LV10493B - Hepatitis b virus surface proteins with reduced host carbohydrate content - Google Patents

Abstract

To produce hepatitis B virus (HBV) surface proteins in the form of particles with significantly reduced content of carbohydrates, the DNA responsible for coding the HBV surface proteins is expressed in the recombined yeast shell, which has insufficient ability to turn proteins into glucose. These HBV surface proteins expose the antigen epitope, which is coded by the HBV shell protein gene S domain reading sequence, and contains a significantly reduced amount of incorporated carbohydrates, compared to the HBsAg particles produced in the natural yeast shells. These particles are applied as a vaccine in prophylaxis or in both active and passive treatment for diseases caused by the HBV or other agents serologically related to the HBV.

Description

LV 10493

10 TITLE QF THE INVENTION

EEPATITIS B VĪRUS SURFACE PROTEĪNS WITH REDUCED HOST CARBOHIDRATE CONTENT

15 BACKGROUNP OF THE INVENTION

Hepatitis B virus (HBV) is the infectious aģent responsible for several vārieties of human liver disease. Many individuāls who are infected by HBV suffer through an acute phase of disease, which 20 is followed by recovery. However, a percentage of infected individuāls fail to clear their infection, thereby becoming chronic carriers of the infection. HBV infection is endemic in many parts of the world, with a high ihcidence of infection occurring 25 perinatally from chronically infected mothers to their newborns who themselves often remain 30 - 2 - - 2 - LV10493 chronically infected. The number worldwide has been estimated at over three hundred million. From this pool of carriers, hundreds of thousands die annually from the long-term conseguences of chronic hepatitis B (cirrhosis and/or hepatocellular carcinoma).

The hepatitis B delta vīrus is an aģent which, during coinfection with HBV, is responsible for an acute fulminating disease with a generally fatal resolution. The delta virus does not encode (from its own genetic material) proteīns which serve as the virion envelope; rather, the virus encapsidates with the envelope proteīns encoded by the coinfecting HBV, thereby sharing a close structural and immunologic relationship with the HBV proteīns which are described below. It is unknown at this time whether other infectious aģents share similar relationships with HBV. However, it is clear that proteīns with expanded breadth of serologic re-activity or enhanced immunogenic potency would be useful in systems for diagnosis or prevention (or treatment) of diseases (or infections) by a class of aģents with even slight or partial antigenic cross-reactivity with HBV.

The HB virion is composed of two groups of structural proteins, the core proteins and the envelope or surface proteins. In addition to being the major surface proteins or the virion, i .e., Dane particle, the envelope proteins also are the major constituents of Australia antigen, or 22 nm pārticies. These envelope proteins are the translational products of the large virai open - 3 - - 3 - LV 10493 reading frame (ORF) encoding at least 389 amino acids (aa). This ORF is demarcated into three domains, each of vhich begins with an ATG codon that is capable of functioning as a translational initiation 5 site in vivo. These domains are referred to as preSl (108 aa), preS2 (55 aa), and S (226 aa) in their respective 5’-3' order in the gene. Tīras, these domains define three polypeptides referred to as S or HBsAg (226 aa), preS2+S (281 aa), and preSl+preS2+S 10 (389 aa), also referred to as p24/gp27, p30/gp33/gp36 and p39/gp42 respectively (as well as the major, middle and large proteīns).

The envelope proteins of HBV are glycoproteins with carbohydrate side chains (glycans) 15 attached by N-glycosidic linkages to defined peptide recognition sites. [Heermann ££. &1. , J. Virol. 52., 396 (1984) and Stibbe fit al., J. Virol. 46, 626 (1983)]. Tīras, the HBV polypeptides produced during natūrai infection comprise the species p24/gp27 (the 20 S polypeptide and its glycosylated derivative), gp33/gp36 (the preS2+S polypeptide glycosylated in the preS2 domain only and the same polypeptide glycosylated in the S as well as the preS2 domain), and p39/gp42 (the preSl+preS2+S peptide and its 25 derivative glycosylated in the preSl domain). Currently available plasma-derived vaccines are composed of proteins containing virtually only the S domain (comprising the p24 monomer and its glycosylated derivative gp27), while yeast-derived 30 vaccines successfully developed to datē are composed exclusively of the S polypeptide (comprising exclusively the nonglycosylated p24 species).

The 22 nm HBsAg pārticies, have been purified from the plasma of chronic carriers. In terms of their plasma being particle-positive, these chronic carriers are referred to as HBs+. If 5 infected persons have mounted a sufficient immune response, they can clear the infection and become HBs“. In terms of their formation of antibodies to BBs, these individuāls are denoted anti-HBs+. In this way, anti HBs+ is correlated with recovery from 10 disease and with immunity to reinfection from disease and with immunity to reinfection with HBV.

Therefore, the stimulation or formation of anti-HBs by HB vaccines has been expected to confer protection against HBV infection. 15 This hypothesis has been testable experimentally. Outside of man, the chimpanzee is one of the few species which is fully susceptible to HBV infection , as reflected in quantifiable markers such as HBs+ and elevated serum Ievels of liver 20 enzyme. Chimpanzees have been vaccinated with three doses of purified HBsAg pārticies and then challenged intravenously with infectious HBV. While mock-vaccinated animals have shown signs of acute HBV infection, the HBsAg-vaccinated animals have been 25 protected completely from signs of infection. Therefore, in this experimental system, HBsAg pārticies, composed of p24 (or p24 and p27), have been sufficient to inducē protective immunity.

Spurred by these observations, several manufacturers 30 have produced HB vaccines composed of HBsAg pārticies.

In order to expand the available supply of HB vaccines, manufacturers have turned to recombinant DNA technology to mediate the expression of virai - 5 - - 5 - LV10493 envelope proteins. Among microbial systems, Escherichia coli and f>. cerevisiae have been used most commonly for the expression of many recombinant-derived proteiiis. Numerous attempts to express 5 immunologically active HBsAg pārticies in 33. coli have been unsuccessful. However, £. cerevisiae has shown great versatility in its ability to express immunologically active HBsAg pārticies. These pārticies (composed exclusively of p24), when 10 formulated into a vaccine, have proven capable of fully protecting chi.mpanzees against challenge with live HBV of diverse serotypes. Furthermore, yeast-derived S pārticies are also immunologically active and as effective in preventing disease or 15 infection in human clinical trials as plasma-derived HBsAg [Stevens akai-, JAMA. 257:2612-2616 (1987)]. Therefore, the utility of £. cerevisiae as a host species for directing the synthesis of recombinant HBsAg is established firmly. In addition, expression 20 of human therapeutic aģents and vaccines in yeast can be very useful for product development, since yeast is free of endotoxin, is nonpathogenic to man, can be fermented to industrial scale, and lacks many of the safety concerns which surround the use of continuous 25 mammalian celi lines (many of which are virally transformed, may be tumorigenic in mice and ali of which contain protooncogenes). £. cerevisiae (bakers* yeast) is a eukaryote which is capable of synthesizing glycoproteins. 30 Protein glycosylation in yeast has been the subject of numerous recent review articles [notably: Kukuruzinska gt ai. , Ann. Rev. Biochem.. (1987) 2L6, 915-44; Tannen at ai-, BBA, (1987) 2M, 81-99]. This glycosylation or additiion of glycans to appropriate receptor amino acids (aa) on the polypeptide occurs either at specific serine (Ser) or threonine (Thr) residues (0-linked) or at specified asparagine (Asn) residues (N-linked). The specificity for 0-linked addition at Ser or Thr residues is not clearly understood and is determined empirically on a case-by-case basis.

The signal sequence for N-linked glycosylation is well defined as either of the amino acid sequences Asn-X-Thr or Asn-X-Ser (wherein X is any amino acid). In addition to synthesizing many autologous, native, glycosylated proteins (among them being those called mannoproteins, or mannopeptides), yeast also are capable of glycosylating heterologous or foreign proteins expressed by recombinant technology (if the heterologous protein contains the appropriate glycosylation signal sequence for either N-linked or 0-linked glycosylation).

The preS2+S polypeptides, which are produced during natūrai infection contain no more than two "core" [ca. 3 kilodaltons (kD) in size] N-linked glycans, one in the S region and a second on the Asn at amino acid residue 4 of the preS2 domain. The recognition site in the S domain is not glycosylated in either Recombivax ΞΒ® or in recombinant preS2+S synthesized in yeast. However, the site at amino acid residue 4 of the preS2 domain is recognized and glycosylated by yeast.

The preSl domain contains an N-linked glycosylation site at amino acid residue 4 of the preSl region and a potential site at aa residue 26 for serotype adw. It is readily apparent to those skilled in the art that arguments set forth for preS2 glycosylation also will follow for diverse sequences - 7 - - 7 - LV 10493 in the preS2 region as veli as for those in the preSl and S domains.

Yeast synthesizing recombinant preS2+S add a "core" glycan which is similar to that added to the 5 native polypeptide during virai infection. However, if the yeast host celi is Mwild-typeM for glycosylation containing the full complement of enzymes reguired for native glycosylation which is the case for virtually ali commonly used yeast 10 strains), a significant number of these glycans are extended with a large number of additional mannose residues in a manner identical to that employed by yeast in making its own structural mannoproteins.

This extended addition of the glycan, when it occurs 15 on a foreign gene product such as the preS2+S polypeptide, is referred to as hyperglycosylation.

It is readily apparent to those skilled in the art that arguments set forth for yeast also will extend to other host celis (£.g,., insect, fungi, etc.) which 20 may be subject to divergent glycosylation patterns.

Furthermore, it has been demonstrated that recombinant forms of 22nm pārticies of HBV surface proteins expressed in wild-type yeast host celis, entrap substantial amounts of yeast celi carbohydrate 25 (deriving at least in part from the structural mannoproteins and mannopeptides of the yeast host celi) within the 22nm particle. This entrapped carbohydrate could pose potential problems in that the entrapped carbohydrate may cause the generation 30 of antibodies against yeast carbohydrate moieties on glycosylated proteins, and a vaccine immunogen containing entrapped yeast carbohydrate would react with anti-yeast antibodies present in most mammalian species thereby potentially diminishing its effectiveness as an immunogen and vaccine.

Hyperglycosylation and entrappment of complete mannoproteins and mannopeptides may be eliminated or glycosylation limited in HBV preS and S 5 polypeptides, and their corresponding pārticies, by any of the following approaches.

Firstly, N-linked hyperglycosylation may be prevented or limited during growth of the recombinaņt host through the presence in the growth medium of an 10 exogenous aģent (e.£., tunicamycin). Seeondly, polypeptides, from recombinant or natūrai sources may be deglycosylated either chemically (£.£. anhydrous trifluoromethane-sulfonic acid or anhydrous hydrogen fluoride) or enzymatically (e.g., with N-glycanase, 15 Endo-F or Endo-H) or physically (£.£. sonication). Thirdly, the recognition site for glycosylation may be changed or deleted by mutagenesis at the DNA Ievel, such that core glycosylation, and thereby hyperglycosylation as well, is prevented. Such 20 modified preS+S ORF's in which the glycosylation recognition sequence has been altered (directed by suitable promoters active in yeast) have been transformed into yeast host celis. The resultant preS+S polypeptides lack glycosylation. Fourthly, 25 host celis may be identified which lack critical enzymes reguired for glycosylation, which illustrates the present invention without however limiting the same thereto. One such yeast strain has been identified (mnn9- mutant) [Ballou, L. al., (1980), 30 J.Biol.Chem., 255. pp 5986-5991] which lacks a critical enzyme in the glycosylation pathway necessary for the elongation (hyperglycosylation) of the N-linked glycans; Chemical studies indicate that this mutant makes mannoproteins vithout outer-chain - 9 - - 9 - LV10493 mannose residues and containing only the "core" carbohydrate [Ballou, C.E. al., (1986), Proc.Natl.Acad.Sci .TJ.S.A.. 81, pp 3081-3085; Tsai, P. fit al..f (1984), J.Biol.Chem. . 21i, pp 3805-3811]. 5 The ORE for the S or preS+S polypeptide (transcription directed by suitable promoters active in yeast) has been used to transform such mnn9-mutant yeast. The resulting preS+S polypeptide contains only "core" glycosylation and lacks 10 hyperglycosylation.

Although the S polypeptides are neither glycosylated nor hyperglycosylated when expressed in yeast, pārticies composed therefrom contain significant Ievels of entrapped carbohydrate deriving 15 from yeast mannopeptide. Therefore, the expression of S polypeptides as well as preS containing polypeptides in yeast celis which cannot hyperglycosylate results in decreased Ievels of carbohydrate in the expressed 22nm pārticies. 20 S.· cerevisiae has shown great versatility in its ability to express immunologically active 22 nm pārticies. These pārticies, when formulated into a vaccine, have proven capable of fnlly protecting chimpanzees against challenge with live HBV. 25 Furthermore, yeast-derived HBsAg has been effective immunologically in human clinical trails as plasma-derived HBsAg. Therefore, the utility of S.. cerevisiae as a host species for directing synthesis of recombinant HBsAg is established firmly. 30 In a variety of recombinant microbial expression systems, the synthesis of many different polpeptides has been shown to be deleterious to the host celi. As a conseguence, there is selective pressnre against the expression of such polypeptides, such that the only celis which accumulate in a scale-up of such a recombinant culture are those which have ceased to express the foreign polypeptide or express so little of the foreign polypeptide that 5 the culture becomes an uneconomical source of that polypeptide. In some cases, the deleterious effect is so strong that when expression is driven by a strong constitutive promoter, newly transformed celis fail to propagate and form colonies on selective 10 plates. These deleterious effects can be overcome by using an inducible promoter to direct the synthesis of such polypeptides. A number of inducible genes exist in £. cerevisiae. Four well-characterized inducible genetic systems are the galactose (GAL) 15 utilization genes, the alcohol dehydrogenase 2 (ΑΡΗ2) gene, the alpha mating factor gene, and the pho5 gene. £· cerevisiae has 5 genes which encode the enzymes responsible for the utilization of galactose as a carbon source for growth. The GALI. GAL2. GAL5t 20 GAL7 and GAL10 genes respectively encode galactokinase, galactose permease, the major isozyme of phosphoglucomutase, a-D-galactose-l-phosphate uridyltransferase and uridine diphospho-galactose-4-epimerase. In the absence of galactose, 25 very little expression of these enzymes is detected.

If celis are grown on glucose and then galactose is added to the culture, these three enzymes are induced coordinately, by at least 1,000-fold, (except for GAL5. which is induced to about 5 fold) at the Ievel 30 of RNA transcription. The GALI GAL2. GAL5. GAL7 and GAL10 genes have been molecularly cloned and - 11 - - 11 - LV 10493 sequenced. The regulatory and promoter sequences to the 5' sides.of the respective coding reģions have been placed adjacent to the coding reģions of the lacZ gene. These experiments have defined those 5 promoter and regulatory sequences which are necessary and sufficient for galactose induction. » £. cerevisiae also has 3 genes, each of which encodes an isozyme of alcohol dehydrogenase (ADH). One of these enzymes, ADHII. is responsible 10 for the ability of cerevisiae to utilizē ethanol as a carbon source during oxidative growth.

Expression of the ADH2 gene, which encodes the ADHII isozyme, is catabolite- repressed by glucose, such that there is virtually no transcription of the ADH2 15 gene during fermentative grovth in the presence of glucose Ievels of -0.1% (w/v). Hpon glucose depletion and in the presence of non-repressing carbon sources, transcription of the ADH2 gene is induced 100- to 1000-fold. This gene has been 20 molecularly cloned and sequenced, and those regulatory and promoter sequences which are necessary and sufficient for derepression of transcription have been defined.

Alpha mating factor is a sex pheromone of £. 25 cerevisiae which is required for mating betveen MATa and MATa celis. This tridecapeptide is expressed as a prepropheromone which is directed into the rough endoplasmic reticulum, glycosylated and proteolytically processed to its final mature form 30 which is secreted from celis. This biochemical pathway has been exploited as an expression strategy for foreign polypeptides. The alpha mating factor gene has been molecularly cloned and its promoter with pre-pro-leader seguence has been utilized to express and secrete a variety of polypeptides. Likewise, the pho5 gene promoter has been shown to be inducible by low phosphate concentrations and this 5 also has utility for physiologically regulated expression of foreign proteins in yeast.

As expected by their traversal of the rough endoplasmic reticulum and Golgi apparatus, foreign proteins can undergo both N- and 0-linked 10 glycosylation events. The alpha mating factor promoter is active only in celis which are phenotypically g. There are 4 genetic loci in £. cerevisiae. known as SIR. which synthesize proteins required for the repression of other normally silent 15 copies of a and g Information.

Temperature-sensitive (ts) lesions which interfere with this repression event exist in the gene product of at least one of these loci. In this mutant, growth at 35eC abrogates repression, resulting in 20 celis phenotypically g/g in which the alpha mating factor promoter is inactive. Upon temperature shift to 23eC, the celis phenotypically revert to g such that the promoter becomes active. The use of strains with a ts SIR lesion has been demonstrated for the 25 controlled expression of several foreign polypeptides.

OBJECTS OF THE INVENTION

It is an object of the present invention to 30 provide a HBV surface protein which forms pārticies with substantially reduced entrapped carbohydrate content. It is another object of the - 13 - - 13 - LV10493 present invention to provide a method of producing in a yeast host, EBV surface proteins which form pārticies and which contains substantially reduced entrapped carbohydrate content. An additional object 5 of the present invention is to provide a vaccine against HBV comprising the HBV surface protein pārticies with substantially reduced entrapped carbohydrate content for both active and passive treatment of prevention of disease and/or infections 10 caused by HBV or other aģents serologically related to HBV. A further object of the present invention is to provide conditions for the large scalē growth of recombinant host celis and the purification of the recombinant HBV surface proteins. These and other 15 objects of the present invention will be apparent from the following description.

STMMARY OF THE INVENTION 20 HBV surface proteins have been expressed at high yield in a recombinant yeast host which is genetically deficient in its ability to glycosylate proteins. The expression of HBV surface proteins in yeast celis results in the formation of the 25 characteristic pārticies. Formation of these pārticies in yeast celis results in the entrapment of yeast celi substances within the pārticies. TJsing "wild-typeM yeast host celis substantial amounts of yeast celi carbohydrate may become entrapped within 30 the HBsAg pārticies. In order to circumvent the production of a HBV vaccine consisting of pārticies which contain substantial amounts of carbohydrate, the HBV surface proteins were produced and purified from a recombinant yeast host which is genetically deficient in its ability to glycosylate proteīns.

The HBV surface proteins produced by such a host form pārticies which contain substantially less carbohydrate than pārticies produced in wild-type yeast celis. These HBV surface proteins are useful as a vaccine for the treatment and/or prevention of HBV related infections, and as an antigen for immunologic diagnosis with reduced reactivity with naturally occuring anti-yeast antibodies.

BRIEF DESCRIPTION OF THE DRAWTNG FIGURĒ 1 shows schematically plasmid pCl/lpGALlOHBsAg-tADH-1 which contains the pGALlO promoter driving transcription of the HBsAg ORF, followed by the tADHl terminator, and the selectable marker LEU2+.

DĒTAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method for the preparation of HBV surface protein pārticies which contain substantially reduced amounts of entrapped carbohydrate, for use as a vaccine against HBV.

Dane pārticies (serotype adw) were utilized as the source of HBV nucleic acid for the isolation of the virai ORFs. It is readily apparent to those skilled in the art that this invention extends to the use of nucleic acid from HBV strains or related viruses with other serologic reactivities which derive from virai genetic diversity. The endogenous polymerase reaction was employed in order to producē - 15 - - 15 - LV10493 covalently-closed circular double-stranded DNA of the HBV genome from the nicked and gapped nucleic acid form that natively resides in the HB virion. The DNA was isolated, digested to completion with EcoRI. and 5 cloned into the EcoRI site of pBR322, thus generating pHBV/ADW-l. The recombinant plasmids containing the HBV genome in a circularly permuted form at the EcoRI site of the PreS region were selected. The complete ORF encoding the 55 amino acids of the preS2 region, 10 and the 226 amino acids of the S region was constructed first by purifying the 0.8 kilobase pair (kbp) fragment obtained following digestion of pHBV/ADW-l with EcoRI and Accl; this fragment encodes the preS2+S polypeptide lacking only the initiation 15 codon, the amino-terminal 3 amino acids, the carboxy-terminal 3 amino acids, and the translational terminator codon.

Oligonucleotides were synthesized and ligated to this fragment, converting it to a HindIII 20 fragment containing a 10 bp yeast-derived non-translated 5’ flanking seguence and the complete preS2+S ORF was chosen such that the termination codon was directly joined to a natūrai HindIII site in the ADH1 transcriptional terminator, thus creating 25 a completely native yeast-derived junction without any additional intervening bases. It is readily apparent to those skilled in the art that for expression of HBV surface and related proteīns, any suitable yeast-active transcriptional terminator may 30 be substituted for ADH1.

The 5' flanking sequence for the construction ACAAAACAAA (SEQIDN0: 1) was chosen to 1 10 correspond to that for the non-translated leader (NTL) of the yeast gene GAP63 (GAP) [Holland, J..

Biol. Chem.. 225. 2596 (1980)] and is also a consensus for the GAP gene family. The construction was made in such manner as to join the NTL directly to the initiation codon of the preS2+S ORF without the intervention of any additional bases. Therefore, it is readily apparent to those skilled in the art that, for expression of HBV surface proteīns, the selection of NTL sequences extends to other sequences which result in suitable expression Ievels. DNA sequence analysis revealed 2 base substitutions which resulted in amino acid differences from the preS2+S sequence encoded by the DNA of pHBpreSGAP347/19T [Valenzuela £t al., Biotechnologv. 3.(4), 317-320 (1985)]. In order to evaluate indentical polypeptides for both constructions, these nucleotide substitutions, which were T instead of C at base 64 of the 846 bp ORF of HBV preS2+S (encoding Phe rather than Leu) and C instead of A at base 352 (encoding His rather than Gln) were changed by site-directed mutagenesis [Zoller e£ al·» Nucleic Acids Research 10:6487-6500 (1982)]. The encoded amino acid sequence for the optimized construction then was verified. It is readily apparent to those skilled in the art that this invention is not limited to this sequence and extends to any sequence wherein the DNA encodes a polypeptides with HBV-related antigenicity.

The large DNA fragment of 3.3kbp which contains pUC19 and the HBsAg coding region was separated from the preS2 encoding DNA fragment after digestion with EcoRI and StvI. and purified by preparative agarose gel electrophoresis. A synthetic DNA oligonucleotide was then ligated with the -17- -17- LV10493 pTJC19-HBsAg fragment. This synthetic DNA oligonucleotide contains 5’ EcoRI and 3' StvI sticky ends as well as providing a HindIII site immediately following the 5' EcoRI site. In addition, the 5 synthetic DNA oligonucleotide contains the HBsAg ATG codon plus the 9 upstream nucleotides and the 21 dbVnstream nucleotides including the StvI site.

This oligonucleotide rebuilds the complete coding region of the HBsAg and allows its subseguent 10 removal intact, from the pUC19 based vector by digestion with HindIII.

The pUC19-HBsAg DNA fragment with the ligated synthetic DNA olgonucleotide described above was used to transform E. coli. Recombinant plasmids 15 were selected which possess the complete reconstructed HBsAg coding region. The complete HBsAg open reading frame (ORE) was removed from the recombinant plasmid by digestion with HindIII followed by isolation and purification of the 0.7kbp 20 HBsAg DNA by preparative agarose gel electrophoresis for cloning into a GAL10 promoter expression vector.

The expression cassette (pGALlO-tADHl) drives expression of foreign genes inserted at a unique HindIII site down stream from the 25 galaetose-inducible GAL10 promoter. The HBsAg ORE (with HindIII termini) described above was ligated into the HindIII site of the vector. This expression cassette was inserted between the Sphl sites of the 1. coli shuttle vector pCl/1 (Beggs, supra) and this 30 vector was introduced into £. cerevisiae strains CF52 or CF54 and transformed clones were selected.

Folloving mutagenesis, the fragment encoding either S or preS+S described above was used to construct an expression cassette, as described p.reviously [Kniskern et &1., Gene 46:135-141. (1986)], which was composed of: (a) £&. 1.1 kbp of the GAP491 promoter, (b) a 10 bp yeast-derived flanking sequence, (c) 1230bp of the virai ORF for 5 preSl+preS2+S or 846 base pairs of the virai ORF for preS2+S or 681 bp of the virai ORF for S, and (d) ca. 0.4 kbp of the yeast APEI terminator.

Three different expression vectors were used to construct HBsAg expression cassettes. The GAP 491 10 promoter expression cassette described previously [Kniskern £t ai-, 1986 Gene 46 ppl35-141], is composed of about 1.1 kbp of the glyceraldehyde-3-phosphate dehydrogenase (ĢAPDH) promoter and about 350bp of the yeast alcohol dehydrogenase I (ADH1) 15 terminator in a pBR322 backbone, with a unique HindIII site between the promoter and terminator.

The HBsAg ORF from Example 2 was ligated in the unique HindIII site, and its presence and orientation confirmed by restriction endonuclease analyses and 20 Southern blot.

Alternately the (0.5kbp) GAL10 promoter (Schultz et. al., 1987, Gene. 54. ppll3-123) was substituted for the l.lkbp GAP promoter in the above construction, or the (1.25 kbp) ADH2 promoter 25 (Kniskern £t al·, 1988 Hepatologv 8, 82-87) was substituted for the GAP promoter (see Figurē 1).

In each case, the expression cassette containing the specific promoter, the HBsAg ORF, and the ADHl terminator was cloned into the shuttle 30 vector pCl/1 (Beggs, supra: Rosenberg, fit. ai·. supra) to create a yeast expression vector which was then used to transform S.. cerevisiae as described below. These transformants were established as frozen stocks for evaluation and subsequent experimentation. - 19 - - 19 - LV 10493

Parental strain CF52 was obtained as follows: The α mating type strain CZ5/LB347-1C Cmnn9~. SUCZ") was mated with the a type strain 2150-2-3 (l£u2*“, adel") by mixing the strains on a TEHD complete media 5 plate. To select for diploids, the mated strains were replica plated onto leu“ minimal medium containing 27« sucrose as the sole carbon source.

After isolating single colonies, the diploids were sporulated, and asci were dissected by Standard 10 techniques. The ΚΗΥ-107 strain was isolated as a single spore and characterized as cir*. adel*, leu2~, and mnn9~ (by Schiff stain technique). ΚΗΥ107 (cir 0) was derived from strain ΚΗΥ107 (£lx*) as described by Broach fMethods in 15 Enzvmologv. 101. Part C. pp 307-325, (1983)]. The cured strain was made nra3~ by integrating a disrupted nra3 gene. The resulting strain, KHY-107ura3A, was grown in rich media to allow the accumulation of spontaneous mutations and a 20 canavanine resistant mutant was selected. The mutant strain, CF55, was shown by complementation tests to be canl~. The GAL10pGAL4 expression cassette was integrated into the HIS3 gene of CF55 (Methods in Enzvmologv. 1990, 185 pp297-309) to yield the final 25 host strain CF52 (Mata leu2-2,112 ura3A canl his3A::GAL10pGAL4-URA3, cir°). These transformants were established as frozen stocks for evaluation and subsequent experimentation.

Recombinant yeast from the frozen stocks was 30 grown in TEHD medium [Carty et al·, J.* Industrial Micro.. 2, 117-121, (1987)]. After growth to stationary phase, yeast celis were harvested.

Lysates were prepared, resolved by sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotted with antibodies to HBsAg. One polypeptide was found with molecular weight of about 24-kD in accord with the predicted molecular weight of the translational product of the 5 S ORF. Furthermore, lysates of recombinant, but not parental, yeast were positive for S by radioimmunoassay (AusriaR). Electron microscopic examination of partially purified yeast lysates showed high densities of typical HBsAg pārticies. 10 The yeast-derived promoter initiates transcription of the HBsAg and related genes. Therefore, it is readily apparent to those skilled in the art that any yeast-active promoter sequence (fi.g. including by not limited to GALI. GAL10. ADH2 15 Pho5. etc.) may be substituted for the GAP491 promoter. It is also readily apparent to those skilled in the art that a suitable assay system, £.g., immunoblot or RIA or enzyme-linked immunoassay (EIA), should be utilized in order to assay 20 expression of HBsAg and related polypeptides in this system, such that the time of harvesting of the culture for attaining a maximal yield can be optimized.

The GAP491 promoter has been useful for the 25 expression in yeast of several foreign proteins, including HBsAg [Bitter ai-, Gene. 32:263-274. <1984); Wampler al., Proc. Nat. Acad. Sci. USA. 82:6830-6834, (1985)]. Based upon our previous results of expressing HBcAg to about 40% of soluble 30 yeast protein (Kniskern et. al.. supra). we have used this promoter to drive the expression of HBsAg and related proteins in suitable yeast host celis.

It is readily apparent to those skilled in - 21 - - 21 - LV10493 the art that the selection of a suitable yeast strain for expression of HBV surface proteins encompasses a vide variety of candidates. Suitable yeast strains include but ar not limited to those vith genetic and 5 phenotypic characteristics such as protease deficiencies, and altered glycosylation capabilities.

In order to control and define the glycosylation of recombinant yeast-expressed HBV proteins, £. cerevisiae strain CF52 (Mata l£n2-2, 112 10 ura3A canl his3A:: GAL10pGAL4-ura3. cir°) vhich vas constructed as described above.

The expression plasmid pCl/lpGAL10HBsAg-tADH-l vas used to transform CF52 (Mata leu2-2. 112 ura3A canl his3A:: 15 GALI0pGAL4-ura3. cir0). Transformed clones were selected on minimal medium (leu-) containing 1M sorbitol. These cloned transformants vere established as frozen stocks in 177» glycerol for subsequent evaluation and further experimentation. 20 To provide a glycosylation wild-type control, the expression plasmid vas also used to transform yeast strain CF54, vhich vas isolated by established techniques from strain CF52, and vhich is a spontaneous revertant to MNN9+ (and thus is 25 vild-type for glycosylation but othervise is of identical genotype to strain CF52). 30

Transformed clonal isolates were established as frozen stocks in 177» glycerol for subsequent evaluation and further experimentation.

Clones of transformed yeast containing the expression plasmids were plated onto leu“ selective agar plates (containing 1M sorbitol for mnn9-transformants) and incubated at 30°C for 2-3 days. These yeast were inoculated into 5-7 mL cultures of complex TEHD (Carty al., supra) medium (containing 0.1-1M sorbitol), plus 2% galactose for GAL10 based plasmids, and the cultures were incubated at 30°C with aeration for 12-18 hours. Flasks containing 50 mL complex TEHD medium with 1M sorbitol (hereafter called YEHDS) were inoculated from the above cultures (to an inital A^qq =0.1) and were incubated at 30eC with shaking (350 rpm) for 48-72 hours to a final A6qo of 10-16· Samples of 10 A^q0 units were dispensed into tubes, and the yeast celis were pelleted by centrifugation at 2000xg for 10 minūtes. Samples either were assayed directly or stored frozen at -70eC. At the time of assay, the pellets were resuspended in 0.4 mL of phosphate- buffered saline (PBS) containing 2mM phenylmethyl sulfonyl fluoride (PMSF) and transferred to 1.5 mL Eppendorf tubes. Teast celis were broken by: 1) the addition of 200-300 mg of washed glass beads (0.45 mm) and agitation on a vortex mixer for 15 minūtes, 2) addition of TRITON Χ-100 to 0.57», 3) agitation on the vortex mixer for 2 minūtes, and 4) incubation at 4eC for 10 minūtes. Cellular debris and glass beads were removed and the supernatant assayed for protein - 23 - - 23 - LV 10493 [by the method of Lowry fii ai., J. Biol. Chem., 193, 265, (1951)] and RIA specific for preS2+S [Hansson sL al.. Infect. Iirominnl. 26: 125-130, (1979), Machida fii ai-, Gastroenterologv 86: 910-918, (1984)] or S (AUSRIAr).

Clones of transformed yeast mnn9- containing tlie expression plasmids were plated onto (leu-) selective agar plates containing 1M sorbitol and incubated at 30°C for 2-3 days. These yeast were inoculated into 5-7 mL cultures of complex IEHDS media (plus 27«. galactose for GAL10 promoter plasmids), and the cultures were incubated from the above cultures (to an initial Agoo = 0*1) and were incubated at 30eC with shaking (350 rpm) for 48-72 hours to a final A^qq of 10-16. Triplicate samples of 10 A^oo units were dispensed into tubes, and the yeast celis were pelleted by centrifugation at 2000xg for 10 minūtes. Samples either were assayed directly as described above or stored frozen at ~70eC.

Immunoblot analysis of the polypeptide derived from ali recombinant clones described above, in host celis with the mnn9- phenotype, showed one band with apparent molecular size of about 24-kD.

For recombinant proteins, the qualitative and quantitative glycosylation patterns are a function of and largely dependent upon the host celi species, and within a species upon the celi line. It is thus readily apparent to those skilled in the art that the selection of a host strain extends to species and celi lines other than £. cerevisiae for which mutations in enzymes in the glycosylation pathway may be identified. It is also readily apparent to those skilled in the art that selection of host strains of £. cerevisiae extends to ali strains in which mutations in enzymes of the glycosylation pathway may be identifed.

The transformed clones were then screened for the presence of the HBsAg DNA and expression of 5 p24 HBsAg. Celis were grown in TEHDS medium (also containing galactose for the GAL10 promoter plasmids to inducē expression following glucose depletion). Lysates were prepared, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 10 and Western blotted to nitrocellulose. A p24 product was found to be specific to S protein by virtue of its presence only in induced transformants and its reactivity with anti-p24 serum. One of these clones was selected for further analysis. Furthermore, 15 lysates of transformants, but no parental £. cerevisiae. were positive for HBsAg by radioimmunoassay.

This highlights the utility of the expression vector which utilizēs the GAL10 promoter 20 to direct the expression of HBsAg and related surface proteins in £. cerevisiae. It is readily apparent to those skilled in the art that the regulatable GAL10 promoter, or GALI. GAL2. GAL7 or MELI promoters which function in an indistinguishable manner, enable the 25 growth of a recombinant £. cervisiae culture to be scaled up to a production-scale volume before synthesis of the recombinant protein is initiated, such that negative effects on the host celi are minimized. Moreover, it is readily apparent to those 30 skilled in the art that an expression vector containing another regulatory promoter, including but not limited to AD52 and alpha mating factor, physiologically inducible or derepressible by other means, can be utilized to direct expression of S and - 25 - - 25 - LV 10493 preS-containing peptides. Furthermore, it is readily apparent to those skilled in the art that a constitutive promoter less potent than GAPDHT including but not limited to CYC1. drives expression 5 of S and pre-S-containing polypeptides to a lower percentage of celi protein, such that the negative physiological effects of overexpression would be obviated. It is readily apparent to those skilled in the art that a suitable assay system, £.g., V/estern 10 blot or radioiranmnoassay, should be utilized in order to assay expression of S and pre-S-containing polypeptides in this system so that the time of harvesting of the culture for attaining a maximal yield can be optimized. 15 An immune-affinity column, bound with goat antibodies which recognize the particulate form of HBsAg, has been utilized to purify S and S-related proteins from transformed £. cerevisiae. The eluted product is positive for HBsAg by radioimnmnoassay, 20 and is of particulate form in electron microscopy.

Such a particulate form which contains both HBsAg and pre-S antigens or HBsAg alone is effective as a HBV vaccine and diagnostic reaģent.

Yeast celis transformed with expression 25 vectors coding for a hepatitis B virus surface protein or variants thereof are grown and harvested. The celis may be stored if desired by washing the 30 celis in a buffer solution, £.g_. PBS, and forming a celi paste which is typically stored frozen at -70°C.

Purification of HBsAg and related proteins typically begins as follows. A batch of fresh or 5 frozen celi paste is suspended in a buffer, preferably TRĪS, at a high pH ranging between about 8.5 and about 11.0, preferrably about 10.5 (the buffer may also contain suitable protease inhibitors). The celis are then disrupted, 10 preferably by mechanical means. The gentle bead breakage method of disruption has been found to be unsuitable for scale-up use. Disruption by a high pressure homogenizer (about 10,000 to 20,000psi, using a Gaulin or Stansted homogenizer) is preferred 15 because of its rapid and efficient operation.

Disruption of the yeast celis results in a crude extract. The crude extract is then pH adjusted. The pH is adjusted to within the range of 8.0 to 11.0, with 10.5 being preferred. 20 It may be desired at this point to add a dētergent to the crude extract. The addition of a detergent will facilitate the separation of yeast celi membranes from unwanted Cellular debris. It has been shown that preS2+S protein, as well as other 25 forms of the surface proteins, may associate with yeast celi membranes. A variety of neutral or non-ionic detergents can be used, including but not limited to detergents of the TRITON-N series, TRIT0N-X series, BRIJ series, TWEEN series or EMASOL 30 series, deoxycholate, octylglucopyranoside or NONIDET-Np-40. Zwitterionic detergents such as CHAPS or CHAPSO are also useful and suitable aģents.

If a detergent is to be used, the preferred - 27 - - 27 - LV10493 detergent is TRITON Χ-100 at a concentration of about 0.5%. It must be stressed that the method of this invention does not reguire detergent use at this step and the use of detergents is optional. 5 The extract then may be heat treated if protease inhibitors are not present during lysis.

Heat treatment is effective over a range of temperatures and for a range of treatment duration. Typically a temperature range of 45eC to 60eC is 10 used, with 50°C as the preferred temperature. The duration of heat treatment typically ranges betveen 20 to 40 minūtes with 30 minūtes as the preferred time. The extract is heat treated in a suitable vessel and the vessel is immersed in a heated bath, 15 or a heat exchanger is used. The material is then cooled to about 10°C, preferably by placing it into an ice-water bath or by using a heat exchanger. It will be readily apparent to those skilled in the art that, according to the method of this invention, the 20 order in which the heat treatment and the debris removal steps are done may be reversed without significant effect on the result of this procedure. Alternatively, whole yeast celis can be heated in a neutral pH buffer, disrupted and detergent added as 25 described above.

Removal of cellular debris from the heat treated crude extract is necessary to prevent physical occlusion during subsequent purification steps. Debris can be removed by centrifugation, 30 microfiltration, or filtration producing a clarified extract. Centrifugation and microfiltration are the most preferred methods. Centrifugation can be done for varying lengths of time at different centrifūgai forces. Centrifugation at about 3,000 x g for 15 mimites at 4°C has been found adeguate. It may also be advantageous to dilute the extract before centrifugation to reduce the typically viscous nature of a crude yeast celi extract. Dilution will not alter any subseguent steps of this procedure.

Microfiltration has an advantage in that filtration and dialysis can be performed simultaneously. Several types of microfiltration units are suitable for use in this step, £.£. KROSFLO by Microgon Inc. or any variety of hollow fiber cartridges by Amicon or A/G Technology. The preferred microfiltration method is to pass the extract through Prostak Durapore (Millipore) membrane, plate and frame microfiltration unit with a pore size of about 0.1 microns to 0.2 microns, at an inlet pressure of about 2 to 7 psi, using a buffer consisting of about 0.1M TRĪS, pH about 10.4 and about 0.1% TRIT0N Χ-100.

The supernatant from centrifugation or the . filtrate from microfiltration may be concentrated prior to the next step of this procedure. Concentration can be achieved by several methods including, but not limited to, dialysis, filtration, lyophilization, ultrafiltration and diafiltration.

The preferred method of concentration of the present invention is to run the clarified extract through a ΙΟ» molecular weight cut off, hollow fiber ultrafiltration system. The volume of the clarified extract is typically reduced by about 6.5 fold for the microfiltration product and about 2 fold for the diluted, centrifuged product, yielding a concentrated retentate. Following concentration, the retentate'is diafiltered to further remove lower molecular weight - 29 - - 29 - LV 10493 contaminants. Diafiltration is performed using a 10*> molecular weight cutoff, hollow fiber system.

If TRITON Χ-100 was added, it can be removed by several conventional methods including, but not 5 limitted to, dialysis, addition of certain organic solvents, refrigeration, chromatographic separation, and contact with a gel or resin which specifically binds detergents, such as Extractogel (Pierce) and XAD resin (Romicon). The preferred method of this 10 invention to remove TRITON Χ-100 is to circulate the heat treated extract containing TRITON Χ-100 through a cartridge of ΧΑΒ-2 or XAD-4 resin (polystyrene divinylbenzene). The heat treated extract is circulated through the XAD cartridge for about 10 15 hours at 4°C and then collected in a suitable vessel, for example, a sealable glass bottle.

If the celis were disrupted in a high pH buffer, the pH of the heat treated extract, or the extract containing protease inhibitors, is then 20 adjusted to between about pH 7.0 to about 7.9 with the preferred pH of about 7.7. Adjusting the pH to about 7.7 folloving heat treatment at a high pH according to the method of this invention, greatly facilitates the adsorption of envelope proteins to 25 the wide pore silica utilized in a subsequent step. Adjustment of the pH of the heat treated extract can be performed prior to the Triton Χ-100 removal step without effecting the outcome of the procedure. Therefore, it will be obvious to those skilled in the 30 art that, according to the method of this invention, the order in which the pH adjustment and the Triton Χ-100 removal steps are done may be reversed vithout significant effect on the result of this procedure.

The HBsAg is then easily separated from the contaminants yielding substantially purified HBsAg. The preferred method of eliminating the contaminants is to adsorb the HBsAg onto vide pore silica. The 5 most preferred method of this invention is to adsorb the HBsAGr onto a vide pore silica vith a pore size range of about 1000 to 1500 angstroms and silica particle size range of about 30 to 130 microns (Amicon). The surface protein readily enters the 10 pores of the silica and is retained. The yeast cellular protein contaminants can therefore be easily vashed avay.

Adsorption of surface protein onto vide pore silica can be done chromatographically or in a 15 non-chromatographic, batchvise fashion.

Chromatographic adsorption is done by passing the pH adjusted extract through a bed of vide pore silica in a column chromatography apparatus. Typically, about one liter of heat treated extract is applied to a 5 20 cm jacketted column apparatus containing about 300 ml (about 100 g dry veight) of vide pore silica beads at a flov rāte of about 200ml/hour.

Non-chromatographic adsorption onto vide pore silica is typically done by mixing the heat 25 treated extract vith the silica in a suitable vessel, £.g. a sealable glass bottle. The preferred method is to add 300 ml of vide pore silica to about one liter of heat treated extract in a glass bottle and incubate vith constant mixing. Adsorption 30 preferrably continues for about 1.5 hours at about 4 - 8eC although different times and temperatures are - 31 - - 31 - LV10493 suitable.

Washing of the surface protein-adsorbed silica free of unadsorbed material can also be done non-chromatographically, or the silica can be poured 5 into a column apparatus, as previously described, for chromatographic adsorption. Batchwise washing is done by draining the heat treated extract from the vide pore silica and adding several volumes of a buffer which will not cause the release of EBsAg 10 adsorbed onto the silica. The preferred buffer is PBS. The silica is drained and the washing steps are Λ * repeated 3 to 5 times.

Chromatographic washihg of the surface protein-adsorbed silica is done by passing PBS 15 through the silica at a flow rāte of about 200ml/hour until the extinction at 280nm is constant.

The HBsAg is eluted from the washed wide pore silica using a buffer solution with a pH between about 8.5 to 9.0. Surface proteīns are preferably 20 desorbed using a buffer solution consisting of about 0.05 M Borate at a pH of about 8.7. Desorption of HBsAg can be facilitated at elevated temperatures over a vide range. Desorption at about 55 °C is preferred. 25 Non-chromatographic desorption is done by mixing 1200 ml of 0.05 M Borate buffer at pH 8.7 with about 700 ml of vashed HBsAg-adsorbed vide pore silica. Desorption continues for about 25 minūtes. The eluate is then collected, the desorption steps 30 are repeated tvice and the eluate is cooled.

Chromatographic desorption is done by warming the jacketted column of washed silica to about 55eC. The 0.05M Borate buffer at pH 8.7 is warmed to 55°C and then applied to the column at a rāte of 500 ml/hour. The eluate is then collected and cooled. The volume of eluate is usually roughly equivalent to the volume of heat treated extract applied to the wide pore silica.

Concentration of the eluted HBsAg is usually desired. The preferred concentration method is to pass the eluate through a 10^ molecular weight cut-off hollow fiber diafiltration system using a 0.05M Borate buffer, pH 8.7. The volume of the eluted surface protein may be generally reduced by as much as 16 fold using this system. The diafiltration retentate can be sterilized by microfiltration if necessary.

The carbohydrate content of the HBsAg is determined by the method of Dubois, M. al., Anal. Chem.. 28, pp.350, 1956. The general principle of this procedure is that simple sugars, oligosaccharides, polysaccharides and their derivatives, including the methyl ethers with free or potentially free reducing groups, give an orange yellow color when treated with phenol and concentrated sulfuric acid. The amount of color produced at a constant phenol concentration is proportional to the amount of sugar present.

To determine the carbohydrate content of a sample of HBV surface proteins, 1 iL of a solution containing between 10 to 70 μg of protein is placed in a tēst tube. A series of carbohydrate standards and blank samples are prepared. One mL of - 33 - - 33 - LV10493 a 5% phenol solution is added to each tube, the tubes are mi^ed, and 5 mL of a 96% sulfuric acid solution is added and mixed. The tubes are incubated at room temperature for 10 minūtes, mixed, and incubated at 5 25 to 30°C for .20 minūtes. The samples are read in spectrophotometer (A490 for hexoses and methylated hexoses, and A^gQ for pentoses, uronic acid, and their methylated derivatives) and the amount of carbohydrate in the HBsAg samples is determined by 10 comparison with the carbohydrate standards. HBsAg produced in "wild-type" recombinant yeast celis (CF54), and HBsAg produced in the CF52 recombinant yeast celis were both analyzed for carbohydrate content as described above. Based on 15 these results, a ratio of the amount of carbohydrate to protein present in each sample was calculated by dividing the micrograms of carbohydrate by the micrograms of protein in the sample. This ratio calculation demonstrated that HBsAg produced in mnn9~ 20 recombinant yeast celis consistently contained one tenth of the carbohydrate content of HBsAg produced in recombinant ,,wild-type" yeast celis. These results show that HBsAg produced in the mnn9~ mutant yeast celis contained substantially reduced amounts 25 of carbohydrate when compared to HBsAg produced in ,,wild-type" yeast celis.

The following examples illustrate the. present invention without, however, limiting the same thereto. The disclosure of each reference mentioned 30 in the following examples is hereby incorporated by reference. a 5% phenol solution is added to each tube, the tubes are mixed, and 5 mL of a 96% sulfuric acid solution is added and mixed. The tubes are incubated at room temperature for 10 minūtes, mixed, and incubated at 25 to 30°C for 20 minūtes. The samples are read in spectrophotometer (A490 for hexoses and methylated hexoses, and A^gQ for pentoses, uronic acid, and their methylated derivatives) and the amount of carbohydrate in the HBsAg samples is determined by comparison with the carbohydrate standards. HBsAg produced in "wild-type" recombinant yeast celis (CF541, and HBsAg produced in the CF52 recombinant yeast celis were both analyzed for carbohydrate content as described above. Based on these results, a ratio of the amount of carbohydrate to protein present in each sample was calculated by dividing the micrograms of carbohydrate by the micrograms of protein in the sample. This ratio calculation demonstrated that HBsAg produced in mnn9~ recombinant yeast celis consistently contained one tenth of the carbohydrate content of HBsAg produced in recombinant nwild-type" yeast celis. These results show that HBsAg produced in the mnn9~ mutant yeast celis contained substantially reduced amounts of carbohydrate when compared to HBsAg produced in "wild~type" yeast celis.

The folloving examples illustrate the. present invention without, however, limiting the same thereto. The disclosure of each reference mentioned in the following examples is hereby incorporated by reference. LV10493 - 34 -EKAMPLE 1 ΓΊ οτιίηρ of HBV DNA in pBR322 HBV Dane pārticies <serotype adw) were isolated and purified from human plasma (carrier), and double-stranded DNA was synthesized by the endogenous polymerase in the Dane pārticies according to the methods of Landers ai-, [l. Viroloev. 13.» 368-376, (1977)3 and Hruska fit sJL·» [sl· Virologv. 21. (1977)]. The DNA was isolated after digestion with Proteinase K in SDS followed by extraction with phenol/chloroform and ethanol precipitation. The HBV genomic DNA was digested with EcoRI. producing a single 3.2 kbp fragment, that was cloned into the EcoRI site of pBR322 to form pHBV/ADW-l. The presence of the HBV DNA was confirmed by EcoRI digestion, Southern blot transfer to nitrocellulose, and hybridization with [^^P]-labelled specific oligonucleotide probes. EXAMPLE 2

Cloning of the preS2+S Gene into the pGAP-tADH-2

Expression Vector_

Plasmid pHBV/ADW-l (described in Example 1) was digested with EcoRI and Accl, and the 0.8 kbp fragment was purified by preparative agarose gel electrophoresis. Also, a pUC plasmid was digested with EcoRI and SsmHI and the linear vector was purified by preparative agarose gel electrophoresis.

To reconstruct the 5‘ portion of the preS2+S ORE, a pair of oligonucleotides was synthesized which EXAMPLE 1

Cloning of HBV DNA in pBR322 HBV Dane pārticies (serotype adw) were isolated and purified from human plasma (carrier), and double-stranded DNA was synthesized by the endogenous polymerase in the Dane .pārticies according to the methods of Landers &1., Cl· Virologv. 23.» 368-376, (1977)] and Hruska et al♦. [i. yirology. 21» (1977)]. The DNA was isolated after digestion with Proteinase K in SDS followed by extraction with phenol/chloroform and ethanol precipitation. The HBV genomic DNA was digested with EcoRI, producing a single 3.2 kbp fragment, that was cloned into the EcoRI site of pBR322 to form pHBV/ADW-l. The presence of the HBV DNA was confirmed by EcoRI digestion, Southern blot transfer to nitrocellulose, and hybridization with [^^P]-labelled specific oligonucleotide probes. EXAMPLE 2

Cloning of the preS2+S Gene into the pGAP-tADH-2

Expression Vector_

Plasmid pHBV/ADW-l (described in Example 1) was digested with EcoRI and Accl. and the 0.8 kbp fragment was purified by preparative agarose gel electrophoresis. Also, a pTJC plasmid was digested with EcoRI and BsmHI and the linear vector was purified by preparative agarose gel electrophoresis.

To reconstruct the 5' portion of the preS2+S ORE, a pair of oligonucleotides was synthesized which - 35 - - 35 - LV 10493 reconstitutes the ORF from the EcoRI site upstream to the ATG through a 10 bp NTL sequence through a HindIII site to an EcoRI terminus. The sequence of these oligonucleotides are: 5 AATTCAAGCT TACAAAACAA AATGCAGTGG (SEQIDN0: 4) 1 10 20 30 GTTCGAATGT TTTGTTTTAC GTCACCTTAA (SEQIDN0: 3) 10 1 10 20 30

To reconstitute the 3' portion of the preS2+S ORF, a second pair of oligonucleotides was synthesized which reconstitutes the ORF from the Accl 15 site through the translational terminator through a HindIII site to a ]3amHI terminus. The sequence of these oligonucleotides are: ATACATTTA AGCTTG (SEQIDN0: 4) 20 1 10 15 TGTAAATTTC GAACCTAG (SEQIDN0: 5) 10 10 18 25 The oligonucleotide pairs were annealled,

then ligated to the pUC EcoRI - BamHI digested vector. The resultant vector (2.8kbp) was purified by preparative agarose gel electrophoresis. The 0.8kbp EcoRI - Accl fragment from above was ligated 30 with this vector. The presence and orientation of the PreS2+S ORF was confirmed by restriction endonuclease analysis and Southern blot. DNA seguence analysis [Sanger Sl·» 1977] revealed two base substitions that resulted in amino acid differences from the sequence encoded by the DNA of plasmid HBpreSGAP347/19T. To evaluate identical polypeptides for both constructions, these substituions, which were T instead of C at base 64 (encoding Phe rather than Leu) and C instead of A at base 352 (encoding His rather than Gln), were changed by site-directed mutagenesis. [Zoller and Smith 1982, Nucleic Acids Research, 10, pp6487-6500]. A plasmid containing the HBsAg coding region without the preS2 coding region was constructed as follows: The pUCHBpreS2+S plasmid (described above) was digested with EcoRI and StvI restriction endonucleases. The large DNA fragment (3.3kbp) which contains pUC and the HBsAg coding region was separated from the preS2 encoding DNA fragment and purified by preparative agarose gel electrophoresis. A synthetic DNA oligonncleotide pair: AATTCAAGCT TACAAAACAA AATGGAGAAC ATCACATCAG 1 10 20 30 40 GATTC (SEQIDN0: 6) 45 GTTCGAATGT TTTGTTTTAC CTCTTGTAGT GTAGTCCTAA 1 10 20 30 40 GGATC (SEQIDN0: 7) 45 - 37 - - 37 - LV10493 was then ligated with the pUCHBsAg fragment. This synthetic oligonucleotide pair contains 5' EcoRI and 3' StvI sticky ends as well as providing a HindIII site immediately following the 5' EcoRI site. In 5 addition, the synthetic DNA oligonucleotide pair contains the HBsAg ATG codon, the upstream lObp non-translated leader sequence, and the 21 downstream nncleotides inclnding the Styl site.

This oligonucleotide pair rebuilds the 10 complete coding region of the HBsAg and allows its subsequent removal intact, from the pUC based vector by digestion with HindIII.

The pUC-HBsAg DNA vector with the ligated DNA oligonucleotide pair described above was used to 15 transform E. coli. Recombinant plasmids were selected vhich possess the complete reconstructēd HBsAg coding region. The complete HBsAg open reading frame (ORF) was removed from the recombinant plasmid by digestion with HindIII followed by isolātion and 20 purification of the (0.7kbp) HBsAg DNA by preparative agarose gel electrophoresis for cloning in to an expression vector. 25 EXAMPLE 3

Cloning of HBsAg ORF into 3 different expression vectors_

Three different expression vectors were used to construct HBsAg expression cassettes. The GAP 491 30 promoter expression cassette described previously [Kniskern £t sX., 1986 Gene 46 ppl35-141], is composed of about 1.1 kbp of the glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) promoter and about 350bp of the yeast alcohol dehydrogenase I (ADH1) terminator in a pBR322 backbone, with a unigue HindIII site betveen the promoter and terminator.

The HBsAg OKF from Example 2 was ligated in the unique HindIII site, and its presence and orientation was confirmed by restriction endonuclease analyses and Southern blot.

Alternately the (0.5kbp) GAL10 promoter (Schultz al., 1987, Gene. 54. ppll3-123) was substituted for the l.lkbp GAP promoter in the above construction, and the (1.25 kbp) ADH2 promoter (Kniskern al., 1988 Hepatologv 8, 82-87) was substituted for the GAP promoter (see Figurē 1).

In each case, the expression cassette containing the specific promoter, the HBsAg ORF, and the ADH1 terminator was cloned into the shuttle vector pCl/1 (Beggs, supra: Rosenberg, gt al., supra) to create a yeast expression vector which was then used to transform £.· cerevisiae as described below. EXAMPLE 4

Construction of yeast £. Cerevisiae CF52 (mmi9-) mutant veast strain_

Teast £. cerevisiae strain ΚΗΥ 107 (cir*. adel+ T leil2“ and mim9-) was constructed as follows: The α mating type strain CZ5/LB347-1C (mnn9~. ŠUCZ“) was mated with the a type strain 2150-2-3 (leu2~'t adel~) by mixing the strains on a YEHD complete media plate. To select for diploids, the mated strains were replica plated onto leu- minimal medium - 39 - - 39 - LV 10493 containing 2% sucrose as the sole carbon source.

After isolating single colonies, the diploids were sporulated, and asci were dissečted by Standard technigues. The ΚΗΥ-107 strain was isolated as a 5 single spore and characterized as cir*. adel*. leu2~, and mnn9~ (by Schiff stain technique). KHT107 (cir 0) was derived from strain ΚΗΥ107 (cir-1") as described by Broach rMethods in Enzvmologv. 101. Part C. pp 307-325, (1983)]. The 10 cured strain was made ura3~ by integrating a disrupted ura3 gene. The resulting strain, KHY-107ura3A, was grown in rich media to allow the accumulation of spontaneous mutations and a canavanine resistant mutant was selected. The mutant 15 strain, CF55, was shown by complementation tests to be canl~. The GAL10pGAL4 expression cassette was integrated into the HIS3 gene of CF55 (Methods in Enzvmologv. 1990, 185 pp297-309) to yield the final host strain CF52 (Mata leu2-2.112 ura3A canl 20 his3A: :GALI0pGAL4-ŪRA3, cir° ) . EEAMPLE 5

Yeast Transformation and Seed Establishment for HBsAg 25 in CF52 mnn9- Mutant Yeast_

The pCl/1 pGAL10HBsAg-tADH-l plasmid described in Example 3 was used to transform cerevisiae strain CF52. Clones were selected on -minimal medium (leu- containing 1M sorbitol), 30 established as frozen stocks (in 17% glycerol) and evaluated as described below. EXAMPLE 6

Growth and Expression of the HBsAg Gene behind the GAL10 promoter in Yeast CF52 (mnn9~)_

Clones of yeast containing the expression 5 plasmid described in Example 5 above were plated oņto leu- selective agar plates containing 1M sorbitol and i'ncubated at 30eC for 2-3 days. These yeast were inoculated with 5-7 mL of complex TEHDS (TEHD + 1M sorbitol) and the cultures were iņcnbated at 30eC 10 with aeration for 12-18 hours. Flasks containing 50 mL YEHDS + 27« galactose media were inoculated from the above culture (to an initial A^qo = and incubated at 30eC with shaking (350 rpm) for 72 hours to a final A^oo of 10-16. Samples of 10 A^oq units 15 were dispensed into tubes, and the yeast celis were pelleted at 2,000 x g for 10 minūtes. The pellets either were assayed directly or stored at -70eC for future assay. At the time of assay, the pellets were resuspended in 0.4 mL of phosphate-buffered saline 20 containing 2mM PMSF. Yeast celis were broken by: 1) the addition of 200-300 mg of vashed glass beads (0.45 mm), 2) agitation on a vortex mixer for 15 minūtes, 3) addition of TritonX-100 to 0.57» (v/v), 4) agitation on a vortex mixer for 2 minūtes, and 5) 25 incubation at 4°C for 10-15 minūtes. Cellular debris and glass beads were removed by centrifugation at 13,000 x g for 10 minūtes. The clarified supernatant fluid was removed and analyzed for protein [by the method of Lowry fit. ai. , !. Eiol. Chem. . 193. 265 30 (1951)] and for HBsAg by (AUSRIAR) assay (Abbott).

Typical assay results are shown below. - 41 - - 41 - LV 10493

TABLE I

P24 LEVEL 5 SAMPLE DESCRITPION AUSRIA. UG/MG PROTEIN BREAKAGE (IMMUNOBLOTi

Shake Flasks mnn9- mutant (0.55, 0.61, 0.53) Glass beads +++ wlld type (1.8) Glass beads + 10 (MŅŅ9+) EXAMPLE 7

Large Scale Growth of J£. cerevisiae (mmi9“) Producing HBsAg in Fermentors_

The. frozen recombinant yeast culture was inoculated onto leu” plates containing 1M sorbitol.

The plates were incubated inverted at 28°C for 2-3 days. The growth on the plates was resuspended in YEHDS and the resuspended growth was transferred into 2-L Erlenmeyer flask containing 500 mL of IEHDS, and 2% galactose. The flask was incubated at 28eC and 350 rpm in a controlled environment shaker incubator for 18-22 hours. These seed cultures then were used 25 to inoculate the production stage vessels.

An inoculum (1-5% v/v) from one or more flasks was transferred into 16-L or 250-L fermentors containing 10-L or 200-L of YEHDS, respectively. The 16-L fermentors were operated at 500 rpm, 5 L/min air, and 28eC. The 250-L fermentors were operated at 160 RPM, 60 L/min air and 28°C. The fermentors were 30 harvested 40-46 hrs after inoculation with the seed culture. Optical density values of 15.0 Ag^g units typically were obtained. Harvesting consisted of concentrating the celis using a hollow fiber filtering device followed by washing the celis in buffered salt Solutions. Celi slurries were assayed as described below or stored frozen at -70eC for further processing and analysis.

Small samples (0.6 mL) of 20% washed celi slurries were broken using glass beads (0.45-0.52 mm) in 1.5-mL Eppendorf tubes. PMSF (6.5 μΐ of 200 mM stock) was added as a protease inhibitor. Aliguots were removed from the tubes after breakage and frozen at -70°C for immunoblot analysis. TRITON Χ-100 was added to the remaining sample in the tubes to a final concentration of 0.5%, and the samples were mixed briefly and incubated at 4°C for 20-40 min. The celi debris was removed by centrifugation and the

R clarified celi extract assayed for antigen (Ausria ) and protein (Lowry). EXAMPLE 8

Purification of S protein in particulate form by means of inunune affinitv chromatographv_

Recombinant £. cerevisiae. constructed as described in Example 5, were grown in either flasks or fermentors. The yeast celis were harvested by microfiltration in an Amicon DC-10 unit, suspended in 30 ml buffer A [0.1M Na2HP04, pH 7.2, 0.5H NaCl], and broken in a Stansted pressure celi for seven passages at 75-85 pounds per square inch. The broken celi - 43 - - 43 - LV10493 suspension (31 gm wet celi weight) was diluted with 120 ml buffer A, Triton Χ-100 was added to a final concentration of 0.5% (w/v), and the suspension was clarified by centrifugation at 10,000 x g for 20 min. 5 at 4eC. The clarified broth was decanted and incubated with Sepharose 4B coupled with antibodies to HBsAg [McAleer ai·, Nature 307: 178 (1984)] for 19 hours at 4°C to adsorb the antigen onto the resin. After the incubation period, the slurry was 10 warmed to room temperature for ali subsequent steps and degassed under vacunm for 15 min. The degassed slurry was poured into a 2.5 x 40 cm column. When the column had been packed fully, -unhonnd protein was washed away with buffer A. The antigen was eluted 15 with 3M KSCN in buffer A. Fractions containing antigen were dialyzed against 0.007M Na2HP04, pH 7.2, 0.15M NaCl at 4eC and pooled to form the Dialyzed Affinity Pool containing 1.08 mg of protein in 20 ml. Sixteen ml of Dialyzed Affinity Pool was diluted 20 to 40 mcg/ml with 5.6 ml 0.006M Na2HP04, pH 7.2, 0.15M NaCl. The product was sterilized by filtration through a Millex-GV 0.22 μ membrane. The identity of the product in the Dialyzed Affinity Pool was verified by the detection of HBsAg by AusriaR 25 reactivity. 30

TABLE II SAMPLE DESCRIPTI0N AUSRIA. UG/MG/ PROTEIN BREAKAGE Fermenters mnn9- (1.13, 1.10, 1.06) Glass Beads mnn9- (3.1, 4.4) Hanton-Gaulin wi1d-type (3.3) Manton-Gaulin EXAMPLE 9

Large Scale Purification of Recombinant·. ITBsAj?

About 250g of frozen celi paste (producing recombinant S protein) was resuspended to 177. wet weight/volume (about 1500 ml) in phosphate buffered saline solution (PBS). The celis were heated to 45eC by immersion in a water bath. The celis were held at 45°C for 15 minūtes and then cooled on ice to about 10eC. The celis were then disrupted by two passages through a Gaulin homogenizer.

Following homogenization, 107« Triton Χ-100 was added to a final concentration of 0.37« and mixed for about 15 minūtes. The celi extract was then centrifuged at 3,600 x g for 20 minūtes at 4eC, and the supernatant was collected. - 45 - - 45 - LV10493

The superantant was then passaged over a column containing about 200g of XAD-2 resin to remove the Triton Χ-100. The effluent was then passaged directly over a column containing about 150g of vide 5 pore silica with a pore size of about 1,500 angstrom and a particle sizē of about 50 microns. The columns used were 5 cm diameter (Pharmacia) and were run at a flow rāte of about 200 ml per hour.

The silica column was vashed with PBS until 10 the A28O returned to baseline.

The S protein was eluted from the silica column using first, cold borate buffer (50 mM, pH 8.7, 4°C) at a flow rāte of about 500 ml per hour, until a rise in the A28O was observed. Once the A28O 15 began to rise the column was heated to 55eC and 55eC borate buffer was run through the column at about 500 ml per hour. The eluate containing S protein (about 1L) was collected on ice. The eluate was then concentrated to about 200 ml by difiltration against 20 50 mM borate buffer at pH 8.7, using a hollow fiber diafiltration unit with a molecular weight cutoff of 105. The S protein was then filtered through a 0.2 micron filter and stored. The product was found to be stable with no significant degradation observed on 25 Western blot analysis. .EXAMPLE 10

Assay of Carbohydrate Content of the Recombinant HBV 30 Surface Proteīns..______

The carbohydrate content of the recombinant HBV surface proteins was determined by the method of

Dubois, M. ai., Anal. Chem.. 28. pp.350, 1956.

The general principle of this procedure is that simple sugars, oligosaccharides, polysaccharides and their derivatives, including the methyl ethers with free or potentially free reducing groups, give an orange yellow color when treated with phenol and c'oncentrated sulfuric acid. The amount of color produced at a constant phenol concentration is proportional to the amount of sugar present.

To determine the carbohydrate content of HBsAg produced in wild-type yeast strain and produced in the mnn9~ yeast strain, 1 mL of a solution containing between 10 to 70 μg of protein was placed in a tēst tube. A series of carbohydrate standards and blank samples were prepared containing various amounts of carbohydrate. One mL of a 57« phenol solution was added to each tube, the tubes were mixed, and 5 mL of a 967» sulfuric acid solution was added and mixed. The tubes were incubated at room temperature for 10 minūtes, mixed, and incubated at 25 to 30°C for 20 minūtes. The samples were read in spectrophotometer (A^gg for hexoses and methylated hexoses, and A4qq for pentoses, uronic acid, and their methylated derivatives) and the amount of carbohydrate in the HBV surface protein samples was determined by comparison with the carbohydrate standards.

Based on these results, a ratio of the amount of carbohydrate to protein present in each sample was calculated by dividing the micrograms of carbohydrate by the micrograms of protein in the sample, which is shown below. - 47 - - 47 - LV10493

Carbohydrate-to-protein ratio of HBsAg 5 Yeast strain Carbohvdrate/Protein mnn9~ 0.05 wild-type for 10 glycosylation 0.56 (Ū9+)

This ratio calculation demonstrated that HBsAg produced in mnn9~ recombinant yeast celis 15 consistently contained one tenth of the carbohydrate content of HBsAg produced in recombinant ”wild-type" yeast celis. These results show that HBsAg produced in the mnn9~ mutant yeast celis contained substantially reduced amounts of carbohydrate when 20 compared to HBsAg produced in "wild-type" yeast celis. 25 30

SEQUENCE LISTING LV10493

- 49 - (2) INFORMATION FOR SEQ 10 NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 base pairs (B) ΤΥΡΕ: nucleic acid (C) STRANOEONESS: single (D) TOPOLOGY: linear 10 (ii) MOLECULE ΤΥΡΕ: DNA (genomic) 15 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1: 10

ACAAAACAAA 20 25 30 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (8) ΤΥΡΕ: nucleic acid (C) STRANDEDNESS: single (0) TOPOLOGY: linear (ii) MOLECULE ΤΥΡΕ: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2: AATTCAAGCT TACAAAACAA AATGCAGTGG 30 (2) INFORMATION FOR SEQ ID N0:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) ΤΥΡΕ: nucleic acid (C) STRANDEDNESS: single (D) TOPOIOGY: linear (ii) MOLECULE ΤΥΡΕ: DNA (genomic) (xi) $EQUENCE OESCRIPTION: SEQ ID N0:3: 30

GTTCGAATGT TTTGTTTTAC GTCACCTTAA - 51 -LV 10493 (2) INFORMATION FOR SEQ ID N0:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) ΤΥΡΕ: nucleic acid (C) STRANOEDNESS: single (0) TOPOLOGV: linear (ii) MOLECULE ΤΥΡΕ: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4: ATACATTTAA GCTTG 15 (2) INFORMATION FOR SEQ ID N0:5: (i) SEĢUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) ΤΥΡΕ: nucleic acid (C) STRANDEDNESS: single (D) TOPOtOGY: linear (ii) MOLECULE ΤΥΡΕ: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5: 18

TGTAAATTTC GAACCTAG (2) INFORMATION FOR SEQ 10 N0:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) ΤΥΡΕ: nucleic acid (C) STRANDEDNESS: single (0) TOPOLOGY: linear (ii) MOLECULE ΤΥΡΕ: DNA (genomic) (xi) SEQUENCE OESCRIPTION: SEQ 10 N0:6: AATTCAAGCT TACAAAACAA AATGGAGAAC ATCACATCAG GATTC (2) INFORMATION FOR SEQ 10 N0:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) ΤΥΡΕ: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE ΤΥΡΕ: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ 10 N0:7:

GTTCGAATGT TTTGTTTTAC CTCTTGTAGT GTAGTCCTAA GGATC - 53 -LV10493 (2) INFORMATION FOR SEQ ID N0:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) ΤΥΡΕ: nucleic acid (C) STRANDEONESS: single (D) T0P0L0GY: linear (ii) MOLECULE ΤΥΡΕ: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8: AATTGTCGAC AGCTAGCTGA ATTCCCGGG 29 (2) INFORMATION FOR SEQ ID N0:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) ΤΥΡΕ: nucleic acid (C) STRANDEDNESS: single (0) TOPOLOGY: linear (ii) MOLECULE ΤΥΡΕ: ONA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:9: 29

AGCTCCCGGG AATTCAGCTA GCTGTCGAC

Claims (16)

An eukaryotic expression system for the production of recombinant polypeptides and proteins, characterized in that it contains a reduced host cell carbohydrate or glycoprotein level. 2. An expression system according to claim 1, wherein the eukaryotic host is yeast. 3. The expression system of claim 2 wherein the yeast is Saccharomyces cerevisiae. The expression system of claim 1, wherein the recombinant peptide or protein is a hepatitis B virus polypeptide or protein. 5. The expression system of claim 4, wherein the hepatitis B virus is. a polypeptide or protein is a shell polypeptide or protein. 6. The expression system of claim 5, wherein the hepatitis B virus polypeptide or protein is HBsAg. 7. Hepatitis B virus surface protein, characterized in that it forms particles with significantly reduced carbohydrate or glycoprotein content. 8. Hepatitis B virus surface protein according to claim 7, characterized in that it is produced in recombinant yeast cells which are genetically unsuitable for glycosylation of the protein. 9. Hepatitis B virus surface protein according to claim 8, characterized in that the yeast cell genetic defect is in the mnn9 gene. 10. Hepatitis B virus surface protein according to claim 7, wherein the purified surface protein, carbohydrate and protein ratio is less than 0.5. A vaccine against hepatitis B virus for use in humans, characterized in that it comprises a hepatitis B virus surface protein that forms particles with a significant reduction in the content of carbohydrates or glycoproteins included. 2 A vaccine according to claim 11, characterized in that it is produced in recombinant yeast cells which are not genetically suitable for glycosylation of the protein. 13. A vaccine according to claim 12, wherein the genetic defect is in the mnn9 gene. . 14. A vaccine according to claim 11, wherein the ratio of carbohydrate to protein in the surface protein is less than 0.5. An immuno-diagnostic reagent, characterized in that it contains a hepatitis B virus protein that forms particles with a significantly reduced host cell carbohydrate or glycoprotein content and a reduced response capacity with a naturally occurring antibody. 16. An immunoassay reagent according to claim 15, wherein the purified surface protein, carbohydrate and protein ratio is less than 0.5. i