,Ν¹,N'-tetra acetic acid), 1% Tween 20. 4 mM PMSF and 10% isopropanol. ¹

The homogenate is adjusted to pH 8.0 or 9.0 respectively and subsequently centrifuged for 45 minutes at 30,000 X g.

To 500 ml of the crude yeast extract, adjusted to pH 7.2, is added 10 gr colloidal Silica (Aerosil 380 Degussa). The colloidal suspension is stirred overbad ORIGINAL $

117 night at 4° and subsequently centrifuged at low speed. The Aerosil pellet is washed three times with 150 ml 0.15 M NaCl for 1/4 hour and then eluted batchwise with 250 ml 10 mM pyrophosphate buffer (pH 9.3) containing

2% Tween-20 at 37° for 3 hours. The desorbate, a yellowish. lightly opalescent solution, is passed over a 50 ml column of phenylboronate Agarose (Amicon) equilibrated in 10 mM pyrophosphate buffer pH 9.0. The flow rate is 15 ml/hour.

The column is further washed with 2 column volumes of equilibration buffer, and is then eluted in the reversed direction with 100 mM sorbitol in 50 mM Tris (pH 7.2) at a flow rate of 15 ml/hour. Eluted peak fractions are pooled and after adjustment to pH 8.1, passed over a DEAE-Fractogel TSK column equilibrated in 50 mM Tris pH 8.1. The particle is eluted in the same buffer containing 75 mM NaCl. After this step residual nucleic acids were removed (<1 pg/20 pg protein).

The balance sheet is shown in Table VI.

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118

Table V. Two step purification of preS2-S cor.'.aininq

particles
		mg S-related protein mg	mg	mg poly-
Fraction	ml		(RIA)	protein	1 ipids	saccharides
Crude extract	500		11.3	14,600	5.286	7.850
Desorbate aerosil	270		5.96	385	554	249
Flow through PBA-columr.	315		2.83*	365	360	267
Eluate PBA-column	70		2.8	2.9	5	2 . 5

*not sufficiently glycosylated to be cetainrd on Ui·.· column.

Material purified from extracts of cells co ntlining pRIT12660 as described in this Example 28, foil'? wed by a CsCl equilibrium centrifugation, was prepared for electron microscope examination after staining wi/.'i uranyl acetate. Examination showed the presence <·< discrete particle structures about 19 microns in diem ter, and that such hybrid particles contained between 5 to 20 pg Tween 20 per 100 pg protein.

119

Example 29. Affinity chromatography on lentil sepharose.

In this example, the phenylboronate agarose chromatography step is replaced by affinity chromatography on lentil sepharose (Pharmacia).

ml of Aerosil-desorbate, obtained as described in Example 28, was passed over a 5 ml column of lentil Agarose equilibrated in equilibration buffer such as Tris buffer 10 mM (pH 7.2). 0.14 M NaCl and 0.3%

Tween. The column was washed with 2 column volumes of equilibration buffer such as Tris Buffer 10 mM (pH 7.2) and then eluted with sugar additive with specificity for lentil such as 0.5 M alpha-methylmannopyranoside in equilibration buffer. The balance sheet is shown in Table VII .

Table VII. Application on lentil Sepharose affinity chromatography in the purification on preS2-S containing particles.

AP 0 0 0 0 5 6

	Volume	yg S-related	Tot.3 1
Fraction	.(.mil	protein (RIA)	F r o t e i n
Crude	100	217 0	3 gc
Desorbate aerosil	50	1700	13 8 mg
Flow through Lentil-sepharose	50	428	-
Eluate Lenti1-sepharose	12.5	925	1.26 mg

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Examination revealed that the hybrid partial·.; purified as described herein contained between 5 to o pg Tween 20 per 100 pg protein.

Example 30. 1mmunogenicity of the preS2-S particle.

The immunogenicity. of particles prepared from extracts of cells containing pRIT12660 (Example 16) was studied in 2 strains of mice: Balb/c (H -dand NMR1 (H₂-Q)

The particles were absorbed on Al(OH) injection and the intraperitoneally (I.P.) with 0.3 reacting material. One month after the immunization, the mice were bled and the sera from the mice of the same group were pooled. The titer of anti-S antibodies 15 was determined in the pools by using the AUSAB kit (Abbott Labs) and the WHO reference. The titers wore converted in mlU/ml by using the Hollinger formula (Hollinger et al., in Szmuness et al., eds. Viral Hepatitis, Philadelphia : Franklin Institute Press, 45120 466, 1982). The anti-preS2 antibodies were determined using a solid phase assay (RIA) in which the synthetic preS2 peptide given in Example 23. is absorbed on plasd e t ec’o d with a goat urn before injected mice (5 mice/group) were pg of El A tic and bound antibodies were 125.

anti-mouse IgG labelled with 25 marized in Table VIII show

PRIT12660 containing cells

I . The results that the particles induce both anti - S anti-preS2 peptide antibodies in both strains of r o m and m 1 c e peptide antibodies The anti-S titer 2 to 3 times higher > I par*, not contain with higher titers of anti-preS2 induced in NMR1 than in Balb/c mice 30 obtained in Balb/c or NMR1 mice is than the titer obtained with a higher hose from pRIT12363 containing cells which do the preS2 sequence. PreS determinants enhance the response to the S determinants. This enhancing effect is 35 not shown if the preS2 synthetic peptide is injected

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121 together with particles which lack the preS2. Moreover, the preS2 synthetic peptide alone is unable to elicit anti-preS2 antibodies.

This indicates that the association of preS2 5 determinants and S determinants on the same particle is required to obtain an anti-preS2 response. Furthermore this association results in an improved anti-S response.

The results of the immunogenicity studies referred to herein related to the PreS2-S particle are summari10 zed in Table VIII.

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122

Table VIII. Immunoqenicity of the preS

Preparation Dose of injected related ant igen (yg)(1)

Titre of

Anti-S antibodi (mlU/ml)(2)

Balb/c

NMR1

Anti - pre32 anti Pod i es ( .· >

Balb/c NMR!

Yeast-derived 1.7 S particle directed by pRIT12363	1404
Yeast-derived 1.55 S particle directed by PR1T12363 4- synthetic. p r e 3 2 peptide: 0.03 yg	748
Synthetic preS2 peptide: 0.03 μ g	2

Yeast-derived 0.3 ( p r e S 2 - S) particle directed by pR1T12 6 6 0

3 344

(1) determined by using the AUSRIA hit (i.o.Ht Lair.) and the Proposed Internationa 1 Referenc; Preparation supplied by Dr. Schild, National Institute of Biological Standardization, London, as soradard.

_ba0 oh«'^nW-

123 (2) determined by using the AUSAB kit (Abbott Labs) and the World Health Organization standard.

(3) determined by using a solid phase radio immuno assay and synthetic preS2 peptide adsorbed to plas5 tic.

The titer is the dilution which gives a significant binding of the antibody (2.5 times the value obtained with a negative control serum).

EXAMPLES OF THE EXPRESSION OF THE ENTIRE PreSlPreS2-S PROTEIN CODING SEQUENCE IN YEAST

Example 31. Construction of plasmid pRIl’12845 that expresses the preSl-preS2-S protein in yeast

The first step was the fusion of the TDH3 promoter region with the pre SI N-terminal region frcm the HBV genome. The starting material was plasmid pRIT12?92 that harbors a 495 bp Ncol-Xbal fragment containing the pre Sl-Pre S2 coding sequence except for the last aspa ragine codon. The Ncol site overlaps the ATG codon of amino acid residue -163 of the HBV pre SI sequence. The sequence of the Ncol-Xbal fragment is shown in Figure 4A.

An essentially similar plasmid to ph IT12'/ 9 . , idea25 tified as pRIT12793 (5940 bp), was deposited in the

American Type Culture Collection, Rockville. Maryland, U.S.A., in accordance with the regulations of the Budapest Treaty under accession number ATCC 67193 on September 5, 1986. From plasmid PRIT12793, a 495 bp

Neo I-XbaI fragment can be recovered which contains a complete pre Sl-pre S2 coding sequence. This frauuent differs from that shown in Figure 4A by one nucleotide having the sequence ACGAACTAATAATCTAGA at the 3' end of the fragment. The nucleotide difference is undersco35 red. In the following examples, pRIT12792 can be

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124 replaced with pRIT12793. Both the Ncol-XbaI fragments contained on pRIT12792 and pRIT12793 were derived by in vitro manipulation of DNA from the PreSl-PreS2 region of the DNA of Hepatitis B virus strain (serotype adw) cloned on plasmid pRIT10616. Plasmid pRIT10616 is available from the American Type Culture Collection under accession number ATCC 39131.

pg of pRIT12792 were digested with the Neo I and XbaI restriction enzymes and treated with Mung Bean nuclease in order to eliminate the 5' extensions. 600 ng (2 pmol) of the treated and purified 495 bp NeoIXbai fragment were ligated to 170 ng (0.02 pmol) of pRIT12314 vector that had been previously opened by BamHI and SmaI enzymes and also was treated with mung bean nuclease. pRIT12314 contains the entire 2 micron fragment from S. cerevisiae and an expression cassette in which the TDH3 promoter is separated from the ARGO terminator by a BamHI-SmaI-EcoRI linker:

BamHI EcoRI

Sma I

...... ATGGATCCCCGGGAATTC ....

pTDH tARG₃

A more detailed description of the construction of pRIT12314 is found in Example 10(B) above. The ligation mix described above was used to transform E. coj i MM294 to ampicillin resistance according to the method of Cohen et al.. cited above. Six transformants containing a plasmid in which the pre Sl-pre S2 fragment was inserted in the correct orientation wore retained. These were the plasmids pRIT12843 (a.....f) (Figure 10).

The second step was the insertion into each plasmid pRIT12843 (a......f) described above, of the s

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125 coding region to reconstitute the entire pre S1-S2-S gene .

This was done as follows:

plasmids pRIT12843 (a.....f) were each digested with

BamHI and Sail enzymes. The BamHI site is placed at the beginning of the pre S2 region, the Sail site is placed behind the ARG3 terminator in pBR 327.

ng (0.012 ymol) of the largest BamHI-Sa11 fragment (10,400 bp) purified from each plasmid pRIT12843 (a.....f) were ligated to 300 ng (0.11 pmol) of the small BamHI-Sall fragment from pRIT12660 (Example 16). The small (4800 bp) BamHI-Sall fragment of pRIT12660 contains part of the pre S2-S coding sequence followed by the ARG terminator sequences and the LEU2 yeast sequence. After ligation and transformation of E. col i MM294 with each of plasmids pRIT12843 (a.....f) according to the method of Cohen et al., cited above, a series of ampicillin resistant clones containing the plasmids named pRIT12845 (a.....f) was obtained.

Figure 18 is a flow sheet illustrating preparation of

PRIT12845 (a.....f). These plasmids -pRIT12845 (a.....f), were used to individually transform the S.

cerevisiae strain 10S44C cir° according to the method of Ito et al., cited above. The screening of cultures grown from individual clones from the yeast transformations was done by testing crude cells extracts in the commercially available AUSRIA test (Abbott). Crude cell extracts were made by disrupting yeast cells resuspended in PBS, containing 0.5% Tween 20, 2 mM

PMSF and 5% isopropanol. One transformant of each of the six separate transformations [with pRIT12845 (a.....f)] was tested. Five out of six tested trans formants gave a positive AUSRIA.

To test whether a protein of the correct size gave the S-related antigenicity detected by the AUSRIA, samAP 0 0 0 0 5 6

126 pies of the crude extracts described above, were sub jected to the immunoblot procedure described in example

20. Four of the five AUSRIA positive tra isCorma nts showed a S-related protein of about 39K. One transformant (extract a) showed a S related protein of 23K. Retained as pRIT12845 was the yeast tranformaut expressing the 39K protein present in extract (e). See, Figure 18.

Example 32. The preSl-preS2-S protein is assembled into particles carrying a receptor for polymerized human serum albumin and a preSl epitope on their surface .

Crude cell extracts from yeast strain 1OS44C cir° harboring either pRIT12845, pRIT1266O or pR.7’i2 377 and yeast strain DC5 cir° harboring pRIT12363 were prepared as described in Example 19. Total protein concentration, HBsAg related antigenicity and detection of a receptor for polymerized human serum albumin were performed as described in Example 19. The result showed a strong binding activity for polymerised human serum albumin present in extracts from strain 1CS44C cir’ containing pRIT12843 and pRIT1266O (see also Example 10) which is absent in extracts from strain 1OS44C cir° harboring pRIT12377 or strain DC5 cir° harboring PRIT12363.

CsCl equilibrium centrifugation of crude cell extracts from 1OS44C cir° cells containing pRIT12845 (described in Example 12) and measurement of HBsAg related antigenicity by AUSRIA assay in the CsCl fractions, slowed the antigen to band around :ho = 1.2 . .

g/cm . This was confirmed by immunoblot analysis of the CsCl fractions. Pre SI antibody binding activity in the CsCl fractions was tested by the ELISA assay described in Example 12. After allowing the antigen to

127 bind to the solid-phase anti-HBsAg. the bound antigen was incubated with monoclonal antibody MA18/7 (Example 31) which was then detected by biotinylated anti-mouse Ig, streptavidin-biotinylated horseradish peroddase com- plex and chromogen as described in Example 12.

The pre SI specific antibody binding activity was found to coequilibrate with the HBsAg related antigenicity in the CsCl gradient. These results show that the pre Sl-pre S2-S protein produced in 10S44C cir° cells harboring pRIT 12845 is assembled into particles like the 22 nm HBsAg particles derived from serum. The particles expose pre SI specific sequences as well as a receptor for polymerised human serum albumin on their surface. From the level of activity of those particles in the AUSRIA test and the intensity of the reaction of the pre Sl-pre S2-S protein in an immunoblot analysis, it can be concluded that the particle's S determinants are partially obstructed or deformed by the presence of pre S sequences in the particle.

Example 33. Two proteins of molecular weight of about 38 and 45 kilodaltons (kD), both carrying preSl, preS2 and S epitopes are expressed in yeast transformed with R1T12845

HBsAg related protein monomers expressed in pRIT12845 containing cells were analysed as described in Example 20.

Migration and immune reactivity of the cellular proteins were compared to the proteins from HBsAg par30 tides (serotype ad) purified from human serum (obtained from Dr. W.H. Gerlich, Department of Medical Micro biology. University of Gottingen. Federal Republic of Germany).

Three immunoreagents were used to analyse the pro35 teins:

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128

- the monoclonal antibody (obtained from H. Thomas Example 20) recognizing a S epitope

- a polyvalent rabbit antiserum raised against the synthetic pre S2 peptide (described in Example 23)

- monoclonal antibody MA18/7 specific for a pre SL epitope as described by Heermann et al. (J. Virol., 52., 396-402. 1984).

The immunoblot showed, among the cellular proteins derived from pRIT12845 containing 10S44C cells, the presence of two proteins reacting specifically with all three immunoreagents described above. The molecular weight of these two proteins was estimated to be about 38,000 and 45.000 daltons. The molecular weight estimation was based on the migration of the proteins from serum derived HBsAg as defined by Heerman et al. (J. Virol., 52.· 396-402. 1984). The 38.000 dalton pre Slpre S2-S protein directed by pRIT12845 migrates slightly faster than the p39 pre Sl-pre S2-S protein derived from HBsAg particles. The p39 protein from human serum particles of serotype ad presumably has a pre SI region of 119 amino acids (Heermann et al., J Virol. , 52, 396-402, 1984). pRIT12845 codes for a prSI region of 108 amino acids.

The above results show that yeast cells harboring pRIT12845 express two proteins of about 38 and 4^!? ! 1' carrying pre SI, pre S2 as well as S epitopes.

Example 34. The 45 Kd preSl-preS2-S protein species carries a N-glycosidically bound high-mannose :ype oligosaccharide chain

Cells harboring pRIT12845 were di. rupte: with ,·η equal weight of glass beads (diameter u.45-0 50 mm) in an equal weight of 10 mM sodium phosphate buffer (pH 7.4) containing 2% SDS and 8 mM EDTA by agitation with a Vortex mixer.

bad OWGWN-

129

After centrifugation, the supernatant was heated for 4 minutes at 100°.

yl of the heated supernatant fluid was diluted with 40 yl 25 mM sodium citrate buffer (pH

5.0), and 2 yl EndoH (74 yg/ml) (see. Example 27) was added. Samples with and without added EndoH were incu- bated for 2.5 hrs at 37°.

The EndoH treated and untreated samples were analysed by SDS-polyacrylamide gel electrophoresis and immunoblotting as described in Example 20.

The blot was analysed with monoclonal antibody 5 (S specific) and MA18/7 (pre SI specific) (see. Example 33 ) .

The immunoblot showed both with monoclonal 6 and 15 monoclonal MA18/7, the disappearance of the 45 kD band and the appearance of a 41 kD band upon Endo H treatment. The migration of the 38 kD band was not affected by the Endo H treatment.

These results show that the 45 kD pre Sl-pre S2-S 20 protein species carries a N-glycosidica1ly bound highmannose type oligosaccharide chain.

Example 35. The 38kD preSl-preS2-S protein contains covalently bound myristic acid present in an amide lin25 kage

Cells from yeast strain 10S44C cir° harboring either pRIT12845, pRIT12660 or pR!T12377 were grown in

YNB to an OD,.. of 0.4.

620nm

Cells (20 ml culture) were labeled for 60 min with 30 1 mCi of 3H-labeled myristic acid (New England Nuclear) and then collected by centrifugation. Col¹ - vn e washed with cold H₂O, resuspended in 100 yl 5 mM tris-HCl (pH 7.4) containing 3 mM (DTT), 1% SDS and L mM PMSF. and broken with 100 mg glass beeds by six loss second spurts of vigorous mixing in a Vortex, cooling

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130 on ice between each mixing. Debris was removed by centrifugation for 1 minute in a Eppendorf 5414 centrifuge (Towler and Glaser, Proc. Natl. Acad. Sci.. ?_2, 28122816. 1986). 20 pi of the extract was treated for

3.5 hrs at room temperature with 7 pi of freshly prepared 4 M hydroxylamine, 20 mM glycine, pH 10.

Hydroxylamine treated and untreated samples were subjected to SDS polyacrylamide gel electrophoresis and fluorography as described in Example 26. The hydroxy10 lamine untreated samples were also subjected to immuno blot analysis with the monoclonal antibody recognizing a S epitope, as described in Example 20.

The immunoblot showed results identical to those described in Examples 20 and 33.

The fluorograph showed a labeled band of 38kD present only in the cellular extract from cells harboring pRIT12845, which was hydroxylamine-stable, indicating that the 3H-myristate label is present in an .imide lin kage. No labeled bands specific for the extracts from cells harboring pRIT 12660 were detected.

These results show that the 38kL' pre SI S2-S pre tein contains covalently bound myristic acid present η an amide linkage. In general, myristate becomes ceo lently bound to proteins via amide linkage to an amino-terminal glycine. This suggests that the init.i tor Met has been removed from the pre Sl-pce S2-S pre tein and that the Gly residue at position 2 gas become available for acylation with myristic acid.

Example 3 6 . Use of preSl vectors for insertion ot functional DNA coding sequences

The starting material is DNA of pi uiiid orit j ;p, ? ; described in Example 31 above. pRIT12793 contains the pre Gl-pre S2 coding sequence on a 495 bp Neo 1-XbaI fragment. DNA of plasmid pRIT12793 is digested with bad original

131

Ncol restriction endonuclease, treated with T4 DNA polymerase to fill in the sticky ends and digested with XbaI endonuclease. The Ncol/T4 DNA polymerase/XbaI fragment is purified by polyacrylamide gel electropho5 resis and electroelution and inserted on vector pUCL2 previously digested with Smal and XbaI endonucleases. The cloning vector pUC12 is commercially available from Amersham (Little Chalfont, Bucks, United Kingdom) and from Pharmacia (Piscataway, NJ). The plasmid pRITX is obtained as shown in Figure 5B. In the pre )l-pre S2 region of plasmid pRITX, a single Ncol site overlaps the ATG start codon of the pre SI region, ar.d a Xba I site is present adjacent to and downstream of the ΤΑΛ stop codon at the C-terminus of the pre-S2 coding

15 sequences. A single	BstX restriction	site end	is of	present	i; ‘
on	the plasmid close	to the	N-termina1	the	pre
SI	coding region.						C -.½
	It will be appreciated	by one skilled	in	the	art	&

that the vector pRITX or similar derivative vectors 20 containing the pre S1-S2 sequences may be used for introduction of further functional DNA coding sequences encoding peptides of interest to create in phase fusions within the pre SI region by insertion at the BstX site.

functional DNA coding sequences nay digestion of pRITX and derivatives or Ps '. I

Furthermore , be introduced by with combinations of Bs tX and BamHI or EcoRl ______ endonucleases and insertion of the functional DNA coding sequence between these sites, thus removing the 30 greater part of the pre SI coding region and the N-terminal part of the pre S2 sequence. Such fusions will, be of the type pre Sl-introduced functional DNA coding sequence pre S2. Once such fusions are made,' they can be recombined onto other plasmids containing the rest 35 of the pre Sl-pre S2 sequences and sequences necessary

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132 for maintenance and replication in E. coli and s. c e r r- _ visiae as exemplified in Example 16A.

Example 37. Fusion of peptide from proteins of HTLVIII virus to the preS2 region of preS2-S sequences to create novel hybrid particles

The preS2 region of a HBV preS2-S coding sequence can also be used for the insertion or fusion of peptide sequences of other viral antigens including those of the causative agent of human AIDS syndrome, the retrovirus known as HIV, HTLV-III or LAV.

The cloned genome of a HIV virus has been described by Ratner L. et al. (Nature, 313, 277-284, 1985 ) and Shaw G. et al. (Science, 226. 1165-1171, 1934).

From this genomic clone various subfragments can be obtained as functional DNA coding sequences which correspond to peptide regions of interest for fusion to the preS2-S sequence to form novel hybrid particles containing epitopes of the HIV virus proteins. Such hybrid particles can serve as the basis of a human vaccine for protection against the infective agent HIV.

Among the peptide regions of interest are the following :

a. C7 peptide. This region corresponds to a stretch of 45 amino acid residues immediately adjacent to the processing site of the envelope precursor and was defined by Starcich B.R. et al. (Cell. 45. 637-64,?. 1986) .

b. Peptide 121. This region correspunds t: a relatively conserved amino acid sequence present in several retroviral transmembrane envelop'·- proteins i ·· Cianciolo G.J. et al. (Science, 2.30., 4 53- 455, 1985 ).

This sequence is thought to participate in the pathogenesis of retrovira 1-induced immunosuppression (Snyder133 man R. and Cianciolo G.J., Immunol. Today, 5, 240.

1984) .

c. Dreesman peptide. A synthethic peptide cor responding to amino acid sequence 735 to 752 of the precursor envelope glycoprotein of HIV was used by Kennedy R.C. et al. (Science. 231, 1556-1559, 1986) to immunize rabbits and rabbit antiserum was used to investigate the recognition of HIV envelope proteins by induced antibodies. The results suggest that this region could induce an immune response directed against the native glycoprotein.

A. Fusion of the DNA sequence corresponding to the C7 peptide from a HTLV-III isolate to the preS2-3 region on pRIT10911.

The starting material was plasmid pBHlO-R2 containing the cloned genome of HIV isolate BH10. This plasmid was obtained from R.C. Gallo. National Institutes of Health. Bethesda, MD. A 3108 bp Sall-Xhol fragment of DNA of viral origin which was excised from pBHlO-R2 and cloned between the Sail and Xhol sites of pUC18 to create plasmid pRIT12901. A restriction map of this Sall-Xhol fragment is shown in Figure IB. Plasmid pUCI8 is described by Norrander J. et al. (Gene, 26, 101-106, 1983) and is commercially available from Pharmacia Inc., Piscataway N.J.

To create the fusion, DNA of pRITl2901 was digested with Bqllland MbolI endonucleases, and the 117 bp fragment isolated by acrylamide gel electrophoresis and electroelution (see. Figure IB). DNA of this fragment was treated with Mung Bean nuclease to remove single stranded extensions and ligated with DNA of pRIT109ll (see. Example 5, above) which had been digested with BamHI endonuclease and treated with Mung Bean nuclease. From this ligation mixture a plasmid,

PRIT12893, was isolated in which the 117 bp Bql 11-Mbo11

APO00056

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134 fragment from pRIT12901 had been inserted on PRIT1O911. The nucleotide sequence across tha fusi junction between the TDH3 promoter and the 117 bp HTLV-III BqllI-Mbo I1. mung bean nuclease treated Crag5 ment insert on pRIT12893 was determined and it was found that 10 nucleotides in excess had beer, removed from the Bqll I end of the fragment. The sequence found was

1.0 5 ' ATGGAGGAGGAGATATGAGGGA 3 ' where the first ATG codon underscored is that of the TDH3 promoter region from pRIT10911 and the second is an ATG codon internal to the C7 peptide fragment. The second ATG condon overlaps a TGA termination codon. This termination codon is in the same reading frame as the first ATG codon. DNA sequence determination of the 3' end of the fusion region on pRlT12893 showed the correct sequence Cor in phase fusion of the Mbo'. I - mung bean nuclease treated end of the HTLV-III fragment to the preS2 sequence on pRIT10911 as shown in Figure ZB.

DNA of pRIT12893 was then digested with Hindlll endonuclease and the 3250, bp fragment isolated and purified by agarose gel electrophoresis and electroelu’L tion. The 3250 bp Hindi 11 fragment was then inserted at the Hindlll site of plasmid YEpl3 to create pRIT12894. DNA of the plasmid was then used to transform cells of yeast strains DC5 and 10S44C to leucine independance by the method of Ito et al. as cited in

Example 10 above.

Transcription and translation in yeast cells of the C7-pre32-S fusion DNA on PRIT12894 will result in synthesis of a small 5 residue peptide commencing at the TDH3 ATG codon and terminating at the TGA c:don underscored above and of the C7-preS2-S fusion peptide bad original

135 commencing at the second ATG codon internal to the C7 sequence. This fusion contains 38 amino acid residues from the C7 peptide in place of the 45 residues from the intact BqIII-MbolI-mung bean nuclease treated C7 fragment. It will be appreciated that other plasmids recovered from the same ligation mixture as pRlT12893 can be examined and sequenced to determine if they carry a fusion corresponding to the complete fragment.

B. Fusion of the DNA sequence corresponding to the

Peptide 121 to the preS2-S region on pRIT10911.

To create the fusion, DNA of pRIT12901 (described in Part A, above) was digested with Hha I and Hind 111 endonucleases to liberate the 230 bp fragment shown in Figure IB. This fragment was purified by acrylamide gel electrophoresis and electroelution and treated with T4 DNA polymerase to fill in single strand extensions. The so treated fragment was ligated with DNA of PRIT10911 which had been digested with BamHI and Mung Bean nuclease. From this ligation mixture, a plasmid was identified as pRIT12897 on which the 230 bp fragment of HIV DNA has been inserted in the correct reading frame for fusion to the preS2 sequence of pRH0911 as shown in Figure 3B. DNA of plasmid pRIT12897 was then digested with Hind 111 endonuclease, and the 3 3 65 bp fragment containing the TDH3 promoter. HIV-preS2-S fusion and the ARG3 terminator was purified by agarose gel electrophoresis and electroelution. This 3365 bp fragment was ligated onto YEpl3 digested with Hind 111 endonuclease to form plasmid pRIT12898. DNA of this plasmid (pRIT 12898) was then used to transform cells of yeast strains DC5 and 1OS44C to leucine independence by the method of Ito et al. as cited in Example 10, above.

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136

C. Fusion of the DNA sequence corresponding to the Dreesmanpeptide to the preS2 region on pRIT10911. A 60 bp fragment of synthetic DNA with the sequence shown below was synthesized by conventional means as two single strands which were then annealed together :

5' GATCTCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGA 3¹3' AGCTGTCCGGGCTTCCTTATCTTCTTCTTCCACCTCTCTCI 5¹

5' GACAGAGACAGATCCCCG 3'

3’ CTGTCTCTGTCTAGGGGCCTAC 5¹

This fragment has 5' BqllI and 3' BamHI single strand extensions. About 200 ng of a double stranded fragment and 300 ng of DNA of plasmid pRITL09ll digested with BamHI were mixed and ligated. From this ligation mixture, a plasmid, pRIT12899, was identified and recovered on which the 60 bp fragment has been inserted at the BamHI site of pRIT10911 so as to create an inframe fusion as shown in Figure 4B. DNA of JRITL2B?? was digested with Hindi 11 endonuclease, and the 3 200 bp Hind 111 fragment was purified by agarose gel electrophoresis and electroelution. This Hi nd I I I fragrent; was inserted at the Hi nd111 site of YEpl3 to create plasmid pRIT129OO. DNA of plasmid pRIT12900 was then used to transform cells of yeast strains DC5 and 10S44c to leucine independence by the method of Ito et al. cited in Example 10, above.

Example 38. Expression of fusion proteins C pRIR12894 and pRIT12898

Yeast strains DC5 or 10S44C harboring either YEpl3. pRIT12363, pRIT10912. pRIT12894 or pRIT12898 were separately grown in liquid medium lacking leucine

137 (YNB + 80 yg/ml histidine or YNB). and cellular proteins were analyzed by the immunoblot procedure as -'escribed in Example 11 using as the first antibody a monoclonal antibody directed against a denaturation and reduction resistant epitope of human derived HBsAg (monoclonal 6 obtained from H. Thomas, Royal Free Hospital, London, England). pRIT10912 is described in Example 5 above. pRIT12894 and pR!T12898 are described in Example 37, above. pRIT12363 carries the S gene of

HBV fused to the TDH3 promoter as the 2900 bp Hind 111 fragment from pRIT12322 (Example 8) inserted on a yeast vector plasmid identical to pRIT12377 (Example 10, part B above) and produces HBsAg under the control of the TDH3 promoter.

The immunoblot showed a protein band related to 3 protein of estimated molecular weight of about 32 kD among the total cellular proteins from yeast strains DC5 or 10S44C transformed with pRIT12894. A protein band related to S protein of estimated molecular weight of about 45 kD was present among the total cellular proteins from yeast strains DCS or 1OS44C harb ring pRIT12898, while a protein band related to 5 protein of estimated molecular weight of about 29 kD was present among the total cellular proteins from yeast strain DCS harboring pRIT10912.

Crude cell extracts from yeast strain DCS harboring either PRIT12363. pRIT10912, or pR!T12894 were prepared as described in Example 12. Protein concentration and AUSRIA activity were measured as described in Example 12. Immunoblot analysis of these extracts was performed as described above.

CsCl equilibrium centrifugation of the crude extracts, (described in Example 12) and measurement of HBsAg related antigenicity by AUSRIA assay in the CsCl fractions, showed the. antigen to band around rho = 1.2

AP 0 0 0 0 5 6

BAD ORIGINAL

138 g/cm . This was confirmed by immunoblot analysis of the CsCl fractions.

These results show that the 32 kD fusion protein produced in yeast strain DC5 cells carrying pRITL2894 is assembled into particles like the 22 nra HBsAg particles derived from human serum. From the level of activity of those particles in the AUSRIA test, and the intensity of the reaction of the 32 kD protein in the immunoblot analysis, it can be concluded that the par10 tides' S determinants are partially obstructed or deformed by the presence of C7 sequences.

From these results it is concluded that yeast cells of strains DC5 and 10S44C transformed with either pRIT12894 or pRIT12898 specifically synthesize a fusion protein of molecular weight of about 32KD or 15 kD respectively, both carrying a S epitope.

It will be appreciated by one of ••.kill in the art that hybrid particles carrying the HIV virus peptide epitopes can be extracted and purified by convention.··!

means from cultures of yeast cells transformed d'.i pRil’12894, pRIT12898 and pRIT12900. Such purified tides may then be formulated in the presence of adju vants such as Al(OH)^ or AlPO^ as vaccines C·· human use. The preferred dose will be in the range >f

1-1000 yg protein and is determined by c onven t ion-· I trials. It will also be understood that the invention as exemplified herein includes other peptide regions from HIV viral proteins which can be fused to the preS2-S region of HBV coding sequences to Com hybrid particles presenting the HIV epitope of interest.

O'

975 MID ΛΓ.Γ'Τ ' ’V ' I’ . ; · ,η; f ” ιπητη Γ’· · ·”··. ,ΛιΙΕ ΪΙΙΛΙ !.·'W7 Cl.Ad'i :2:- 139 -

Claims

1. A recombinant DNA molecule comprising a functional DNA coding sequence fused, in phase, to a pora HBV Pre S2-S protein functional DNA coding coding sequence, PreSl tion of the Pre S2 region of coding sequence wherein the sequence comprises the PreS2 coding sequence or entire PreSl-PreS2 coding sequence, the circumsporozoite protein coding sequence of Plasmodium. or a HIV coding sequence such as a HIV envelope gene sequence such as the HIV C7 protein coding region, the HIV Peptide 121 coding region or the HIV Dreesman peptide coding region.

2. A recombinant vector which comprises a recombinant DNA molecule operatively linked to a regulatory region, wherein the DNA molecule contains a functional DNA coding sequence fused, ..in phase, to a portion of the Pre S2 region of a HBV Pre S2-S protein coding sequence wherein the regulatory region comprises the TDH3 promoter.

AP 0 0 0 o 5 6

3. A recombinant vector which comprises a recant binant DNA molecule operatively linked to a r o<.,u 1 a t o r y region, wherein the DNA molecule contains a function:'. DNA coding sequence fused, in phase, to a portion of the pre S2 region of a HBV Pre 52 5 protein coding sequence wherein the functional DNA coding sequenco comprises the PreS2 coding sequence, PreSl coding sequence or entire PreSl-PreS2 coding sequence, circumsporozoite (CS) protein coding sequence of P1 amod ' η-η or a HIV coding sequence such as a HIV envelope gene sequence such as the HIV C7 protein, the HIV Peptide 121 protein or the HIV Dreesman peptide.

BAD ORIGINAL

140

4. The vector of Claim 3 which comprises a 192 bp Sau3A fragment coding for 16 tetrapeptide repeats of the CS protein of P. falciparum wherein such fragment is one which could be derived from Sau3A digestion of a 1215 bp Stul - Rsa I fragment of plasmid WR20^ containing the P. falciparum CS protein coding sequence minus the first 52 bp of such sequence.

5 Ala Phe Gly Ala Asn Set Asn Asn Pro Asp

TGG GAC TTC AAC CCC ATC AAG GAC CAC TGG

Trp Asp Phe Asn Pro lie Lys Asp His Trp

5 20. A transformed host cell comprising the Pre SI protein coding region of Claim 18.

21. Protein encoded by the Pre SI protein coding region of Claim 18.

22. A vaccine comprising an immunoprotective amount of the protein encoded by the Pre SI protein coding region of Claim 18.

15 23. A HBV Pre S2 protein coding region or a HBV

Pre S2-S protein coding region wherein the Pre S2 portion thereof codes for the following amino acid sequence:

Thr

20 -55 -50 or

Met-Gln-Trp-Asn-Ser-Thr-Ala-Phe-His-Gin-Ala-Leu-Gin-Asp-40 -30

Pro-Arg-Val-Arg-Gly-Leu-Tyr-Phe-Pro-Ala-Gly-Gly-Ser-Sec25 Ser-20

Gly-Thr-Val-Asn-Pro-Ala-Pro-Asn-Ile-Ala-Ser-His-Ile-Ser-10

30 Ser-Ser-Ser-Ala-Arg-Thr-Gly-Asp-Pro-Val-Thr-Asn

24. A recombinant DNA vector comprising the Pre S2 or the Pre S2-S protein coding region of Claim 23 operatively linked to a regulatory region.

AP 0 0 0 o 5 6

146

25. A transformed host cell comprising the Pre S2 or the Pre S2-S protein coding region of Claim 23.

26. Protein encoded by the Pre S2 or the Pre S2-S protein coding region of Claim 23.

27. A vaccine comprising an immunoprotective amount of protein encoded by the Pre S2 or the Pre S2-S protein coding region of Claim 23.

28. A particle comprising protein encoded by the Pre S2-S protein coding region of Claim 29.

29. A vaccine comprising an immunoprotective amount of a particle encoded by the Pre S2-S protein coding region of Claim 23.

30. A micelle containing the particle of Claim 2Q and polysorbate.

31. A vaccine comprising an immunoprotective amount of the micelle of Claim 30.

32. A recombinant DNA vector comprising a Pre SIPre S2-S protein coding sequence operatively linked to a regulatory region wherein the Pre SI - Pre 32 portion of such sequences codes for the following amino acid coding sequence:

PRE-SI REGION

-163

ATG GGG ACG AAT CTT TCT GTT CCC AAC CCT CTG

Met Gly Thr Asn Leu

Ser Val Pro Asn Pro Leu

147

GGA TTC TTT CCC GAT CAT CAG TTG GAC CCT

Gly Phe Phe Pro Asp His Gin Leu Asp Pro

GCA TTC GGA GCC AAC TCA AAC AAT CCA GAT

5 recombinant vector, wherein said vector is according to Claim 5.

5 His He Ser Ser Ser Ser Ala Arg Thr Gly

GAC CCT GTG ACG AAC

Asp Pro Val Thr Asn.

10

5. The vector of Claim 4 which comprises additional Sau3A fragments of CS protein DNA.

6. A recombinant vector which comprises a recombinant DNA molecule operatively linked to a regulatory region, wherein the DNA molecule contains a functional DNA coding sequence fused, in phase, to a portion of the Pre S2 region of a - HBV Pre S2-S protei n coding sequence wherein the functional DNA coding sequence comprises all or part of the Pre S2 protein coding sequence .

7. The vector of Claim 6 wherein the functional· DNA coding sequence comprises the folic .ng amino <- encoding sequence:

PRE-S2 REGION

ATG CAG TGG AAT TCC ACT GCC TTC CAC CAA Met Gin Tr p Asn Set Thr Ala Phe His Gin GCT CTG CAG GAT ccc AGA GTC AGG GGT CTG Ala Leu Gin Asp Pro Arg Val Arg Gly Leu TAT TTT CCT GCT GGT GGC TCC AGT TCA GGA Tyr Phe Pro Ala Gly Gly. Ser Ser Ser Gly

BAD ORIGINAL

141

ACA GTA AAC CCT GCT CCG AAT ATT GCC TCT

Thr Val Asn Pro Ala Pro Asn lie Ala Ser

CAC ATA TCG TCA AGC TCC GCG AGG ACT GGG

8. The vector of Claim 6 wherein the functional

DNA coding sequence comprises all or part of the Pre Sl-Pre S2 protein coding sequence.

9. The vector of Claim 8 wherein the functional

15 DNA coding sequence comprises the following amino acid coding sequence:

PRE-SI REGION

-163 20 ATG GGG ACG AAT CTT TCT GTT CCC AAC CCT Met Gly Thr Asn Leu Ser Val Pro Asn Pro GGA TTC TTT CCC GAT CAT CAG TTG GAC CCT Gly Phe Phe Pro Asp His Gin Leu Asp P r o 25 GCA TTC GGA GCC AAC TCA AAC AAT CCA GAT Ala Phe Gly Ala Asn Ser Asn Asn Pro Asp TGG GAC TTC AAC CCC ATC AAG GAC CAC TGG 30 Trp Asp Phe Asn Pro He Lys Asp His Tr p CCA GCA GCC AAC CAG GTA GGA GTG GGA GCA Pro Ala Ala Asn Gin Val Gly Val Gly Ala

APO 0 0 0 5 6

142

TTC GGG CCA GGG CTC ACC CCT CCA CAC GGC Phe Gly Pro Gly Leu Thr Pro Pro His Gly GGT ATT TTG GGG TGG AGC CCT CAG GCT CAG Gly He Leu Gly Trp Ser Pro Gin Ala Gin GGC ATA TTG ACC ACA GTG TCA ACA ATT CCT Gly He Leu Thr Thr Val Ser Thr He Pro CCT CCT GCC TCC ACC AAT CGG CAG TCA GGA Pro Pro Ala Ser Thr Asn Arg Gin Ser Gly AGG CAG CCT ACT CCC ATC TCT CCA CCT CTA Arg Gin Pro Thr Pro I le Ser Pro Pro Leu AGA GAC AGT CAT CCT CAG GCC Arg Asp Ser His Pro Gin Ala

PRE-S2 REGION

-55 ATG CAG TGG AAT TCC ACT GCC TTC CAC CAA Met Gin Trp Asn Ser Thr Ala Phe His Gin GCT CTG CAG GAT CCC AGA GTC AGG GGT CTG Ala Leu Gin Asp Pro Arg Val Arg Gly Leu TAT TTT CCT GCT GGT GGC TCC AGT TCA GGA Tyr Phe Pro Ala Gly Gly Ser Ser Ser Gly ACA GTA AAC CCT GCT CCG AAT ATT GCC TCT Thr Val Asn Pro Ala Pro Asn He Ala Ser CAC ATA TCG TCA AGC TCC GCG AGG ACT GGG His He S e r Ser Ser Ser . Ala Arg Thr Gly

143

GAC CCT GTG ACG AAC Asp Pro Val Thr Asn.

10 CCA GCA GCC AAC CAG GTA GGA GTG GGA GCA

Pro Ala Ala Asn Gin Val Gly Val Gly Ala

TTC GGG CCA GGG CTC ACC CCT CCA CAC GGC

Phe Gly Pro Gly Leu Thr Pro Pro His Gly

GGT ATT TTG GGG TGG AGC CCT CAG GCT CAG

Gly lie Leu Gly Trp Ser Pro Gin Ala Gin

GGC ATA TTG ACC ACA GTG TCA ACA ATT CCT

20 Gly lie Leu Thr Thr Val Ser Thr lie Pro

CCT CCT GCC TCC ACC AAT CGG CAG TCA GGA

Pro Pro Ala Ser Thr Asn Arg Gin Ser Gly

25 AGG CAG CCT ACT CCC ATC TCT CCA CCT CTA

Arg Gin Pro Thr Pro He Ser Pro Pro Leu

AGA GAC AGT CAT CCT CAG GCC

Arg Asp Ser His Pro Gin Ala

PRE-S2 REGION

ATG CAG TGG AAT TCC ACT GCC TTC CAC CAA 35 Met Gin Trp Asn Ser Τΐις Ala Phe His Gin

AP000056

148

GCT Ala CTG Leu CAG Gin GAT Asp CCC Pro AGA Arg GTC Val AGG Arg GGT Gly CTG Leu TAT TTT CCT GCT GGT GGC TCC AGT TCA GGA 5 Tyr Phe Pro Ala Gly Gly Ser Ser Ser Gly ACA GTA AAC CCT GCT CCG AAT ATT GCC TCT Thr Val Asn Pro Ala Pro Asn He Ala Ser 10 CAC ATA TCG TCA AGC TCC GCG AGG ACT GGG His lie Ser Sec Ser Ser Ala Arg Thr Gly GAC CCT GTG ACG AAC Asp Pro Val Thr Asn

33. A yeast host cell transformed with the vector of Claim 32.

34. Protein prepared by the PreSL-PreS2-S sequence 20 of the vector of Claim 32.

35. A vaccine comprising an immunoprotective amount of the protein of Claim 34.

25

36. A particle comprising protein encoded by a

PreSl-PreS2-S protein coding region.

37. A vaccine comprising an immunoprotective amount of the particle of Claim 36.

38. A micelle comprising the particle cf Claim 3 and polysorbate.

39. A vaccine comprising an immunoprotective 35 amount of the micelle of Claim 38.

bad original

149

40. A Pre Sl-Pre S2 protein coding sequence which

comprises the following amino acid sequence : Pre SI Region 5 -163 ATG GGG ACG AAT CTT TCT GTT CCC AAC CCT Met Gly Thr Asn Leu Ser Val Pro Asn Pro GGA TTC TTT CCC GAT CAT CAG TTG GAC CCT 10 Gly Phe Phe Pro Asp His Gin Leu Asp Pro GCA TTC GGA GCC AAC TCA AAC AAT CCA GAT Ala Phe Gly Ala Asn Ser Asn Asn Pro Asp 15 TGG GAC TTC AAC CCC ATC AAG GAC CAC TGG Trp Asp Phe Asn Pro He Lys Asp His Trp CCA GCA GCC AAC CAG GTA GGA GTG GGA GCA Pro Ala Ala Asn Gin Val Gly Val Gly Ala 20 TTC GGG CCA GGG CTC ACC CCT CCA CAC GGC Phe Gly Pro Gly leu Thr Pro Pro His Gly GGT ATT TTG GGG TGG AGC CCT CAG GCT CAG 25 Gly He Leu Gly Trp Ser Pro Gin Ala Gin GGC ATA TTG ACC ACA GTG TCA ACA ATT CCT Gly lie Leu Thr Thr Val Ser Thr He Pro 30 CCT CCT GCC TCC ACC AAT CGG CAG TCA GGA Pro Pro Ala Ser Thr Asn Ar g Gin Ser Gly AGG CAG CCT ACT CCC ATC TCT CCA CCT CTA Ar g Gin Pro Thr Pro lie Ser Pro Pro Leu

AP000056

150

AGA GAC AGT CAT CCT CAG GCC Arg Asp Ser His Pro Gin Ala Pre S2 Region -55 ATG CAG TGG AAT TCC ACT GCC TTC CAC CAA Met Gin Tr p Asn Ser Thr Ala Phe His Gin GCT CTG CAG GAT CCC AGA GTC AGG GGT CTG Ala Leu Gin Asp Pro Arg Val Arg Gly Leu TAT TTT CCT GCT GGT GGC TCC AGT TCA GGA Tyr Phe Pro Ala Gly Gly Ser Ser Ser Gly ACA GTA AAC CCT GCT CCG AAT ATT GCC TCT Thr Val Asn Pro Ala Pro Asn lie Ala Ser CAC ATA TCG TCA AGC TCC GCG AGG ACT GGG His lie Ser Ser Ser Ser Ala Arg Thr Gly GAC CCT GTG ACG AAC As p Pro Val Thr Asn

41. A recombinant DNA vector comprising the Pre Sl-Pre S2 protein coding sequence of Claim 40 operati vely linked to a regulatory region.

42. A yeast host cell transformed with the vectr? of Claim 41.

43. Protein encoded by the Pre Sl-Pri coding region of Claim 40.

pro· - 1 :

151

44. A vaccine comprising an immunoprotective amount of protein encoded by the Pre Sl-Pre S2 protein coding region of Claim 40.

5

45. A recombinant DNA vector comprising a Pre Si functional DNA coding sequence - Pre S2-S protein coding sequence operatively linked to an expression control sequence.

10

46. A yeast host cell transformed with the vector of Cla im 45.

47. Protein expressed by the host of Claim 46.

10. A eukaryotic host cell transformed with a

11. The host of Claim 10 which is a yeast cell.

10

12. A method of preparing a hybrid particle containing HBsAg protein which contains and/or presents a peptide encoded by a functional DNA coding sequence which comprises culturing a eukaryotic host cell transformed with a vector in appropriate culture media and

15 isolating the particle from a cell lysate or extract of such host's culture, wherein said vector is according to Claim 5.

13. A particle prepared by the method of Claim 12.

14. A hybrid immunogenic particle comprising HBsAg protein wherein said particle contains and/or presents a peptide encoded by a functional DNA coding sequence.

25 15. A vaccine comprising an immunoprotective amount of a particle according to Claim 14.

16. A micelle comprising the particle according to Claim 14 and polysorbate.

17. A vaccine comprising an immunoprotective amount of a micelle according to Claim 16.

18. A HBV Pre SI protein coding region which codes 35 for the following amino sequence:

AP 0 0 0 o 5 6

BAD ORIGINAL

144

-163

ATG GGG ACG AAT CTT Met Gly Thr Asn Leu

GGA TTC TTT CCC GAT Gly Phe Phe Pro Asp

GCA TTC GGA GCC AAC Ala Phe Gly Ala Asn

TGG GAC TTC AAC CCC Trp Asp Phe Asn Pro

CCA GCA GCC AAC CAG Pro Ala Ala Asn Gin

TTC GGG CCA GGG CTC Phe Gly Pro Gly Leu

GGT ATT TTG GGG TGG Gly lie Leu Gly Trp

GGC ATA TTG ACC ACA Gly lie Leu Thr Thr

CCT CCT GCC TCC ACC Pro Pro Ala Ser Thr

AGG CAG CCT ACT CCC Arg Gin Pro Thr Pro

AGA GAC AGT CAT CCT Arg Asp Ser His Pro

TCT GTT CCC AAC CCT Ser Val Pro Asn Pro

CAT CAG TTG GAC CCT His Gin Leu Asp Pro

TCA AAC AAT CCA GAT Ser Asn Asn Pro Asp

ATC AAG GAC CAC TGG He Lys Asp His Trp

GTA GGA GTG GGA GCA Val Gly Val Gly Ala

ACC CCT CCA CAC GGC Thr Pro Pro His Gly

AGC CCT CAG GCT CAG

Ser Pro Gin Ala Gin

GTG TCA ACA ATT CCT

Val Ser Thr lie Pro

AAT CGG CAG TCA GGA Asn Arg Gin Ser Gly

ATC TCT CCA CCT CTA

He Ser Pro Pro Leu

CAG GCC

Gin Ala

CTG

Leu bad

145

19. A recombinant DNA vector comprising the Pre SI protein coding region of Claim 18 operatively linked to a regulatory region.

15 48. A vaccine comprising an immunoprotective amount of the protein of Claim 47.

d .T ’ '··:’ of January 19·°3,