HRP940048A2 - Process for the preparation of polypeptides having the antigenicity or antigen specificity of hepatitis b viral antigens - Google Patents

Description

The application was separated from our approved patent application P-3127/79.

The field of technology

The invention is from the field of methods for obtaining polypeptides. The designation according to the International Classification of Patents is C12N 15/00; C07K 1/00; C12Q 1/70; A61K 37/02.

Technical problem

The invention solves the technical problem of the procedure for obtaining polypeptides that are produced by expression of DNA sequences of structural genes, which code for at least one polypeptide that shows the antigenicity or antigenic specificity of the core antigen or surface antigen of the hepatitis B virus in the respective host organisms. The polypeptides obtained by the process according to the invention can be used in the detection of the hepatitis B virus in humans and in the stimulation of the production of antibodies against this virus.

State of the art

The virus that causes hepatitis B, or serum hepatitis, appears to infect only humans. Hepatitis B virus ("HBV") infection in humans is widespread. In the United States, Great Britain and Western Europe, approximately 0.1% of all blood donors are chronic carriers of HBV. Although the mortality from viral hepatitis is not high (in 1975 in Great Britain it was 3 per million), there are indications that as many as 5% of the population in Great Britain and 15% of the population in the USA are infected. In many African and Asian countries, up to 20% of the population are chronic carriers of HBV, and over 50% of all adults in these countries have been or are infected with HBV.

Hepatitis infection is transmitted by three general mechanisms: (1) parenteral inoculation of infected blood or blood fluid, either in large quantities such as through an accidental skin wound; (2) by close family or sexual contact; and (3) by means of some substance, which causes infection during pregnancy, so that the virus is transmitted to the newborn. Transmission by inhalation occurs rarely, if at all.

Many HBV infections are subclinical. Clinical illness usually lasts three to six weeks and varies in severity from mild to acute explosive hepatitis followed by cirrhosis or death. Recovery from clinical or subclinical HBV infections is usually complete. However, in some cases serious long-term consequences arise: (1) approximately 5% of acute infections lead to chronic carriage of hepatitis B virus antigens with prolonged potential for infecting others and prolonging liver damage; and (2) it is likely that previous infection with HBV may be partially or fully responsible for initiating a significant number of HBV-seronegative cases of chronic active hepatitis, cirrhosis, and primary liver cancer.

Recent advances in molecular biology have enabled the introduction of DNA coding for specific non-bacterial enkaryotic proteins into bacterial cells. Basically, with DNA made by other means than chemical synthesis, the construction of recombinant DNA molecules involves the steps of producing a single-stranded DNA copy (cDNA) of a purified messenger (mRNA) that is a template for the desired protein, converting cDNA to double-stranded DNA ; linking the DNA at the appropriate site in the appropriate carrier for cloning and transformation of the appropriate host with the recombinant DNA molecule. Such a transformation enables the host to produce the desired protein. Furthermore, at least in the case of ovalbumin DNA, it is known that appropriate fusion of certain DNA to a strong bacterial promoter or to an expression control sequence produces larger amounts of the desired ovalbumin protein, i.e., about 0.5 to 1% of the total protein mass of an E. coli cell (0. Mercereau-Puijalon et.al., "Synthesis of an Ovalbumin-Like Protein by Escherischia coli K12 Harboring A Recombinant Plasmid", Nature. 275, pp 505-510 (1978) and T.H. Fraser & B.J. Bruce, "Chicken Ovalbumin is Synthesized and Secreted by Escherichia coli". Proc. Natl. Acad. Sci. USA. 75, pp 5936-5940 (1978).

Several non-bacterial proteins and genes have been synthesized using recombinant DNA technology. These include antigenic determinants similar to rat proinsulin protein (L. Villa-Komaroff et.al. "A Bacterial Clone Synthetozing Proinsulin", Proc. Natl. Acad. Sci. USA. 75, pp 3727-3731 (1978), rat growth hormone ( P.H. Seeburg et.al.. "Synthesis of Growth Hormone by Bacteria", Nature. 276, pp. 795-798 (1978), mouse dihydrofolate reductase (A.C. Y. Chang et.al.. "Phenotypic Expression in E. coli of a DNA Sequence Coding for Mouse Dihvdrofolate Reductase", Nature. 275, pp 617-624 (1978), somatostatin (K. Itakura et.al.. "Expression in Escherichia coli of a Chemically Synthesized Gene for the Hormone Somatostatin", Science. 198, 1056-1063 (1977), United Kingdom Patent Applications 2,007,657A, 2,007,676a and 2,008,123a and related applications in other countries) and the A and B polypeptide chains of human insulin (D.V. Goeddel et.al.. "Expression in Escherichia coli of Chemically Synthesized Genes for Human Insulin", Proc.Natl.Acad.Sci.. USA 76, pp. 106-110 (1979) and applications from Great Britain and other related patent applications, supra).

None of the previous references, however, are directed as in the present invention to the synthesis of recombinant DNA of such viral proteins as hepatitis B virus antigens.

It is known that HBV infections cause the development of antibodies against virus antigens. These antibodies are the body's defense mechanism against HBV infection. Development of such antibodies prior to exposure or rapidly in case of potential exposure should substantially reduce and control virus growth and spread in the patient.

However, the problem with the artificially stimulated development of antibodies against hepatitis B virus antigens is the limited host interval for the virus. For example, although it is highly infectious to humans, experimental infection with hepatitis B virus has been achieved in only a few other primates. And, the virus was not propagated in the tissues. This limited host interval and inability to infect tissue culture cells has greatly slowed both the characterization of the virus and the pathology of its infection and the development of rapid means of detection and effective means of infection control and prevention.

Although there have been attempts to use authentic HBV viral antigens, isolated from victims of HBV infection, to develop antibodies and detect infection, these treatments have generally not been affordable due to the limited source of active specificities. Furthermore, the use of human sources for these antigens is not advantageous because of the well-known contamination problems in the use of human isolates.

Description of the solution to the technical problem

The present invention solves the problems discussed by providing a method according to the invention for the production of polypeptides having the antigenicity of hepatitis B viral antigens. The method of the present invention is characterized by culturing a host transformed with a recombinant DNA molecule having a structural gene coding for at least one polypeptide which exhibits HBV antigenicity and has, operably linked to itself, an expression control sequence, and the polypeptide is then harvested from the culture.

By means of our invention it is possible to obtain these HBV antigens in essential and uncontaminated quantities for making a vaccine and for use in the detection of viral infection as well as for determining its pathology and molecular biology. Until now, such sources have not been accessible due to the narrow host range of the virus and its inability to be cultivated in tissue culture. These products can also be identified and characterized and are useful either as produced in the host or after certain derivatization or modification in preparations and procedures for improving the production of these products themselves as well as for the detection of HBV infection, which allows pathology to be monitored and stimulates the production of HBV antibodies in humans.

The method of our invention is also characterized by linking a DNA sequence made from the endogenous DNA of a given fragment to another DNA sequence made from a source other than the given fragment.

Our method may differ from the earlier methods mentioned above in that none of the previous methods use the natural gene or DNA for a particular protein to construct a recombinant DNA molecule and produce that protein or gene. Instead, they use either synthetic genes made by chemical synthesis or artificial genes made by enzymatically copying mRNA isolated from a donor cell to produce cDNA sequences.

One of the reasons that natural DNA has not previously been used directly in recombinant DNA protein synthesis is that natural DNA from many higher organisms and at least some animal viruses contain "introns" or complementary nucleotide sequences as part of the gene. These introns do not form part of the final message of the gene. Instead, they are removed in vivo in higher organisms under the action of special processing enzymes that act on the primary transcription product so that the obligatory message (mRNA) of the gene is obtained. It is assumed that bacteria are not capable of processing such introns, so natural DNA cannot be expected to be expressed in bacterial hosts, and these bacteria cannot be expected to produce the desired proteins.

Figure 1 is a simplified diagram representing the structure of the endogenous DNA of Dane delić. It shows two complementary DNA strands, strand a and b, a nick in strand a and a gap in strand b, and closure of that strand using a DNA polymerase reaction.

Figure 2 is a schematic representation of a preferred embodiment of the present invention: fragments of endogenous DNA isolated from the Daje fragment are fused to the E. coli penicillin gene on plasmid pBR322 at the Pst I site. After transformation into E. coli HB101, the recombinant DNA molecule pBR322-Pst I dG: HBV-KpnI dC is directed to the synthesis of polypeptides showing HBV antigenicity.

Figures 3-9 show the nucleotide sequence determined for a portion of the hepatitis B virus genome according to the present invention. The sequence is shown with stop codons in three boxes as indicated above. It is arbitrarily numbered from the initiation codon for the gene encoding HBcAg as defined in the present invention. Nucleotides - 80 to -1 represent the leader sequence preceding this gene. Nucleotides 1-549 represent the nucleotide sequence for the gene encoding the internal antigen of the hepatitis B virus, while the amino acid sequence (marked box 1) of this antigen is indicated above that sequence. Nucleotides 1437-2114 represent the nucleotide sequence determined for the surface antigen gene of the hepatitis B virus, while the amino acid sequence (designated box 3) of this antigen is described above that sequence. The various restriction endonuclease recognition sites in these genes are also indicated in Figures 3-9.

Figure 10 is a schematic representation of a process of the present invention in which a recombinant DNA molecule capable of producing a polypeptide expressing the internal hepatitis B virus is fragmented and its fragment is joined to an improved expression control sequence, the lac system.

Figure 11 is a schematic representation of a process of the present invention in which a recombinant DNA molecule of the present invention is fragmented and joined to a phage DNA fragment for use in lysogenizing host cells to increase gene copy number.

Figure 12 is a schematic representation of another embodiment of the method of the present invention: a recombinant DNA molecule of the present invention that does not produce a polypeptide expressing the internal antigen of the hepatitis B virus is fragmented so that the structural gene for HBsAg is separated and this structural gene is used to produce a DNA molecule that produces HBsAg in the appropriate host.

In order to more fully understand the invention described herein, a detailed description is now provided.

The following terms are used in the description:

Nucleotide - Monomeric unit of DNA or RNA that contains a sugar species (pentose), a phosphate and a nitrogenous heterocyclic base. The base is attached to the sugar species via the glycosidic carbon (1' carbon of the pentose) and this combination of base and sugar is a nucleoside. A base characterizes a nucleotide. The four DNA bases are adenine ("A"), guanine ("G"), cytosine ("C") and thymine ("T"). The four RNA bases are A,G,C and uracil ("U").

DNA sequence - A linear sequence of nucleotides connected to each other by means of phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses.

Codon - DNA sequence of three nucleotides (triplet) that encodes one amino acid, a translation initiation signal or a translation termination signal via mRNA. For example, the nucleotide triplets TTA, TTG, CTT, CTC, CTA and CTG code for the amino acid leucine ("Leu"), TAG, TAA and TGA are translational stop signals and ATG is the translational start signal.

Indicated box - A group of codons during the translation of mRNA into amino acid sequences. A proper indicated frame must be maintained during translation. For example, the sequence GCTGGTTGTAAG can be translated into three indicated frames or phases, each yielding a different amino acid sequence:

GCT GGT TGT AAG - Ala-Gly-Cys-Lys

G CTG GTT GTA AG - Leu-Val-Val

GC TGG TTG TAA A - Trp-Leu-(STOP)

Polypeptide- A linear sequence of amino acids linked to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent amino acids.

Genome - The entire DNA of a substance. It includes inter alia gene structures that code for substance polypeptides, as well as operator, promoter and ribosome binding and interaction sequences including sequences such as Shine-Dulgano sequences.

Structural gene - a DNA sequence that encodes through its template or messenger ("mRNA") an amino-acid sequence that is characteristic of a specific polypeptide.

Transcription- The process of producing mRNA from a structural gene.

Translation - The process of producing polypeptides from mRNA.

Expression- The process a structural gene undergoes to produce a polypeptide. It is a combination of transcription and translation.

Plasmid- A double-stranded, nonchromosomal DNA sequence that encompasses an intact "replicon" so that the plasmid is replicated in the host cell. When a plasmid is placed in a single-celled organism, the characteristics of that organism are changed or transformed as a result of the plasmid DNA. For example, a plasmid carrying the gene for tetracycline resistance (TetR) transforms a cell previously sensitive to tetracycline into a cell resistant to it. A cell that has been transformed by plasma is called a "transformant".

Phage or Bacteriophage - Bacterial viruses, many of which include DNA sequences encapsulated in a protein coat or coating ("capsid"). In a unicellular organism, the phage replicates by a process called transvection.

Cloning carrier - Plasmid, phage DNA, or other DNA sequences that can be replicated in a host cell that are characterized by a small number of endonuclease recognition sites at which such DNA sequences can be cut in a specific manner without concomitant loss of the essential biological function of the DNA, e.g., by replication , by the production of a coat protein or by loss of promoter or binding sites, and containing a marker useful in the identification of transformed cells, eg resistance to tetracycline or ampicillin. A cloning medium is often called a vector.

Cloning- Process of asexual reproduction.

Recombinant DNA molecule - A hybrid DNA sequence comprising at least two nucleotide sequences, where the first sequence is not normally found in nature together with the second.

Expression Control Sequence - A DNA sequence of nucleotides that controls and regulates the expression of structural genes, when operably linked to those genes.

In Figure 1, we have shown a simplified diagram of the endogenous DNA of Dane djelica. Dane particles can be detected in the blood plasma of some patients with hepatitis B viral infections (D.S. Dane and C.H. Cameron, "Virus-Like Particles in Serum of Patients with Australia-Antigen Associated Hepatitis", The Lancet. 1, pp. 695-698 ( 1970). This fragment is thought to be identical or closely related to the infectious hepatitis B virion. Although its size is known (42 nanometers in cross-section), its structure is not fully understood. Basically, the Dane fragment is believed to consist of an outer layer and an inner part. The outer layer of the particle mainly contains a polypeptide known as hepatitis B surface antigen ("HBsAg"). The inner part (27 nanometers in cross-section) contains another polypeptide, hepatitis B internal antigen ("HBcAg") as well as an endogenous, circular A double-stranded DNA molecule of 2.1 x 106 daltons, containing a single-stranded gap of varying length. A third polypeptide, the "e" antigen ("HBeAg") can also track Dane particles. Furthermore, it is believed that D ane fragment involves an endogenous DNA-dependent DNA polymerase that partially closes the single-stranded gap in the endogenous DNA polymerase reaction using DNA as a "template/template. The structure and properties of Dana delič and its DNA were the subject of several analyses. For example W.S. Robinson, "The Genome of Hepatitis B Virus", Ann.Rev.Microbiol.. 31, pp. 357-377 (1977). However, the actual structure of the various antigens or their genes has not been determined.

Endogenous DNA of Dane djelic was isolated by extraction from Dane djelic. It consists of one nucleotide chain of constant length that has a small notch or opening in it (Figure 1, chain a, ~ 3000 nucleotides) and another nucleotide chain of somewhat variable length (Figure 1, chain b, ~ 2000 nucleotides). The second strand is complementary to the first and overlaps its cut to complete the circular structure by hydrogen bonding between the complementary bases in standard Watson Crick fashion. This overlap can be seen in Figure 1. Dane Delić's DNA polymerase appears to use the second strand of endogenous DNA as a template and the first strand as a template so that it fills in the variable gap with nucleotides that are complementary to the nucleotides of the first strand and produces a double-stranded DNA with about 3000 nucleotide pairs.

Human serum from one HBsAg positive, HBeAg positive donor (serotype adym) was diluted with an equal volume of buffer (0.1 M tris-HCl, 0.1 M saline (NaCl), 0.1% (w/v) 2-mercaptoethanol, 0.1% bovine serum albumin, 0.001 M EDTA) at pH 7.4. This is centrifuged at 35,000 rpm for 2 hours at 4°C. The granule thus obtained is resuspended in 200 μl of the same buffer and stratified on top of a centrifuge tube containing 20% sucrose. The suspension is centrifuged at 40,000 rpm for 2 hours at 4°C. The thus obtained granule is again resuspended in 100 ml of buffer (0.1 M tris-HCl, 0.1 M saline) at pH 7.5.

The resulting granule containing DNA is then labeled with 3H or 32P as described by P.M. Kaplan et.al.. "DNA Polymerase Associated with Human Hepatitis B Antigen", L ViroL, 12, pp. 995-1005 (1973) to facilitate its follow-up at later stages of the proceedings. This labeling is produced by the reaction of concentrated Dane particles and 3H or 32P-labeled deoxynucleoside triphosphates (dNTPs) for 4 hours at 37°C. After this DNA polymerase reaction leading to partial closing of the DNA single-strand gap (see Figure 1), the labeled DNA material is layered on top of a centrifuge tube containing 30% sucrose and centrifuged at 42,000 rpm for 3.5 hours at 4°C.

The DNA is then extracted with phenol from the resulting pellet using the procedure of L.I. Lutwick and W.S. Robinson,, "DNA Synthesized in the Hepatitis B Dane Particle DNA Polymerase Reaction", J.Virol.. 21, pp. 96-104 (1977). The extracted DNA is then dialyzed against a solution of 0.01 M tris-HCl, 0.001 M EDTA (pH 8.0) to eliminate the phenolic solvent. The isolated DNA is then ready to undergo the action of a restriction enzyme. It shows a specific radioactivity of 108 cpm/ug. The process described above is shown schematically in Figure 2.

A wide range of cloning vehicle and host combinations can be successfully used to clone double-stranded DNA isolated as described above. For example, chromosomal, non-chromosomal and synthetic DNA sequences, such as various known bacterial plasmids - pBR322, other E. coli plasmids and their derivatives, as well as plasmids applicable to a wider range of hosts - can be used as cloning carriers, among others. RP4, phage DNA such as numerous phage derivatives, eg, NM899 and vectors derived from combinations of plasmids and phage DNA, such as plasmids modified to use phage DNA control expression sequences. Hosts that can be used include bacterial hosts such as E. coli X1776, E. coliX2282, E. coli HB101 E. coli MRC1, Pseudomonas strains. Bacillus subtilis and other bacilli, yeasts and other fungi, plant or animal hosts, such as plant and animal cell cultures and other hosts. Of course, not all hosts have to be equally efficient. The choice of a particular combination of host and carrier for cloning should be made by a specialist taking into account the above principles, without departing from the scope of the invention.

In addition, different sites within each vector can be chosen for insertion of isolated double-stranded DNA. These sites are usually determined by the restriction enzyme or endonuclease that cuts them. For example, in pBR322, the Pst I site in the penicillinase gene is located between the triplets of nucleotides encoding amino acids 181 and 182 of the penicillinase protein. This site was used by Villa-Komaroff et.al. above. in the synthesis of a protein showing the antigenic determinants of rat proinsulin. The Hind II endonuclease recognition site is between the triplet coding for amino acids 101 and 102 and the Tag site is on the triplet coding for amino acid 45 of that protein in pHBR322. Similarly, the Eco RI endonuclease recognition site for this plasmid is located between the genes coding for resistance to tetracycline and ampicillin. This site was used by Itakura et.al. and Goedel et al. in their recombinant DNA synthetic schemes, supra. Figures 2 and 11 show for illustrative purposes some recognition sites in plasmid pBR322 and phage NM899.

The specific site chosen for insertion of the selected DNA fragment in the cloning vehicle to create a recombinant DNA molecule is determined by various factors. These include the size and structure of the polypeptide to be expressed, the susceptibility of the desired polypeptide to endoenzymatic degradation by host cell components and contamination by its proteins, expression characteristics such as the location of start and stop codons, and other factors that will be understood by those skilled in the art. Since the expression process is not fully understood, none of these factors absolutely independently controls the choice of insertion site for a particular polypeptide. Rather, this site is affected by the balance of these factors, and for a given protein, not all sites have to be equally efficient.

Although several methods are known in the art for inserting foreign DNA into a cloning vehicle to create a recombinant DNA molecule, the preferred method according to the present invention is shown in Figure 2. It is characterized by cleavage of the DNA fragment with a restriction endonuclease, joining the poly-deoxyC sequences to the 3' terminals (named by convention from the deoxyribose carbon of the DNA sugar backbone) of the cleaved DNA and ligating the elongated DNA to a cloning support that had been cut at a specific site by a restriction endonuclease and extended at the 3' termini of that cut with polydeoxyG sequences, which are complementary to poly-dC sequences of interrupted DNA. The complementary character of the 3' tails of the DNA and the cloning support enables the cohesion of these terminals.

To be useful in the method of the present invention, the selected HBV DNA cleavage restriction enzyme should not cleave HBV DNA within a substantial portion of the gene encoding polypeptides exhibiting HBV antigenicity, and most preferably should cleave the DNA at a limited number of sites. Restriction enzymes that have been used in this invention include Kpn I, Bgl II, Bam HI, Ava I and Eco RI. Other restriction endonucleases may similarly be used according to the present invention. Their selection can be made by those skilled in the art based on consideration of the factors presented above without departing from the scope of this invention. Figures 3-9 show some restriction endonuclease recognition sites in part of the HBV genome.

Of course, other known methods for inserting DNA sequences into cloning vehicles to generate recombinant DNA molecules are equally useful in the present invention. These include, for example, direct ligation in which the same restriction endonuclease is used to cleave HBV DNA and the cloning vehicle. This procedure necessarily ensures complementary ends for cohesion.

It should of course be understood that the nucleotide sequence of the gene fragment inserted at the selected restriction site of the cloning vehicle may include nucleotides that are not part of the actual structural gene for the desired protein or may include only a fragment of that structural gene. For polypeptide production, it is only necessary, regardless of which DNA sequence is inserted, that the transformed host produces a polypeptide that exhibits HBV antigenicity.

A recombinant DNA molecule containing a hybrid gene can be used to transform a host so that it enables that host (transformant) to express the structural gene or a fragment thereof and to produce the polypeptide or part thereof encoded by the hybrid DNA.

The recombinant DNA molecule can also be used to transform the host by allowing the host to produce complementary recombinant DNA molecules during replication as a source of HBV structural genes and its fragments. The choice of an appropriate host for any of these applications is controlled by a number of scientifically accepted factors. These include, for example, compatibility with the chosen vector, co-product toxicity, ease of isolation of the desired polypeptide, expression characteristics, safety and cost. Again, as the mechanism of expression is not fully understood, absolute host selection for a particular recombinant DNA molecule or polypeptide cannot be made based on any one of these factors taken alone. Instead, the balance of these factors must be evaluated in the context of the understanding that not all hosts may be equally efficient at expressing a particular recombinant DNA molecule.

In the present synthesis, the preferred cloning vehicle is the bacterial plasmid pBR322, and the preferred restriction endonuclease site therein is the Pst I site (Figure 2). This plasmid is a small (molecular weight approximately 2.6 megadaltons) plasmid that carries genes for antibiotic resistance to ampicillin (Amp) and tetracycline (Tet). The plasmid has been fully characterized (F. Bolivar et. al. "Construction and Characterization of New Cloning Vehicles II. A Multipurpose Cloning System", Gene, pp 95-113 (1977). The preferred host of this invention is E.coli HB101.

1. Creation of dC-elongated DNA by Dana Djelic

In the actual practice of the preferred embodiment of the present invention, DNA isolated as above from the given fragment is digested with restriction endonuclease Kpn I (approximately 20 ng of DNA in 10 µl of 10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 mM 2-mercaptoethanol, 40 mM NaCl, 0.5 μl of enzyme preparation) at 37°C for 90 minutes and the restriction enzyme was inactivated by heating at 70°C for 5 minutes. Poly-deoxyC sequences (described for illustrative purposes only as CCC in Figure 2) were fused to the 3' termini of the digestion products by standard reaction with polynucleotide terminal transferase after removal of residual protein by extraction with phenol and chloroform (20 μl each). Phenol is removed from the aqueous phase by extraction with ether and the DNA is precipitated by adding 3M sodium acetate, pH 6.5 μl) and cold ethanol 0.1 ml. After storage at -70°C for one hour, the settled DNA is isolated by centrifugation at 10,000 rpm for 20 minutes and the pellet is dissolved in 10 mM Tris-HCl pH 7.5 (10 μl). To this is added 3 μl of 400 mM potassium cacodylate, pH 7.0, 4 mM cobalt chloride, 4 mM deoxycytosine triphosphate (dCTP), 200 μl/ml bovine serum albumin and the mixture is incubated at 27°C for 10 minutes with 0, 5 µl of polynucleotide terminal transferase (6000 u/ml) and the reaction was stopped by adding 50 mM EDTA (1 µl).

2. Generation of Pst I-cleaved, dG-elongated pBR322

Plasmid pBR322 was digested with restriction endonuclease Pst I (for which the plasmid contains one target) and the products were purified by phenol extraction and ethanol precipitation as described above for HBV DNA. Poly-dG sequences (described only as GGG in Figure 2 for illustrative purposes) were added to the 3' termini of linear pBR322 molecules using terminal transferase as described for addition of poly-dC sequences to HBV except that the reaction was performed at 37°C.

3. Cohesion of G-elongated pBR322 and C-elongated HBV DNA

Equimolar amounts of pBR322-poly-dG and HBV DNA-poly-dC were mixed together and the complementary sequences were allowed to join by incubation in 100 mM NaCl, 50 mM Tris-HCl, pH 7.5, 5 mM EDTA (TNE), (50 μl ) at 65°C for one hour, and then at 47°C for one hour, 37°C for one hour and 20°C for one hour and then an equal amount of TNE and 20 μl of 100 mM MgCl2, 100 mM Ca Cl2, 100 mM tris-HCl, pH 7.5.

4. Transformation of E. coli HB 101

Cultures of E.coli. Transformation-competent HB 101s were made as described in E.M. Lederberg and S.H. Cohen: "Transformation of Salmonella typhimurium by Plasmid Deoxyribonucleic Acid", J.Bacteriol. 119, pp. 1072-1074 (1974). Aliquots of 0.1 ml of cells were mixed with 25 μl of the supernatant DNA preparation and incubated at 0°C for 20 min and then at 20°C for 10 min before plating onto L-agar plates containing tetracycline (50 μg/ml) for overnight incubation. at 37°C. As plasmid pBR322 includes a gene for resistance to tetracycline, E.coli colonies that have been transformed with the plasmid will grow in cultures containing that antibiotic unlike those E.coli colonies that have not been transformed. Therefore, growth of a tetracycline-containing culture allows the selection of properly transformed hosts.

5. Examination of E. coli colonies using hybridization

Bacterial colonies cultured on incubated L-agar plates containing tetracycline were tested for sensitivity to ampicillin. Plasmid pBR233 includes the ampicillin resistance gene. This gene is the proposed site for the insertion of the hybrid gene. Therefore, colonies that have been transformed with a plasmid having DNA inserted at the chosen recognition site will be sensitive to ampicillin but retain their resistance to tetracycline. E.coli. colonies that were sensitive to ampicillin but resistant to tetracycline were collected on a Millipore cellulose nitrate filter disk supported on L-agar plates containing tetracycline. After overnight growth at 37°C, the Millipore filter was transferred to fresh L-agar plates containing tetracycline and chloramphenicol (150 μg/ml) and incubated for a few more hours at 37°C in order to amplify the number of plasmid copies in the cells ( picture 2). Millipore filters were then used to hybridize colonies as described in M. Grunstein and D.S. Hogness, "Colony Hybridization: A Method for the Isolation of Cloned DNAs that Contain a Specific Gene", Proc. Natl.Acad.Sci. USA. 72, pp. 3961-3965 (1975) with 32 P-labeled HBV DNA made previously as a probe. Radioautography of the filter revealed the presence of colonies containing DNA sequences complementary to authentic HBV DNA.

6. Detection of colonies that synthesize polypeptides with HBV antigenicity

Colonies that were tetracycline-resistant, ampicillin-susceptible, and hybridized with authentic HBV DNA were tested for their ability to produce at least one polypeptide exhibiting antigenic specificity or antigenicity of HBV. Colonies were collected again on Millipore filters supported on L-agar plates containing tetracycline and incubated at 37°C overnight. Another L-agar plate was covered with a culture of E.coli C600 (0.1 ml in 2 ml of soft agar) infected with a virulent type of bacteriophage λ vir (multiplicity of infection about 1) and also growth confluently lysed (i.e. essentially all host cells had burned walls) using phage (λ vir). A Millipore filter containing cells transformed according to this invention was lifted from its plate and placed, colony side down, in contact with the surface of an E. coli C600 L-agar plate that had been lysed with X vir. This contact was continued for about 10 min to allow infection of the cells lying on the Millipore filter with λ vir. The Millipore filter was then transferred to a fresh L-agar plate incubated at 37°C for another 5 hours. Meanwhile polyvinyl discs were coated with HBV antibodies as described in S.Broome and W. Gilbert "Immunological Screening Method to Detect Specific Translation Products", Proc.Natl.Acad.Sci. USA. 75, pp. 2746-2749 (1978). The Millipore filter on which the transformed bacterial colonies were now apparently lysed to bring the colonies into contact with the coated polyvinyl discs and kept at 4°C for 3 hours. The effect of this contact is that all HBV antigens lysed from the cells on the Millipore filter bind with the HBV antibodies of the disc. The coated disks are separated from Millipore filters and then washed and incubated with 125I-labeled HBV antibodies. This incubation leads to radiolabeling of those sites on the disc where the HBV antigens from the Millipore filter have previously become bound by the disc's HBV antibodies. After washout and for autobiography, as described by Broome and Gilbert, supra. colonies that produced polypeptides with HBV antigen specificity were located using the radioautograph as a reference.

The use of polypeptides and genes derived from recombinant DNA molecules in the detection of the presence of HBV antibodies in humans

Polypeptides showing HBV antigenicity and structural genes and fragments that code for them can be used in procedures and equipment sets intended to detect the presence of HBV antibodies in humans, thus identifying people and blood samples infected with this virus.

For example, HBcAg produced by hosts that have been transformed with the recombinant DNA molecules of the present invention can be used in immunological diagnostic tests currently available for the detection of hepatitis B virus, i.e., radioimmunoassay or ELISA (enzyme-linked immunosorbent assay). In one type of radioimmunoassay, an anti-internal antigen antibody, cultured in a laboratory animal, binds to a solid phase, for example, the inside of a test tube. HBcAg is then added to the test tube so that it binds with the antibody. A sample of the patient's serum is added to a tube coated with the antigen-antibody complex, along with a known amount of HBV anti-intrinsic antibody labeled with such a radioactive isotope as radioactive iodine. Any HBV antibody in the patient's serum will compete with the labeled antibody for free binding sites on the antigen-antibody complex. After allowing the serum to react in excess once, the liquid is separated, the tube is washed, and the amount of radioactivity is measured. A positive result, i.e. that the patient's serum contains HBV antibody, is indicated by a low radioactivity count. In one type of ELISA, a microtiter plate is coated with HBcAg and a patient's serum sample is added. After an incubation period that allows any antibody to interact with the antigen, the plate is washed and the anti-human antibody preparation is cultured in a laboratory animal, some enzymatic marker is added to this, incubated to allow the reaction to take place, and the plate is then washed again. After that, the enzyme substrate is added to the microtiter plate and incubated for a period of time that allows the enzyme to work on the substrate, and then the absorbance of the final preparation is measured. A large change in adsorptivity indicates a positive result.

In vivo activity of polypeptides produced from recombinant DNA molecules

To test the biological activity of antigenic polypeptides translated from recombinant DNA molecules in E.coli and in this invention, sterile extracts of bacterial cells that showed expression of HBeAg were injected into rabbits after mixing with an equal volume of Freund's adjunct. Two animals received the crude bacterial extract, and two were given samples of the same extract after fractionation on a Sephadex 650 column. Additional injections of the same sample, which had been frozen and stored (retaining full antigenic activity) were given two and five weeks after the first injection. Animals were bled at intervals of several weeks after the initial injection.

Immunodiffusion experiments were performed using the procedure described in O. Ouchterlony, "Immunodiffusion and Immunoelectrophoresis", in Handbook of Experimental Immunology (D.W. Weir ed.) (Blackwell Scientific Publications, Oxford and Edinburgh) chap. 19 (1967) for comparison of rabbit serum (antibody) with internal human hepatitis B virus antibody ("HBcAb") using HBcAg derived from human liver (BJ. Cohen and Y.E. Cossart, "Application of a Screening test for Antibody to Hepatitis B Core Antigen ", J.Clin.Path.. 30, pp. 709-713 (1977). All four rabbit sera gave precipitin lines with HBCAg identical to those formed between HBcAg and HBcAb derived from human sources. Therefore, the internal antigen was synthesized in E. coli with recombinant DNA molecules of the present invention serologically active in vivo. This activity establishes the practicality of preparations and methods using viral antigens synthesized in microbial cells to stimulate antibody formation in humans. Such preparations and methods are characterized by polypeptides that are produced in transformed hosts using recombinant DNA molecules made according to the invention These polypeptides will be used alone or with a well-known pharmaceutical agent flexible carriers such as saline solutions or other additives that are scientifically accepted for use in preparations and procedures for the treatment and prevention of viral infections in humans.

As previously indicated, restriction enzymes other than the Kpn I/Pst I combination described above may be usefully employed in making the recombinant DNA molecules of the present invention. Other restriction sites in plasmid pBR322 and part of the HBV genome are described in Figures 2 and 3. In illustration of this alternative, but in less preferred embodiments, the following examples are shown.

BAH HI, ECO RI, BGL II-PST I combinations

HBV DNA fragments produced using Bam HI Eco RI and Bgl II, unlike those produced using Kpn I, could not be conveniently traced directly for cohesion due to the presence of short 5' single-stranded overhangs. They were therefore treated with X exonuclease to remove these projections before adding poly-dC sequences at the 3' termini. This was done by incubating restriction DNA (10 μl solution as described before) with 15 μl 100 mM sodium glycinate, pH 9.5, 10 mM MgCl2, 100 μg/ml bovine serum albumin, 5 μl λ exonuclease at 0°C for 1.5 hours. The mixture is then extracted with phenol and chloroform and the process is continued. In the preparation in which Bam HI was used, subsequent radioimmunoassay confirmed the production of polypeptides with HBV antigenic specificity.

Direct ligation: ECO RI-ECO RI AND BAM HI-BAM HI

Instead of tracing DNA fragments for cohesion, as described above, direct ligation of gene fragments can be used in the methods of the present invention. For example, in two further preparations, HBV DNA (20 ng) was restricted with restriction endonuclease Eco RI or BAM HI in 10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 mM 2-mercaptoethanol, 50 mM NaCl (10 μl ) at 37°C for 1.5 hours and was mixed with an excess of pBR322 that had been incubated with the same enzymes under identical conditions. Concentrated buffer solution (660 mM Tris-HCl, pH 7.5, 100 mM MgCl2, 10 mM EDTA, 100 mM 2-mercaptoethanol, 400 mM NaCl, 1 mM ATP) (2 μl) was added and the mixture was incubated with T4 DNA ligase ( 0.5 μl) at 10°C for 3 hours, after which at 0°C for at least 24 hours. The solution was diluted to 0.1 ml with 10 mM Tris-HCl, pH 7.5 and used to transform competent cultures of E.coli HB101 as described above. In the case of Bam HI-made recombinant DNA molecules, colonies were tested, prior to the hybridization assay, for sensitivity to tetracycline, not ampicillin, because the target for Pam HI in pBR 322 is within the gene encoding tetracycline resistance, hence the successful insertion on this at the recognition site leads to loss of this resistance instead of resistance to ampicillin as in the case of insertion at the Pst I site.

In the case of recombinant DNA molecules made via Eco RI sites, colonies were tested, prior to hybridization testing, for both ampicillin and tetracycline resistance because the target for Eco RI in pBR322 is between the genes encoding tetracycline and ampicillin resistance or tetracycline resistance after of hybrid DNA insertion at this site.

Examples of microorganisms made using the procedures described herein are cultures deposited in the Culture Collection of the Microbiological Research Establishment at Porton Dwn on December 15, 1978 and identified as pBR322-HBV-A to F.

Specifically, these cultures are characterized as follows:

A: E.coli HB 101/pBR322 -Pst I dG:HBV -Kpn I dC:

TetR AmpS HBV+ VA+

B: B.coli HB 101/pBR322 -Pst I dG:HBV -Bani HI dC:

TetR AmpS HBV+ VA+

C: E.coli HB 101/pBR322 -Pst I dg: HBV -Bgl II dC:

TetR AmpS HBV+

D: E.coli HB 101pBR322 -Pst I dg:HBV -Eco RI dC:

TetR AmpS HBV+

E: E. coli HB 101/pBR322 - Bam HI: HBV - Barn HI

TetR AmpS HBV+

F: E.coli HB 101/pBR322 -Eco RI-.HBV -Eco RI

TetR AmpS HBV+

Portions of these identical cultures were also deposited in the Culture Collection of the National Collection of Industrial Bacteria, Aberdeen, Scotland, 19 December 1979.

The above nomenclature for cultures is a description of that culture as grayed out: Host/cloning carrier with indication of nucleotide elongation (if present): restriction site in hepatitis B virus with indication of elongation (if present): resistance (R) or sensitivity (S) to tetracycline (Tet) and ampicillin (Amp), positive hybridization to HBV DNA in the colony hybridization test, supra. (HBV+) and the production of a polypeptide showing HBV antigenicity, supra. (VA+). Using this nomenclature, the pBR322-HBV-A culture means an E.coli HB 101 culture containing as a plasmid a recombinant DNA molecule comprising pBR322 interrupted at the Pst I site and extended with a poly-dC tail at the 3' termini, whereby the culture shows resistance to tetracycline , sensitivity to ampicillin, positive HBV DNA hybridization test and produces a polypeptide that shows HBV antigenicity.

Of course, it should be understood that hybrid microorganisms, recombinant DNA molecules and methods applicable thereto are not limited to those described in the preferred embodiment above. Instead, hybrid organisms of recombinant DNA molecules and polypeptides can be modified during production or later using known methods to good advantage. For example, more efficient expression control sequences can be used for transcription of HBV genes or hybrid genes, mutations can be introduced to reduce synthesis of unwanted gene products, protease levels in host cells can be reduced, thermo-inducible lysogens containing HBV genes can be integrated into the host chromosome or other modifications and procedures can be carried out to increase the number of gene copies in the cell or to increase the productivity of the cell in the production of the desired polypeptides.

In addition to their use for the production of polypeptides exhibiting HBV antigenicity, the hybrid microorganisms of the present invention are also useful for the production of large quantities of DNA containing all or part of the hepatitis B virus genome. For example, amplification, eg by adding chloramphenicol to the growth medium when the cell density has reached a suitable level, or by using mutations to prevent the cleavage of bacteriophage hybrids containing HBV sequences allows the production of HBV DNA in quantities not previously accessible.

HBV DNA made in this way can be used to determine the nucleotide sequence of the genome and, based on that, the sequence of the genes and amino acids of the HBV antigen and the structural genes themselves. Knowledge of these sequences aids in understanding hepatitis B virus biology and allows the modifications described above to be used to best advantage.

Creating a DNA fragment map and determining the nucleotide sequence

1. Hepatitis B internal antigen

A series of recombinant DNA molecules, made according to the procedures described above, were chosen for sequence analysis, which gave their host cells the ability to synthesize HBcAg as detected by solid phase radioimmunoassay. Fragments were made from these recombinant DNA molecules by digestion with appropriate restriction enzymes and the fragments were labeled at their 5' termini with λ -32P) ATP and T4 half-nucleotide kinase. The nucleotide sequences of each fragment were then determined using well known chemical degradative procedures (A.M.Maxam and W.Gilbert, "A New Method for Sequencing DNA", Proc.Nat.Acad.Sci.USA. 74, pp. 560-564, (1977 ). The resulting nucleotide sequence is described in Figures 3-9. For reference, the sequence is numbered from A ATG to the translation initiation codon of the internal gene. The nucleotide sequence of the gene for HBcAg a and the amino acid sequence of the polypeptide derived from this gene (indicated box 1) is described in Figures 3-9 between nucleotides 1-549. The size of the polypeptide encoded by this gene is close to the molecular weight of 19000 observed for internal antigens from the Dana fragment. However, the current structural determination does not exclude the possibility that some amino acids may be cleaved with of the amino terminus of this polypeptide in vivo during authentic antigen formation.This structure also does not account for any other further modifications to the polypeptide caused by by its interaction with other human enzymes, for example with those enzymes that carry out protein glycosylation. Therefore, the polypeptide structure as determined here may be identical to that of HBcAg found in vivo. but will still induce a very similar, if not identical, immune response.

All tested recombinant DNA molecules had HBV DNA inserted such that the internal antigen gene was maintained in the same translation phase as the penicillin resistance gene of pBR322. Moreover, in various recombinant HBV DNA the insert started within one or two nucleotides of the same part in the HBV sequence. Because these recombinant DNA molecules are derived from HBV DNA that has been digested with various restriction enzymes, e.g., Bam HI and Kpn I, this unique junction is surprising and could have arisen by inadvertent cleavage or polynucleotide tracing of BHV DNA at a nick in the endogenous DNA of the fragment. (picture 1). Recombinant DNA molecules having an HBV DNA insert in a different translational phase did not express the HBcAg gene and no polypeptide showing HBV antigenicity was detected in hosts transformed with such recombinant DNA molecules. Of course, it should be understood that such hosts that do not express the gene and recombinant DNA molecules are still useful for the production of HBV DNA according to the present invention.

Although it was expected that HBcAg or its fragments produced by expression of HBV DNA inserts would be fused to the product (β-lactamase) of the penicillinase resistance gene via a small number of glycine residues, this was not the case. Instead, the β-lactamase was in each case fused over 5-8 glycines for the same peptide sequence, but this sequence ended after 25 amino acids. In this indicated frame (Frame 1, Figure 3) the stop codon (TAG) is followed by three nucleotides and later by the initiation codon, from which translation continues unhindered so that a polypeptide with a length of 183 amino acids is obtained (Figures 3-8). Therefore, the activity of the internal antigen lies in a polypeptide of about 21,000 daltons translated de novo from the HBV sequence within the mRNA transcribed from the recombinant DNA molecule. This polypeptide remains inside the host cell except that it is not bound to the secreted penicillinase carrier protein due to the arrangement of the stop and start codons of the HBV DNA insert.

2. Hepatitis B surface antigen

The nucleotide sequence of the HBsAg gene and the amino acid sequence of the polypeptide derived from this gene (Marked Box 3) is also described in Figures 6-8 between nucleotides 1437-2114. The amino acid sequence of this polypeptide begins at the N terminus with the sequence met-glu-asn-ile-thr-ser. This initial amino acid sequence was determined by D.L. Peterson et al. "Partial Amino Acid Sequence of two Major Component Polypeptides of Hepatitis B Surface Antigen". Proc. Natl. Acad.Sci.USA. 74, pp. 1530-1534, (1977) from authentic human HBsAg. The amino acid sequence of this protein continues in Figures 6-8 to a stop codon of amino acids 226. This 226 polypeptide sequence corresponds to a 25,400 dalton protein.

The HBsAg amino acid sequence and length were independently confirmed by sequencing the corresponding recombinant DNA molecules in P.Valenzuela et.al.. "Nucleotide Sequence of the Gene Coding for the Major Protein of Hepatitis B Virus Surface Antigen", Nature. 280. pp. 815-819 (1979).

The nucleotide sequence determined for the recombinant DNA molecules of this invention shows that none of the investigated nucleotides expressing the HBcAg gene can have the HBsAg gene in a position from which expression in the corresponding host cell can also be expected. In fact, no HBsAg was detected in extracts of cells transformed with those plasmids found to express the HBcAg gene. However, as indicated above, knowledge of the nucleotide sequences of these genes allows modification of the expression process to improve yield and effect and to favor gene expression and production of polypeptides not previously expressed or detected.

Creation of improved recombinant DNA molecules for HBV antigen production

The gene and amino acid sequences of these antigens are useful in planning procedures for increasing the level of antigen or gene production per bacterial cell.

The level of protein production is controlled by two main factors: the number of copies of its gene in the cell and the efficiency with which these gene copies are transcribed and translated. The efficiency of transcription and translation (which together constitute expression) again depends on the nucleotide sequence, which normally precedes the desired coding sequence. These nucleotide sequences or expression control sequences define, among other things, the location at which RNA polymerase interacts to initiate transcription (promoter sequence) and at which ribosomes bind and interact with mRNA (transcription product) to initiate translation. Not all such sequences function to control expression with equal efficiency. Thus, it is preferred to separate the specific coding sequences for the desired protein from their adjacent nucleotide sequences and to condense them, rather than to known expression control sequences, so that higher levels of expression are favored. When this is achieved, the newly constructed DNA fragment can be inserted into a plasmid for multicopying the bacteriophage derivative in order to increase the number of gene copies in the cell and thus further improve the yield of the expressed protein.

For illustrative purposes, the sequence determined for the HBcAg gene was used in this way due to the improvement of HBcAg production in E.coli. One such procedure is described in Figure 10.

Inspection of the HBcAg DNA sequence in Figure 3 reveals that this gene is preceded by a target for Alu I at nucleotide-26 (AGCT) and contains targets for Eco RI* at nucleotides 22 and 1590, for Eco RII at 209 (CCTGG) for Hae III at 246 and 461 (GGACC, GGTCC). Given this complexity, it is convenient to transfer the internal antigen coding sequence to more efficient sequences to control expression in two phases. For example, digestion of a recombinant DNA molecule, made according to the present invention, having an HBV DNA insert of about 2350 base pairs (nucleotides), i.e., from nucleotides -80 to about 2270 of Figure 3, with both Alu I and Eco RI* gives, among others, the fragment between nucleotides -26 and 22 (fragment A), while digestion only with Eco. RI* gives a fragment from nucleotides 23 to 1589 (Fragment B) and a fragment from nucleotides 23 to 576 (Fragment B) in low yields. These fragments can be easily identified in Figures 3-4 and are described schematically in Figure 10. Fragments A and B are fractionated by gel electrophoresis to purify them and joined via their Eco RI* cohesive ends to give a combined (Fragment C). Fragment C is now fused to a new expression control sequence as needed either by direct ligation to maintain correct translation phase, by the 3' tracing process as described earlier, or by synthesis of oligonucleotide linkers. This splicing not only provides a better expression control sequence to improve protein production but also allows the gene fragment encoding HBcAg to be fused closer to the expression control sequence itself so that expression control of that gene fragment is enhanced. In a similar manner, Fragments A and B' can be joined or a number of other fragments can be made and the resulting fragment used as above. This procedure demonstrates that only the portion of the gene that codes for a particular polypeptide should be used in the recombinant DNA molecules of the present invention.

To further shorten the distance between a particular expression control sequence and the initiation codon of a selected gene fragment, the particular fragment can easily be treated with a combination of nucleases that act specifically at or near its terminus or used in exonuclease and polymerase splicing reactions to remove some or all of nucleotide fragments preceding the start codon of the fragment. Alternatively, a fragment such as the Alu I fragment resulting from cleavage of nucleotides -26 and 30 can be similarly truncated by treatment with exonuclease or polymerase ligation side reactions and then cleaved with Eco RI* to produce a fragment from between nucleotides -26 and 1 and nucleotide 22 to allow condensation for fragment B prior to splicing for the expression control sequence. A further method could involve hybridization of fragment B, or an equivalent fragment, to a suitable single-stranded template from the basic recombinant DNA molecule for serial extension reactions with DNA polymerase and with a limited number of nucleoside triphosphates so that the strand of the fragment can be reconstructed at the beginning of the coding sequence. This template will then be cleaved at the site of its junction with the extended fragment by S1 endonuclease and the resulting fragment binds to the expression control sequence.

Several expression control sequences can be used as described above. This includes the operator, promoter and ribosome binding and interaction sequences (including sequences such as Shine-Dulgano sequences) of the E.coli lactose operon ("lac system"), the corresponding sequences of the E.coli tryptophan synthetase system ("trp system"), the major operator and promoter regions of phage λ (OLOL and ORPRI) and the control region of phage fd protein coating. DNA fragments containing these sequences are cut by restriction enzyme digestion from DNA isolated from transducing phages λ or fd. These fragments are then manipulated in the manner described for the HBV antigen sequences in order to obtain a limited population of such molecules such that essential control sequences can be joined very closely, or

exactly opposite the initiation codon of the coding sequence for the desired antigen as described above for Fragment C.

The condensation product is then inserted as before into a cloning vehicle for transformation into appropriate hosts and the level of antigen production is measured by radioimmunoassay after cell disruption. Thus, the cells that give the most efficient expression can be chosen. Alternatively, cloning vehicles can be used that carry a lac, trp or λ PL control system fused to the initiation codon and fused to a fragment containing the coding sequence for a polypeptide expressing HBV antigenicity such that the structural gene fragment is correctly translated from the initiation codon cloning carrier.

Although these experiments refer to the production of specific HBV internal antigen, they can be applied to improve the expression of other genes such as HBsAg and HBeAg genes and their fragments.

Further increases in cellular yield of these particular antigens depend on increasing the number of genes that can be used in the cell. This was achieved for illustrative purposes by inserting recombinant DNA molecules constructed in the manner described earlier into the mold bacteriophage λ (NM899), most simply by digesting the plasmid with a restriction enzyme, eg Eco. RI or Hind III, so that a linear molecule is obtained which can then be mixed with a restrictive phage cloning carrier (eg of the type described in N.E.Murrav et.al. "Lambdoid Phages that Simplify the Recovery of In Vitro Recombinants", Molec .gen.Genet.. 150, pp. 53'61 (1977) and N.E.Murray et.al. "Molecular Cloning of the DNA Ligase Gene from Bacteriophage T4". J.Mol.Biol.. 132, pp. 493-505 (1979) and recombinant DNA molecules produced by incubation with DNA ligase. Such a procedure is described in Figure 11. The desired recombinant phage is then selected by radioimmunoassay for a specific antigen or by hybridization with radiolabeled HBV DNA sequences and then used to lysogenize the host species E. coli.

Particularly suitable λ carriers for cloning contain a temperature-sensitive mutation in the cI repressive gene and suppressive mutations in the S gene, the product of which is required for host cell disruption, in the E gene. whose product is the main capsid protein of the virus. With this system, lysogenic cells are cultured at 32°C and then heated to 45°C to induce prophage cleavage. Prolonged growth at 37°C leads to high levels of antigen production, which is retained in the cells, since these are not cleaved by the phage gene product in the normal way, and since the phage gene insert is not encapsulated, it remains accessible for transcription. Artificial disruption of the cells then releases the antigen in high yield (Figure 11).

In addition to the E.coli system to which these examples mainly referred, the same type of manipulation can be performed in order to increase antigen production on other microbial cells, such as B.subtilis. thermoplastic bacteria or yeasts and fungi, or in animal or plant cells in culture. In the case of B.subtilis a plasmid carrying determinants for penicillinase isolated from B.licheniformis provides a useful expression control sequence for these purposes.

Construction of improved recombinant molecules for expression of the gene coding for HBsAg

Certain HBsAg gene and amino acid sequences are useful in designing methods for enabling expression of the HBsAg gene using the method of the present invention. Such expression has not previously been observed in hosts transformed with recombinant DNA molecules producing polypeptides expressing HBV antigenicity.

The nucleotide sequence of Figure 6-8 shows the gene coding for HBsAg between nucleotides 1437 and 2114. This gene is in a different translational phase (marked box 3) than the gene coding for HBcAg (marked box 1) in the HBV genome Figure 3-9. Therefore, a recombinant DNA molecule that did not produce HBcAg when used to transform a suitable host, but contained an HBV DNA insert of about 2350 nucleotides (corresponding to approximately nucleotides -80 to 2270 of the sequence from Figure 3-8) was chosen for use in the production of HBsAg.

The selected recombinant DNA molecule contained, among other things, the entire gene for coding HBsAg. Examination of the nucleotide sequence of the HBV DNA insert of this recombinant DNA molecule reveals several targets for the restriction endonuclease, which allows cutting of the fragments containing the gene coding for HBsAg. For example nucleotide 1409 (Xho), nucleotide 1410 (Tag), nucleotide 1409 (Ava I) and nucleotide 1428 (Hhal) (Figure 6). Of these, the latter (Hha I) is particularly useful because cleavage of the HBV DNA insert occurs between nucleotides before the translational initiation codon (AGT) of the HBsAg gene itself. In all four cases, the excised fragment will extend beyond the selected HBV DNA insert toward a target for a particular restriction endonuclease located within the nucleotide sequence of the pBR322 portion of the recombinant DNA molecule. Due to the use of these restriction endonucleases and others similarly useful, alone or in combination, it allows cutting the gene that codes for HBsAg close to, but before its initiation codon, and after its translational termination codon. Of course, it should be clear that as illustrated in the case of the HBcAg gene, only fragments of the entire gene should actually be used in recombinant DNA molecules that exhibit HBsAg antigenicity in appropriate hosts.

HBV DNA fragments derived from such digests were treated in a series of reactions analogous to those described for the gene and gene fragments coding for the internal antigen. For example, a gene fragment can be inserted into the penicillin resistance gene of pBR322 (eg, the Pst I recognition site, Figure 2) to produce a polypeptide that exhibits HBsAg antigenicity in association with β-lactamase (the product of the penicillase gene), gene fragments can be inserted between the genes encoding penicillin resistance and tetracycline resistance in pBR322 (Eco RI or Hind III recognition sites, Figure 2) and joined to produce a polypeptide that exhibits HBsAg antigenicity in association with /3-galactosidase protein of the lac system, the gene fragment can be inserted into the cloning vehicle as close as possible to the selected expression control sequence itself as described above for the gene encoding HBcAg, or the gene fragment can be cloned in another way as described according to the present invention.

For illustrative purposes, one such scheme is depicted in Figure 12. Here, a selected recombinant DNA molecule having an HBV DNA insert extending between about nucleotides -80 and 2270 (Figures 3-8) is fragmented by digestion with Hha I. The resulting fragment is inserted by the 3' tracing procedure as described previously on Pst and site pBR322. Hosts transformed with this recombinant DNA molecule produced polypeptides expressing HBsAg antigenicity.

Other fragments (eg, Ava I, Taq. Xho. or Pst I (partial digest)) were also inserted at the Pst I recognition site or other sites in pBR322 to produce recombinant DNA molecules useful for the production of polypeptides expressing HBsAg antigenicity or gene fragments coding for HBsAg.

Furthermore, such gene fragments were inserted into pBR322 which had been cleaved with Pst I and the resulting fragment of the gene encoding penicillinase was then cleaved from the codon encoding amino acid 182 of penicillinase or β-lactamase in one case to the codon encoding amino acid 32 and in the second case to the codon that codes for amino acid 13 of that polypeptide. These derivative plasmids pBR322 -Pst I' were also used with HBsAg gene fragments extended from nucleotides 1447 to 1976 (Figures 6-7) to produce polypeptides expressing HBsAg antigenicity.

The nucleotide sequence between nucleotides 948 and 1437 immediately preceding the structural gene encoding HBsAg can also be included in fragments used to produce useful recombinant DNA molecules for the production of polypeptides exhibiting the HBsAg antigenicity of the structural gene for HBsAg. This nucleotide sequence is called the precursor sequence of the gene. Gene fragments including both this precursor sequence and the structural gene for HBsAg can be excised with Alu I (nucleotide 939), Hha I (nucleotide 900), or Hae II (nucleotide 899). In the case of Alu and Hha, both partial digestion and separation of the resulting fragments, eg by gel electrophoresis, are necessary because the recognition sites for these enzymes also occur within the precursor sequence at nucleotide 1091 for Alu I and at nucleotide 1428 for Hha II.

Although we have shown here a number of embodiments of the invention, it is clear that our basic construction can be modified to provide other embodiments that use the process of this invention. Therefore, it should be understood that the scope of this invention is limited by the appended claims and not by the specific embodiments set forth in the examples hereinbefore.

Examples of microorganisms obtained by the process described herein are cultures deposited in the Culture Collection of the National Collection of Industrial Bacteria, Aberdeen, Scotland, December 19, 1979 and identified as pBR322 HBV-G-L and characterized as follows:

G: E.coli HB 101/pBR322-Pst I dG: HBV -Kpa I dC:

(herein late "pHBV114"): TetR AmpS HBV+

H: E.coli HB 101/pBR322-Pst I' dG: pHBV114-Pst I:

TetR AmpS HBV+ VA+

I: E.coli HB 101/((pBR322-Bco RI, Bam HI : lac promoter sequence)-Hind III:

Binding elements:

HBV114-HhaI Bam HI:

TetS AmpR HBV+ VA+

J: E.coli HB 101/pUR2* (see note)-Bco RI:HBV114-HaIBcoRI

connecting elements:

TetS AmpR HBV+ VA+

K: E.coli HB 101/pBR322-Pst I dG: pHBV114-Avo I dC

TetR AmpS HBV+ VA+

L: E.coli HB 101/pBR322-Pst I dC: pHBV114 Tag dC:

TetR AmpS HBV+ VA+

note: a derivative of pBR322 having the DNA sequence including the gene encoding the tetracyclic resistance polypeptide replaced with a smaller DNA sequence including the E.coli lac system.

Claims (11)

1. A method for obtaining polypeptides that have the antigenicity or antigenic specificity of the core antigen or the surface antigen of the hepatitis B virus, characterized in that it consists in the cultivation of a host transformed with a recombinant DNA molecule containing a DNA sequence, wherein said DNA sequence is operatively linked to the sequence for control of expression in a recombinant DNA molecule so that, during the cultivation of said host transformed with said molecule, expression of a polypeptide, or a part of a polypeptide, is carried out, which shows the antigenicity or antigenic specificity of the core antigen or the surface antigen of the hepatitis B virus. 2. The method according to claim 1, characterized in that the DNA sequence, which codes for a polypeptide that shows the antigenicity or antigenic specificity of the core antigen or the surface antigen of the hepatitis B virus, is selected from the endogenous DNA of Dane's corpuscle, its fragments that code for a polypeptide that shows antigenicity or antigen specificity of the core antigen or surface antigen of the hepatitis B virus, or its synthetically derived equivalents. 3. The method according to claim 1, characterized in that the DNA sequence that codes for a polypeptide that shows the antigenicity or antigenic specificity of the core antigen or surface antigen of the hepatitis B virus is obtained by isolating a recombinant DNA molecule from a host that has been transformed by it and then removing said DNA sequence or its fragment from the said molecule. 4. The method according to claim 1, characterized in that the polypeptide showing the antigenicity or antigenic specificity of the hepatitis B virus is selected from the polypeptide of the formula: MetAspIleAspProTyrLysGluPheGlyAlaThrValGluLeuLeuSer PheLeuProSerAspPhePheProSerValArgAspLeuLeuAspThrAla AlaAlaLeuTyrArgAspAlaLeuGluSerProGluHisCysSerProHis HisThrAlaLeuArgGlnAlaIleLeuCysTrpGlyAspLeuMetThrLeu AlaTHrTrpValGlyThrAsnLeuGluAspProAlaSerArgAspLeuVal ValSerTyrValAsnThrAsnValGlyLeuLysPheArgGlnLeuLeuTrp PheHisIleSerCysLeuThrPheGlyArgGluThrValLeuGluTyrLeu ValSerPheGlyValTrpIleArgThrProProAraTyrArgProProAsn AlaProIleLeuSerThrLeuProGluThrThrValValArgArgArgGly ArgSerProArgArgArgThrProSerProArgArgArgArgSerGlnSer ProArgArgArgArgSerGlnSerArgGluSerGlnCys, as well as their fragments that show antigenicity or antigenic specificity of hepatitis B virus core antigen. 5. The method according to claim 1, characterized in that the polypeptide showing the antigenicity or antigenic specificity of the hepatitis B virus is selected from the polypeptide of the formula: MetGluAsnIleThrSerGlyPheLeuGlyProLeuLeuValLeuGlnAla GlyPhePheLeuLeuThrArgIleLeuThrIleProGlnSerLeuAspSer TrpTrpThrSerLeuAsnPheLeuGlyGlyThrThrValCysLeuGlyGln AsnSerGlnSerProIleSerAsnHisSerProThrSerCysProProThr CysProGlyTyrArgTrpMetCysLeuArgArgPheIleIlePheLeuPhe IleLeuLeuLeuCysLeuIlePheLeuLeuValLeuLeuAspTyrGlnGly MetLeuProValCysProLeuIleProGlySerSerThrThrSerThrGly SerCysArgThrCysThrThrProAlaGlnGlyIleSerMetTyrProSer CysCysCysThrLysProSerAspGlyAsnCysThrCysIleProIlePro SerSerTrpAlaPheGlyLysPheLeuTrpGluTrpAlaSerAlaArgPhe SerTrpLeuSerLeuLeuValProPheValGlnTrpPheValGlyLeuSer ProIleValTrpLeuSerValIleTrpMetMetTrpTyrTrpGlyProSer LeuTyrSerIleLeuSerProPheLeuProLeuLeuProIlePhePheCys LeuTrpAlaTyrIle, as well as their fragments that show the antigenicity or antigenic specificity of the hepatitis B virus surface antigen. 6. The method according to claim 1, characterized in that the host is selected from E.coli species. Pseudomonas. Bacillus subtilis. other Bacillus. yeasts, other fungi, or animal cells in culture. 7. The method according to claim 1, characterized in that the expression control sequence is selected from the lac system, trp. system, the main operator and promoter regions of phage λ and the control region of the phage coat protein fd. 8. The method according to claim 1, indicated by that. which is the DNA sequence selected from the DNA sequences of the formula: \y ATGGACATTGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTCTCGT TTTTGCCTTCTGACTTCTTTCCTTCCGTACGAGATCTTCTAGATACCGCCGC AGCTCTGTATCGGGATGCCTTAGAGTCTCCTGAGCATTGTTCACCTCACCAT ACTGCACTCAGGCAAGCAATTCTTTGCTGGGGAGACTTAATGACTCTAGCTA CCTGGGTGGGTACTAATTTAGAAGATCCAGCATCTAGGACCTAGTAGTCAG TTATGTCAACACTAATGTGGGCCTAAAGTTCAGACAATTATTGTGGTTTCAC ATTTCTTGTCTCACTTTTGGAAGAGAAACGGTTCTTAGGTATTTTGGTGTCTT TTGGAGTGTGGATTCGCACTCCTCCAGCTTATAGACCACCAAATGCCCCTAT CCTATCAACGCTTCCGGAGACTACTGTTGTTAGACGACGAGGCAGGTCCCCT AGAAGAAGAACTCCCTCGCCTCGCAGACGAAGATCTCAATCGCCGCGTCGCA GAAGATCTCAATCTCGGGAATCTCAATGT ; fragments thereof that code for polypeptides showing the antigenicity or antigenic specificity of the hepatitis B virus core antigen; and DNA sequences that are degenerate as a result of the genetic code to any of the preceding DNA sequences and that encode polypeptides that exhibit the antigenicity or antigenic specificity of the hepatitis B virus core antigen. 9. The method according to claim 1, characterized in that the DNA sequence is selected from DNA sequences of the formula: ATGGAGAACATCACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCG GGGTTTTTCTTGTTGACAAGAATCCTCCAATACCGCAGAGTCTAGACTCG TGGTGGACTTCTCTCCAATTTTCTAGGGGGAACTACCGTGTGTCTTGGCCAA AATTCGCAGTCCCCAATCTCCAATCACTCACCAACCTCCTGTCCTCCAACT TGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATCTTCCTCTTC ATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTATCAAGGT ATGTTGCCCGTTTGTCCTCTAATTCCAGGATCATCAACCACCAGCACGGGA TCCTGCAGAACCTGCACGACTCCTGCTCAAGGAATCTCTATGTATCCCTCC TGTTGCTGTACAAAACCTTCGGATGGAAACTGCACCTGTATTCCCATCCCA TCATCCTGGGCTTTCGGAAAAAATTCCTATGGGAGTGGGCCTCAGCCCGTTC TCTTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCC CCCATTGTTTGGCTTTCAGTTATATGGATGATGTGGTATTGGGGGCCAAGT CTGTACAGCATCTTGAGTCCCTTTTTACCGCTGTTACCAATTTTCTTTTGT CTTTGGGCATACATT ; fragments thereof that code for polypeptides that show the antigenicity or antigenic specificity of the surface antigen of the hepatitis B virus; and DNA sequences that are degenerate as a result of the genetic code to any of the preceding DNA sequences and that encode polypeptides that exhibit the antigenicity or antigenic specificity of the hepatitis B virus surface antigen. 10. The method according to claim 1, characterized in that the recombinant DNA molecule is selected from recombinant DNA molecules contained in transformed E.coli species. HB101, identified under NGIB accession numbers 11548, 11549, 11559, and 11560. 11. The method according to claim 1, characterized in that the transformed host is selected from the transformed species E.coli HB101, identified under NCIB accession numbers 11548, 11549, 11599 and 11560.