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WO1988010301A1 - Vaccin contenant un antigene de surface de l'hepatite b - Google Patents

Vaccin contenant un antigene de surface de l'hepatite b Download PDF

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Publication number
WO1988010301A1
WO1988010301A1 PCT/EP1988/000551 EP8800551W WO8810301A1 WO 1988010301 A1 WO1988010301 A1 WO 1988010301A1 EP 8800551 W EP8800551 W EP 8800551W WO 8810301 A1 WO8810301 A1 WO 8810301A1
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Prior art keywords
peptide
hbv
dna molecule
recombinant dna
host cell
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PCT/EP1988/000551
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English (en)
Inventor
Hans A. Thoma
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Mccormick And Jones Engineering Ltd.
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Priority to PCT/EP1988/000551 priority Critical patent/WO1988010301A1/fr
Priority to KR1019890700312A priority patent/KR970007153B1/ko
Priority to AU19958/88A priority patent/AU619753B2/en
Publication of WO1988010301A1 publication Critical patent/WO1988010301A1/fr
Priority to US08/484,408 priority patent/US6117653A/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32411—Hepatovirus, i.e. hepatitis A virus
    • C12N2770/32422—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32611—Poliovirus
    • C12N2770/32622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/82—Proteins from microorganisms
    • Y10S530/826—Viruses

Definitions

  • the invention relates to Hepatitis B surface antigen ("HBs antigen” or "HBsAG”) particles which are composed of polypeptides prepared by recombinant DNA processes, DNA sequences coding for these polypeptides end cell lines for the expression of the same.
  • HBs antigen or "HBsAG”
  • the present invention relates especially to new particles having increased immunogenicity.
  • polypeptide antigens produced in vivo are heavily glycosilated (Gerlich, 1984: J. Virol.: 52 (2), 396).
  • glycosilation is not an essential process so that polypeptides produced by genetically engineered bacteria are either not glycosilated or are incompletely glycosilated.
  • polypeptides corresponding to HBsAg when expressed in bacteria, do not raise antibodies which will see HBsAg sufficiently well for an effective vaccine.
  • yeast as a eukaryotic host is capable of more complete glycosilation, polypeptides corresponding to HbsAg expressed in yeast share the same deficiency as in the case of bacterial expression.
  • the eukaryotic structural gene of the HBsAg is in most cases not efficiently transcribed. Furthermore the structure and function of the eukaryotic HBsAg gene product may be dependent on the additional post-translational processes of the linkage of disulfide bonds which can not be accomplished by the bacterial host.
  • the expressed polypeptide is rarely secreted from the bacterial host cells. They must be lysed to harvest the expressed polypeptide. During the purification process bacterial wall components may contaminate the polypeptide and cause serious allergic reactions or lead to anaphylactic shock in patients.
  • eukaryotic promoters usually do not work in bacteria and must be substituted by a bacterial promoter which can result in modification of the polypeptide expressed.
  • HBV Hepatitis B virus
  • HBV-virion (Dane particle), which is thought to be the infectious material, - the filaments, and the 20 or 22 nm particles (hereinafter "20 nm particle”) which consist only of a protein envelope.
  • the most interesting form for an efficient vaccine is the 20 nm particle because 1) the coding sequences are entirely known, 2) it is completely uninfectious, and 3) it causes some useful immunogenicity in a human organism.
  • the three known components of HBV particles differ in their relative amounts of the protein composition. There are three monomers called the major protein with 226 amino acids, the middle protein with 281 amino acids , and the large protein with 389 or 400 amino acids, depending on the subtype ayw and adw. respectively.
  • the large protein is encoded by the complete sequence of the pre-S 1 -, pre-S 2 - and S-regions, whereas the middle protein is derived from only the pre-S 2 - and S- regions, and finally the major protein from only the S-region (Tiollais et al., 1985: Nature, 317, 489; Dubois et al., 1980: PNAS, 77, 4549; McAlzer et al., 1984: Nature, 307, 178).
  • the infectious virion of HBV contains 40-80 times more of the high molecular monomers - the pre-S 1 and Pre-S 2 peptides - compared to the 20 nm particle. It is now known that these pre-S polypeptides may be associated with some biological and clinical implications.
  • the polyalbumin receptor on the pre-S polypeptides can bind polymerized albumins from humans and chimpanzees which are susceptible to HBV (Thung et al., 1983: Liver, 3, 290; Machida et al., 1984: Gastroenterology, 86, 910).
  • Literature data would also suggest a better protection against the infectious Dane-particle where the pre-S, epitope is present in much higher ratio than on the envelope particles.
  • the vaccine obtained from natural sources which causes a limited immunogenic protection, contains (almost) none of the pre-S proteins; this is due to two different reasons.
  • the 20 nm particles are isolated from sera of anti-HBE positive carriers (Hevac B, HepaVac B) or are digested by proteases during the purification process. This proteolytic digestion has been shown to cut the pre-S-polypeptides leaving only the S monomers. As a result these vaccines contain none or very little pre-S polypeptides.
  • EP-A-72 318 describes the expression of HBsAg in yeast cells, which have been transformed by a vector comprising a yeast replicon, a yeast promoter and a DNA sequence coding for the S peptide,
  • Laub et al. J. Virol., Vol. 48, No. 1, pp. 271-280, 1983, disclose the construction of a vector starting from simian virus 40 into which the HBsAg including the 163 codon precursor sequence was incorporated. Laub et al. report that CV-1 cells transformed with said vector yield a better expression when the vector contains only the coding sequence for the S protein as compared to the above vector which comprises additionally also the 163 codon precursor sequence.
  • Japanese Patent Application No. J5-8194-897-A describes the expression of the entire pre-S and S peptides. Reference is also made to the expression of the adw subtype.
  • DE-OS 34 39 400 describes the expression of an immunogenic polypeptide sequence of Hepatitis B virus.
  • Said sequence represents a partial sequence of the pre-S. polypeptide, comprises 108 or 119 codons and starts with the first starting codon of HBsAg, and terminates 281 codons in front of the stop codon.
  • EP-A-154 902 discloses a Hepatitis B vaccine which contains a peptide with an amino acid chain of at least six consecutive amino acids within the pre-S chain coding region of the envelope of Hepatitis B virus. This vaccine is free of an amino acid sequence corresponding to the naturally occurring envelope proteins of Hepatitis B virus.
  • the object of this invention is the development of protein particles which contain an amount of the pre-S polypeptide epitopes comparable to the natural composition of the surface structure of the infectious Dane particle.
  • HBsAg in mammalian cells. This requires overcoming known difficulties where expression of the desired peptide in a mammalian cell can result in: - different regulatory mechanisms for the three translational/(transcriptional) products - promoter-promoter inhibition - different strength of the start codons - not all peptides expressed.
  • HBV S peptide refers to the peptide encoded by the entire S region of the HBV genome.
  • HBV pre-S, peptide refers to the peptide encoded by the entire pre-S 2 and S regions of the HBV genome.
  • HBV pre-S 1 peptide refers to the polypeptide encoded by the entire pre-S 1 , pre-S 2 and
  • epitope refers to a sequence of at least six consecutive amino acids encoded by the designated genome region (e.g., a "HBV pre-S, epitope” refers to a sequence of at least six amino acids encoded by the pre-S 2 region of the HBV genome).
  • antigenicity means the ability to provoke an immune response (e.g., acting as a vaccine or an antigen), the ability to cause the production of antibodies (e.g. acting as an antigen) and/or the ability to interact with a cell surface receptor so as to enhance an immune response or production of antibodies (e.g., reacting with a T-cell surface receptor to enhance immune response).
  • HBV means any subtype of the virus, particularly adw, ayw, adr and ayr, described in the literature (P. Valenzue Nature Vol. 280, p. 815 (1979), Gerlich, EP-A-85 111 361, Neura EP-A-85 102 250). Examples of peptide sequences thereof, from which the epitopes of this invention can be derived, are shown in
  • recombinant DNA molecules which comprise a first DNA sequence an a second DNA sequence.
  • the first DNA sequence encodes for expression of an amino acid sequence a portion of which displays the antigenicity of an epitope selected from the grou consisting of an HBV pre-S 1 epitope and an HBV pre-S 2 epitope.
  • the second DNA sequence encodes for expression of a peptide which upon secretion will form particles which are at least 10 nm in diameter. These particles are believed to be the smallest particles which will effectively form a good vaccine.
  • the peptide which upon secretion will for particles which are at least 10 nm in diameter is either HBV S peptide, HBV core antigen, polio surface antigen.
  • HBV S peptide HBV core antigen
  • HBV core antigen HBV core antigen
  • polio surface antigen HBV S peptide
  • Hepatitis A surface antigen Hepatitis A core antigen
  • HIV surface antigen HIV surface antigen
  • HBV S peptide A substantial portion or all of the HBV S peptide is especially preferred as the peptide encoded by the second DNA sequence.
  • the recombinant DNA molecules encoding for the first and second DNA sequences must be (1) in the same reading frame, (2) encode for respective discrete regions of a single peptide, and (3) be operatively linked to an expression control sequence. Finally, these recombinant DNA molecules are free of DNA sequences encoding for the expression of the entire HBV pre-S 1 peptide or HBV pre-S 2 peptide.
  • the first DNA sequence comprises a nucleotide sequence corresponding to the nucleotide sequence of (I) the HBV pre-S 1 and pre-S 2 regions from which the pre-S 2 start codon ATG has been deleted, (2) the HBV pre-s 1 and pre-S 2 regions and wherein the sequences flanking the pre-S- 1 ATG have been changed from the natural sequence, (3) the HBV pre-S 1 and pre-S 2 regions and wherein the sequences flanking the pre-S 2 ATG have been changed from the natural sequence, (4) the HBV pre-S 1 and pre-S 2 regions and wherein the 5' terminus of the pre-S 1 region has been deleted, (5) the HBV pre-S 1 and pre-S 2 regions and wherein the 5' terminus of the pre-S 2 region has been deleted, (6) the HBV pre-S 1 region and wherein the 3' terminus of the pre-S 1 region has been deleted, (7) the HBV pre-S 2 region
  • Host cells transfected with the recombinant DNA molecules of the present invention are also disclosed.
  • transfected or transfection refer to the addition of exogenous DNA to a host cell whether by transfection, transformation or other means.
  • Host cells include any unicellular organism capable of"transcribing and translating recombinant DNA molecules including without limitation mammalian cells, bacteria and yeast.
  • Host cells of the present invention may also be cotransfected with a second recombinant DNA molecule comprising a DNA sequence encoding for expression of an ammo acid sequence corresponding to a substantial portion or all of the amino acid sequence of the HBV s peptide.
  • Peptides comprising a first discrete region and a second discrete region.
  • the first region displays the antigenicity of an epitope of an HBV prs-S 1 epitope or an HBV pre-S 2 epitope.
  • the second region correspond to a substantial portion of a peptide which upon secretion will form particles which are at least 10 nm in diameter.
  • the peptide which upon secretion will form particles which are at least 10 nm in diameter is either HBV S peptide, HBV core antigen, polio surface antigen.
  • Hepatitis A surface antigen Hepatitis A core antigen, HIV surface antigen and HIV core antigen.
  • a substantial portion or all of the HBV S peptide is especially preferred.
  • the first region is located closer to the N-terminus of the peptide than the second region.
  • Immunogenic particles ara also disclosed which comprise a plurality of first peptide monomers. Each of said first peptide monomers comprises a first discrete region and a second discrete region which can be the same as the first and second discrete regions of the peptides described . above. Immunogenic particles are also disclosed which further comprise a plurality of second peptide monomers and wherein the first and second peptide monomers are bound together by interactive forces between the monomers. Each of said second peptide monomers comprising an amino acid sequence corresponding to a substantial portion of or all of the amino acid sequence of the HBV S peptide.
  • Immunogenic particles are also disclosed which contain substantially more than one percent, preferably more than five percent, of the pre-S. epitope.
  • a particle "contains one percent" of a designated epitope if peptide monomers having the designated epitope constitute one percent of all protein in the particle.
  • Immunogenic particles which contain substantially more than ten percent, preferably more than fifteen percent, of the pre-S 2 epitope are also disclosed.
  • compositions and preparations useful for production of antibodies comprising the above-described immunogenic particles in sufficient concentration to elicit an immune response upon administration of said preparation and a suitable carrier are also disclosed.
  • Suitable carriers are known to those skilled in the art and may include simple buffer solutions.
  • Other preparations useful for production of antibodies are disclosed comprising the above-described immunogenic particles in sufficient concentration to elicit an immune response upon administration of said preparation and a suitable carrier.
  • Suitable carriers are known to those skilled in the art and may include simple buffer solutions.
  • a process for producing a transfected host cell comprises providing host cells which have been made competent for uptake of DNA, exposing the host calls to a first preparation of DNA comprising one of the above-described recombinant DNA molecules, allowing under suitable conditions the host cells to take up DNA from the first preparation of DNA, and selecting for host cells which have taken up exogenous DNA.
  • the process may further comprise exposing the host cells to a second preparation of DNA comprising a DNA molecule encoding for a peptide includin ⁇ the amino acid sequence of the HBV S peptide and allowing under suitable conditions the host cell's to take up DNA from the second preparation of DNA.
  • the exposure and uptake of the second preparation of DNA can be done before or after exposure to and uptake of the first DNA preparation.
  • the first DNA preparation can also include a DNA molecule encoding for a peptide including the amino acid sequence of the HBV S peptide.
  • a method for producing a peptide comprises preparing an above-described recombinant DNA molecule, transfecting a host cell with the recombinant DNA molecule, culturing the host cell under conditions allowing expression and secretion of protein by the host cell, and collecting the peptide produced as a result of expression of DNA sequences within the recombinant DNA molecule.
  • the peptide produced by such method can contain less than the entire amino acid encoded by the coding region of the recombinant DNA molecule. This may result from transcription and/or translation of only a portion of the coding region of the recombinant molecule or by deletions made in the peptide after translation.
  • a method of producing immunogenic particles comprising preparing an above-described recombinant DNA molecule, transfecting a host cell with the recombinant DNA molecule, culturing the host cell under conditions allowing expression and secretion of protein by the host cell, and allowing under suitable conditions the aggregation of peptide monomers produced as a result of expression of exogenous DNA sequences within the host cell.
  • a method of producing immunogenic particles is also disclosed which further comprises transfecting (cotransfection) the host cell with a DNA molecule encoding for a peptide including the amino acid sequence of the HBV S peptide. The cotransfection can occur before, after or simultaneous with the transfection of the above-described recombinant DNA molecule. Presence of peptides encoded by the cotransfected DNA molecule are necessary to obtain more than trace amounts of particles secreted from the host cell.
  • Methods of manufacturing a pharmaceutical preparation and a preparation useful for production of antibodies comprising preparing an above-described recombinant DNA molecule, transfecting a host cell with the recombinant DNA molecule, culturing the host cell under conditions allowing expression and secretion of protein by the host cell, allowing under suitable conditions the aggregation of peptides produced as a result of expression of DNA sequences within the host cell to form immunogenic particles, and combining the immunogenic particles with a suitable carrier such that the immunogenic particles are present in sufficient concentration to cause production of antibodies upon administration of a preparation to an individual.
  • Host cells used in these methods can also be cotransfected as previously described.
  • Figures I to X show gene constructs according to the present invention.
  • Figures XI and XII show the results obtained by caesium chloride sedimentation of immunogenic particles according to example 10.
  • Figure XIII shows a prior art construct shown in Table XI.
  • Figure XIV shows the characterisation of particles derived from a construct according to a further embodiment of the present invention.
  • Figure XV shows the oligo sequence of Figure X-2.
  • Figure XVI to XX show examples of peptide sequences from which the epitopes of this invention can be derived.
  • Preferred DNA constructs of the present invention are characterized by the presence of a selection marker selected from the group consisting of dhfr (dihydrofolate reductase), MT-neo (a neomycin resistance sequence coupled to a methallothionein and MT-ecogpt (a resistance seguence coupled to a methallothionein promoter).
  • a selection marker selected from the group consisting of dhfr (dihydrofolate reductase), MT-neo (a neomycin resistance sequence coupled to a methallothionein and MT-ecogpt (a resistance seguence coupled to a methallothionein promoter).
  • the expression rate may be further enhanced by adding to the constructs a dhfr gene as an amplification gene.
  • HBV nucleotide sequences used in certain constructs of the present invention can be formed or isolated by any means including isolation and ligation of restriction fragments and synthetic oligonucleotides.
  • Constructs specifically described herein were formed by the ligation of synthetic oligonucleotides to a 5' Xbal-Bglll 3' fragment from the S region of the HBV genome shown in Figure IX (hereinafter the "Xbal-Bglll fragment") which is derived from a Bglli-Bglll HBV fragment including the entire pre-S 1 -pre-S 2 -S regions (the "BgllI-Bglll Fragment"), The pre-S 1 pre-S 2 -S region of the HBV genome is shown in Figure IX. Oligonucleotides used in making such constructs are summarized in Table I.
  • oligonucleotides in Table I were combined with the Xbal-Bglll fragment to produce constructs with desired features.
  • adapter oligonucleotide sequences (Table II) were used to create proper matching sticky ends on the oligonucleotides and other construct components.
  • adapter sequences may be used to combine desired oligonucleotides from Table I with the Xbal-Bglll fragment, other restriction fragments, oligonucleotides and other construct components.
  • the necessary sequences of such other adapter sequences will be readily apparent to those skilled in the art from consideration of tables of restriction sites [e.g., that found at pages 121-128 of Mathods in Enzymology. volume 152, "Guide to Molecular Cloning Techniques," ed, Berger and Kimmel (Academic Press 1987) which is incorporated herein in its entirety by reference] and the sequences of tha various nucleotides to be combined.
  • Adapter sequences can also ba used to introduce additional restriction sites into constructs of the present invention. It should be noted that adapter sequences must be selected or designed so that the proper reading frame is maintained throughout the HBV sequence.
  • Each of the constructs shown in Figures I-VIII contain, in addition to a HBV sequence, a neomycin selection marker with the MT promoter, an ampicillin selection marker, a dhfr selection/amplification gene and a promoter for the HBV sequence.
  • the promoter for the HBV sequence is preferably the U2 promoter, the MT promoter or the H2K promoter. Isolation of fragments containing the various promoters, the selection markers and amplification gene is described below.
  • Tha HBV sequences in the constructs of Figures I-VIII are schematically- represented by a rectangular bar in each figure which indicates the oligonucleotides and/or adapter sequences from Tables I and I ⁇ which were combined with the Xbal-Bglll fragment. Shaded areas within the bar indicate generally regions of the entire pre-S 1 -pre-S 2 -S region which are not found in the specific construct. Oligonucleotides from Table I which can be used to construct each type of HBV sequence are indicated in the figures,
  • Figure X depicts two additional constructs for expression of peptides including sequence from the pre-S2 region under the control of the MT promoter.
  • the above-cited promoters are specially preferable when their use is coupled with a modulation method using the dhfr gene and methotrexate to enhance the expression. This is achieved when in addition to the selection marker the dhfr minigene is also introduced into the plasmid sequence. It is essential that the dhfr gene is located on the same plasmid together with the structural gene to be expressed. An enhancement of the expression rate of the structural gene can then be obtained by adding methotrexate in the micromolar concentration range. Thereby a rnanyfold enhancement of the expression rate is achieved.
  • Suitable cells are e.g. VERO cells (monkey kidney cell line), 3T3-cells (murine fibroblast line), C127-cells (murine fibroblast line), L-cells and CHO - cells (Chinese hamster cells, which are either positive or negative in dehydrofolate reductase).
  • HBV protein refers generically to any protein produced in accordance with the present invention which corresponds to HBsAg sequences.
  • the supernatant of HBV protein producing cultures was collected and split into portions of 2,400 ml. To each portion 144 g of PEG 6000 (Serva) were added and dissolved by stirring at room temperature for 20 minutes and was stirred for another 6 hours at 4oC. The precipitate was separated by centrifugation in 500 ml bottles in a GS 3 rotor at 9,000 rpm (15,000 x g) for 30 minutes at 10 C The supernatant was collected and 144 g of PEG 6000 were addsd and dissolved as described above. The solution was stirred at 4 C for 3 hours. The precipitate from this solution was harvested as described above except that centrifugation was continued for 60 minutes.
  • the radioactivity remaining on the beads was counted in a gamma scintillation counter.
  • the plasmid pBPV-342-12 (commercially available from ATCC) was digested with the endonucleases Bglil and BamHI. Three DNA molecules were generated.
  • the fragment of interest contains the methallothionein promoter and a pBR322 sequence comprising 4.5 kb and is easily detectable from the other fragments (2.0 kb and 7.6 kb).
  • the reaction was performed in a total volume of 200 ul of reaction buffer at a final concentration of 0.5 ug/ul DNA including 100 units of each restriction enzyme. The completion of the digestion was checked after incubation at 37°C for three hours by agarose gel electrophoresis at a 0.8% agarose gel. The reaction was stopped by adding 4 ul 0.5 M EDTA.
  • the 4 .5 kb fragment was separated from the other f ragments by preparative 1.2% agarose gel electrophoresis.
  • the DNA was eluted from the agarose gel on DE-81 Whatman filter paper from which the DNA was removed in a high salt buffer.
  • the DNA was purified by a phenol/chloroform extraction and two ethanol precipitations. 2) Ligation of the 2.3 kb HBV BgllI-Bglll fragment
  • a 2.3 kb Bglli-Bglll fragment containing the HBV pre-S 1 ,pre-S 2 and S coding regions was isolated from HBV-containing DNA.
  • the 2-3kb fragment was ligated together with the 4.5 kb fragment (obtained as described in Cl) containing the methallothionein promoter,
  • the ligation mixture was added to 150 ul competent bacterial cell suspension for DNA up-take. After the DNA up-date the bacterial cells were spread on LB agar plate containing 50 ug/ml ampicillin at volumes of 50 to 300 ul cell suspension per plate. The agar plates were incubated at 37oC overnight. Single isolated bacterial colonies were screened for the presence of a plasmid containing the desired fragments.
  • the plasmid resulting from (3) above was digested with the endonucleases Bglll and Xbal. Two molecules were expected, one 550 bp fragment and one 6.250 kb fragment which was isolated after agarose gel electrophoresis. The 6.250 kb fragment was ligated together with oligomecleotide No.55 from Table I. The ligation mixture was added to 150 ul competent bacterial cell suspension for DNA up-take. single isolated bacterial colonies were screened for the presence of the desired plasmid. The new plasmid was proved by a digestion with the endonucleases EcoRI and Bglli. Two molecules were expected, one 1.9 kb and one 4.450 kb.
  • the plasmid resulting from (4) above was linearized by digestion with the restriction enzyme EcoRI.
  • the reaction was performed in a total volume of 50 ul and a final concentration of 1 ug/ul plasmid DNA. 50 units of EcoRI were added and the digestion was proved after incubation at 37oC for three hours by agarose gel electrophoresis.
  • the reaction was stopped by adding 1 ul of 0.5 M EDTA and the DNA was precipitated with a final concentration of 0.3 M sodium acetate and 3-4 volumes of ethanol at -80°C for 30 minutes.
  • the precipitated DNA was dissolved in 50 ul distilled water.
  • 2 ul of the linearized plasmid were mixed with 3 ul of the DNA fragment containing the methallothionein promoter and the neomycin selection gene [isolated from the plasmid pMT-neo-E (available from. ATCC ) by digestion with the endonuclease EcoRI as a 4kb fragment], and ligated together. Single bacterial colonies were screened for the presence of the desired plasmid.
  • the plasmid pdhfr3.2 (available from ATCC) was digested with the restriction endonuclease Hindlll. Two molecules were generated, one of 3,000 bp containing the dhfr gene sequence and one of 3,400 bp. The 3,000 bp fragment was isolated and ligated into the plasmid resulting from (5) above which was previously opened by digestion with Hindlll. The resulting plasmid is represented by Fig. I-2.
  • the plasmid pUC-8-42 (available from Exogene ) was digested with the restriction endonucleases EcoRI and Apal. Two DNA molecules were generated.
  • the fragment of interest contains the U2-promoter comprising 340 bp and is easily detectable from the other fragment (3160 bp).
  • the digestion was performed in a total volume of 200 ul of reaction buffer at a final concentration of 0.5 ug/ul DNA including 100 Units of each restriction enzyme.
  • the completion of the digest was checked after incubation at 37oC for three hours by agarose gel electrophoresis in a 0.7% agarose gel.
  • the reaction was stopped by adding 4 ul 0.5 M EDTA.
  • the 340 bp fragment was separated from the plasmid DNA by preparative 1.2% agarose gel electrophoresis.
  • the DNA was eluted from the agarose gel on DE-81 Whatman filter paper from which the DNA was removed in a high salt buffer.
  • the DNA was purified by a phenol/chloroform extraction and two ethanol precipitations.
  • the plasmid pSP165 (commercially available from Promega Biotec) containing a polylinker sequence (containing the following restriction sites: EcoRI, SacI, Smal, Aval, BamHI, Bglll, Sail, Pstl, Hindlli) was linearized with the restriction enzyme EcoRI. The reaction was performed in a total volume of 50 ul and a final concentration of lug/ul plasmid DNA. 50 Units of EcoRI was added an the digestion was proved after incubation at 37oC for three hours by agarose gel electrophores.
  • the reaction was stopped by adding 1 ul of 0.5 M EDTA and the DNA was precipitated with a final concentration of 0.3 M sidium acetate and 3-4 volumes of ethanol at -80oC for 30 minutes.
  • the precipitated DNA was dissolved in 50 ul distilled water.
  • 2 ul of plasmid DNA were mixed with 10 ul of the fragment DNA containing the V2 promoter sequence, and ligated together in a total volume of 25 ul of ligation buffer containing 2 units T4-DNA ligase and 2 mM ATP at 14oC overnight. Thereafter the DNA was purified by phenol/ chloroform extractions followed by two ethanol precipitations and dissolved in 10 ul distilled water. The resulting sticky ends of EcoRI and Apal had to be converted into blunt ends and ligated.
  • the blunt ends were converted by a removing reaction with the Mung bean nuclease as follows: to 25 ul DNA (1 ug/ul concentration) reaction buffer, 20 units of enzyme and a final concentration of 1% glycerol to the reaction volume of 35 ul were added. After an incubation for 30 minutes at 30 C tha DNA was purified by phenol/chloroform extractions followed by two ethanol precipitations. The DNA was dissolved again in 5 ul distilled.water. The resulting blunt ends were ligated together in 15 ul reaction volume containing 10 x more T4 ligase then used above and 2 mM ATP at 14°C overnight.
  • the ligation mixture was added to 150 ul competent bacterial cell suspension for DNA up-take. After the DNA up-take the bacterial cells were spread on LB agar plates containing 50 ug/ml ampicillin at volumes of 50 to 300 ul cell suspension per plate. The agar plates were incubated at 37oC overnight. Single isolated bacterial colonies were screened for the presence of a plasmid containing the desired U2-promoter fragment.
  • the plasmid pBPV-342-12 (commercially available from ATCC) was digested with the endonucleases EcoRI and BamHl. Two molecules were isolated, one containing the MT promoter together with the neomycin selection gene of 4,000 bp and the plasmid of 10,000 bp.
  • the plasmid resulting from (3) above was linearized with EcoRI and ligated together with the 4,000 bp fragment containing the MT-promoter together with tha neomycin selection gene. The resulting sticky ends were also converted into blunt ends and ligated together as described above.
  • the plasmid resulting from (4) above was linearized with Bglll.
  • the 2.3 kb-Bglll-Bglll fragment was ligated together with the linearized plasmid.
  • Bacterial colonies were analysed to find the resulting plasmid.
  • the plasmid-DNA was digested with EcoRI and two resulting fragments were obtained, a 700 bp fragment (containing the promoter and a part of the HBV-sequence) and a 8,700 bp fragment (containing the rest of the HBV-sequence, MT-neo and plasmid).
  • the plasmid resulting from (5) above was digested with the endonucleases Bglll and Mstll. Two molecules were generated, one of 300 bp containing part of the pre-S sequence and the other (9,100 bp) which was eluted as described above. This 9,100 bp fragment was ligated to another Bglll/Mstll 216 bp fragment (sequence AGATCTACAGCATGGGGCAGAATCTTTCCACCAGCAATCCTCTGGGATTCTTTCCCGA Bglll S1 CCAGTTGGATCCAGCCTTCAGAGCAAACACCGCAAATCCAGATTGGGACTTCAATCC
  • the desired plasmid was digested with EcoRI and two resulting fragments were isolated, a 616 bp fragment and a 8,700 bp fragment.
  • the H2K promoter was isolated as an EcoRI/Bglll fragment (2kb) from psp65H2 (available from Exogene).
  • the fragment containing the methallothionein promoter and the egpt-selection gene was isolated by digestion of the plasmid pMSG (available from Pharmacia) with the . restriction enzyme EcoRI as a 3.6 kb fragment.-
  • mammalian cells In order to achieve secretion of substantial amounts of the HBV peptides encoded by constructs of the present invention, mammalian cells must be transfected with both the construct of the present invention and a construct which will express entire S protein.
  • the cotransfection was performed in two steps (i.e., a separate transfection for each construct) or in a single step (i.e., one transfection using preparation of both constructs). Cotransfection was confirmed either by use of different selection markers on the two constructs or by detection of secretion of expression products of both constructs by immunoassay.
  • a sequence encoding the HBV peptide sequence of the present invention and a separate sequence encoding the entire S protein could be combined in a single construct.
  • restriction endonucleases used were: Bglll, BamHl, Hindlll, EcoRI, xbal, Mstil, Xhol, PflMI commercially available from Gibco/BRL with their respective restriction buffers (10x).
  • restriction digests were performed and fragments were isolated as follows. Reactions typically contained 1-5 ug DNA. distilled water was added to the DNA in an eppendorf tube to a final volume of 8 ul
  • agarose gel normally contains 0.8% agarose 1 x running buffer (TBE, Maniatis). Where a fragment (about 100-1000bp) was isolated from an agarose gel the agarose was increased to 1.2 to 1.4%.
  • OD 600 0.7 which was reached within 2 hours with vigorous shaking in a 500 ml Erlenmeyer flask. Growth was stopped by chilling the culture on ice for 10 minutes. From this culture, 3 ml were taken for harvesting the exponential bacterial cells at 3,000 rpm for 5 minutes. The cells were resuspended in 1.5 ml of 50 mM CaCl 2 in 10 mM Tris, pH 8.0, and incubated on ice for another 15 minutes. The cells were harvested once more by centrifugation at 3,000 rpm for 5 minutes and resuspended in 200 ul of 50 mM CaCl 2 in 10 mM Tris, pH 8.0, and used directly.
  • the DNA to be transformed was suspended in 10 mM Tris, pH 7.5, 1 mM EDT 70 ul and added to the 200 ul bacterial cell suspension for DNA take-up. The mixture was incubated on ice for 30 minutes and then 1 ml L-broth was added. The mixture was incubated at 42°C for 2 minutes and at 37oC for 40 minutes.
  • the cells were spread on agar plates containing 50 ug ampicillin/ml agar at volumes of 50-300 ul cell suspension per plate.
  • the agar plates were incubated at 37oC overnight. After this incubation period, single isolated bacterial colonies were formed.
  • plasmid-bearing cells 1 liter was grown to 0.5 OD 600 in L-broth and amplified for 20 hours with 200 ug/ml chloramphenicol. The culture was then centrifuged at 4,000 rpm for 20 minutes in JA-10 rotor, 4 °C . The pellet was resuspended in 18 ml cold 25% sucrose, 50 mM Tris, pH 8.0, transferred to a 250 ml Erlenmeyer flask and kept on ice. 6 ml 5mg/ml lysozyme in 250 mM Tris, pH 8.0 was added and the mixture was left to stand 10-15 minutes.
  • Pronase was added to supernatant fluid to 250 ug/ml and incubated 30 minutes, 37oC.
  • the solution was extracted with phenol once with 1/2 volume phenol equilibrated with 10 mM Tris, pH 8.0, 1 mM EDTA.
  • the aqueous layer was removed.
  • Sodium acetate was then added to a final concentration of 300 mM, followed by the addition of 3volumes cold 100% ethanol and thorough mixing. The mixture was stored at -20oC overnight.
  • the mixture was thawed and centrifuged.
  • the pellet was resuspended in 6 ml 10 mM Tris, 10 mM EDTA, pH 8.0.
  • 9.4 g CsCl and 0.65 ml of 6 mg/ml ethidium bromide were added and the volume was brought up to 10 ml with sterile double-distilled water.
  • the 10 ml alignots were put into Beckman heat-sealable gradient tubes and centrifuged, 50,000 rpm, 48 hours in Ti70.1 Beckman rotor.
  • Plasmid bands were visualized with UV and removed with syringe and 18 gauge needle by piercing the side of the tube. Ethidium bromide was removed from the plasmid fractions by 3 successive extractions with equal volumes of isobutanol. Fractions were then (1) dialyzed against one 2-liter lot of 10 mM Tris, pH 7.4, 1 mM EDTA, pH 7.5, 5 mM NaCl for 2 hours or more at 4 °C; and (2) phenol extracted once with 1/3 volume phenol equilibrated as above. Sodium acetate was then added to a final concentration of 300 mM, followed by addition of two volumes of 100% ethanol. Precipitate formed at -20oC overnight, or at -70oC for 30 minutes. 5) Mini-Plasmid Preparation
  • Tris pH 8.0
  • 10 mM EDTA pH 8.0
  • 200 ul of 0.2 N NaOH, 1% SDS was added, mixed by vortex and incubated for 5 minutes on ice.
  • 150 ul 3 M Sodium acetate (pH 4.8) was added, mixed by vortex and incubated for 5 minutes on ice.
  • After centrifugation for 5 minutes at 13,000 rpm the supernatant was decanted into a fresh eppendorf tube. 3 volumes of 100% ethanol were supplemented, mixed well and incubated for 30 minutes at -80oC, then centrifuged for 10 minutes at 13,000 rpm. The ethanol was removed, the pellet washed with 70% ethanol, lyophilized and dissolved in 20 ul distilled water. 5 ul of this plasmid DNA solution were used directly for restriction analysis.
  • chromosomal DNA from cell lines producing particles of this invention were isolated and digested with the appropriate restriction enzyme(s) and analysed by the method of Southern (J. Mol. Biol., Vol. 98, pp. 503-517, 1975), which is incorporated herein by reference, using a 32 P-labeled DNA probe.
  • the resulting fragments were separated by 0.7% agarose gel electrophoresis. Thereafter, the DNA was denatured by exposing to 366 nm UV light for 10 minutes and by incubation in a solution of 0.5 M NaOH and 1 M NaCl for 45 minutes. The gels were neutralized by incubation in 0.5 M Tris, 1,5 M NaCl, pH 7.5 for 60 minutes. The DNA was transferred to a nitrocellulose filter by soaking in 3 M NaCl, 0.3 M Sodiumcitrate (20 x SSC) for 20 hours through tha gel by covering the top of the nitrocellulose filter with a staple of dry paper towels. The nitrocellulose filter was kept for 2 hours in a vacuum oven at 80 C. A radioactive DNA probe from the Bglll fragment of the pHBV (2.3 kb) was prepared by nick translation.
  • the nitrocellulose filter was sealed in a plastic bag containing 10 ml of prehybridization mixture: 50% formamide, 5 x SSC, 50 mM Sodiumphosphate, pH 7.0, 5 x Denhardt's solution, 250 ug/ml denatured salmon sperm DNA.
  • the filter was incubated in this mixture for 4 hours at 45°C, after which the pre-hybridization mixture was replaced by the hybridization mixture: 50% formamide, 5 x SSC, 20 mM Sodiumphosphate, pH
  • the filter was dried at 60oC for 10 minutes and exposed to two X-ray films (XAR-5, KODAK) between two intensifying screens and kept at -80oC.
  • the first X-ray film is developed after 3 days' exposure; the second film after 7 days' exposure.
  • the recipient cells (C127 or CHO-cells available from ATCC)we seeded in normal growth medium (DMEM+10% Fetal Calf Serum,Glyco Glutamin) into petri-dishes (1-2 x 10 cells per dish, 4 10 cm) at day 1. The next day the medium was removed (4 hours before the DNA precipitate was added onto the cells), and the cells were washed twice with 1 x PBS. Then 8 ml DMEM without FCS were added. 4 hours later the DNA precipitate (prepared as described below) was added to tha cells.
  • DMEM+10% Fetal Calf Serum,Glyco Glutamin normal growth medium
  • petri-dishes 1-2 x 10 cells per dish, 4 10 cm
  • Glycerol-Mix 50 ml 2.x TBS buffer, 30 ml glycerol, 120 ml distilled water
  • the Glycerol-Mix was immediately removed after an incubation at 37oC for 3 minutes and the cells were washed with 1 x PBS.
  • the cells were cultivated overnight with 8 ml of DMEM with 10% FCS.
  • the calls ware recovered from the dish by- treating With Trypsin-EDTA-Solution (0.025% Trypsi ⁇ + 1 mM EDTA). Afterwards, to remove the Trypsin-EDTA the cells were washed with 1 x PBS, suspended in DMEM with 10% FCS and distributed into 24 costar-well-plates (cells from one dish into four 24-well-plates).
  • selection medium concentration 0.5 - lmg/ml of neomycin,or xanthine: 250 ⁇ g/ml, hypoxanthine: 15 ⁇ g/ml (or adenine: 25 ⁇ g/ml), thymidine: 10 ⁇ g/ml, aminopterine 2 ⁇ g/ml mycophenolic acid: 25 ⁇ g/ml for eco-gpt, for example).
  • the medium was changed every week. The first growing cell colonies were seen after 2 weeks.
  • TE-buffer 10 mM Trix-HCl, 1 mM EDTA, pH 7.05 was added to a final volume of 440 ul and mixed together with 60 ul 2 M CaCl 2 . Then the same amount of 2x TBS (Hepes 50 mM, NaCl 280 mM, Na 2 HPO 4 1.5 mM, pH 7.05) was added and mixed well. The precipitation solution was incubated for 30 minutes at 37oC and added directly to the cells which should be transfected.
  • 2x TBS Hepes 50 mM, NaCl 280 mM, Na 2 HPO 4 1.5 mM, pH 7.05
  • Tables III - X give some of the results of ELISA analysis of immunogenic particles of the present invention as described below:
  • Table III shows the ELISA data of the purified HBs antigen particle produced from any HBV sequence construct of the present invention including the pre-S 1 region with total deletion of pre-S 2 and deletions upstream of the pre-S 2 ATG and the S region with deletion of the S ATG and downstream the S ATG through the XBal site (e.g. the construct of Fig. 1-1) with the anti-pre-S 1 monoclonal antibody MA 18/7.
  • the fractions 9-15 (Fig. XI) were pooled after CsCl sedimentation.
  • Table IV shows the ELISA data of the purified HBS antigen particle produced from any HBV sequence construct of the present invention including the pre-S 1 region with total deletion of pre-S 2 and deletions upstream of the pre-S 2 ATG and the S region with deletion of the S ATG and downstream the S ATG through the XBal site (e.g., the construct of Fig. 1-1) with the anti-pre-S 2 monoclonal antibody MQ 19/10.
  • the fractions 9-15 (Fig. XI) were pooled after CsCl sedimentation.
  • Table V shows the ELISA data of tha purified HBs antigen particle produced from an HBV sequence construct of the present invention including the pre-S 2 region with none of the pre-S 1 region and deletions upstream of the S ATG and downstream of the S ATG through the XBal site, and the S region with deletion of the S ATG (e.g. the construct of Fig. II-1), with the anti-pre-S 1 monoclonal antibody MA 18/7.
  • Table VI shows the ELISA data of the purified HBS antigen particle produced from an HBV sequence construct of the present invention including the pre-S 2 region with none of the pre-S 1 region and deletions upstream of the S ATG and downstream of the S ATG through the XBal site, and the S region with deletion of the S ATG (e.g. the construct of Fig. II-l) with the anti-pre-S 2 monoclonal antibody MQ 19/10.
  • the fractions 9-15 (Fig. XII) were pooled after CsCl sedimentation.
  • Table VII shows the ELISA data of the purified HBs antigen particle produced from any HBV sequence construct of the present invention including the pre-S 1 region with total deletion of pre-S 2 and deletions upstream of the pre-S 2 ATG and the S region with deletion of the S ATG .
  • Table VIII shows the ELISA data of the purified HBs antigen particle produced from any HBV sequence construct of the present invention including the pre-S 1 region with deletions upstream of the pre-S 2 ATG with deletion of the S ATG (e.g., the construct of Fig. VI-4) with the anti-pre-S 2 monoclonal antibody MQ 19/10.
  • the fractions 9-15 (Fig. XT) were pooled after CsCl sedimentation.
  • Table IX shows the ELISA data of the purified HBs antigen particle produced from an HBV sequence construct of the present invention including the pre-S 2 region with none of the pre-S 1 region and deletions upstream of the S ATG . and the S region with deletion of the S ATG
  • Fig. XII the fractions 9-15 (Fig. XII) were pooled after CsCl sedimentation.
  • Table X shows the ELISA data of the purified HBs antigen particle produced from an HBV sequence construct of the present invention including the pre-S 2 region with deletions upstream of the S ATG with deletion of the S ATG (e.g., the construct of Fig. VII-4) with the anti-pre-S 2 monoclonal antibody MQ 19/10.
  • the fractions 9-15 (Fig. XII) were pooled after CsCl sedimentation.
  • Table XI shows the ELISA data of purified HBs antigen particles produced by construct including the entire pre-S 1 - pre-S 2 - S region under control of the LTR region of rous sarcoma virus, after stimulation with stimulating substances (e.g. PMA) and the additional cotransfection with S (Fig. XIII).
  • stimulating substances e.g. PMA

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Abstract

Des particules d'antigène de surface servant de vaccin de l'hépatite B, préparées par des techniques d'ADN recombinant, sont composées d'épitopes provenant du groupe des peptides de surface et/ou des peptides de noyau des virus de l'hépatite de type non A et non B, du virus de l'hépatite A et/ou du virus de l'hépatite B. Des particules respectives se caractérisent en particulier par une composition de différents épitopes choisis parmi des peptides de type pré-S et de type S. Des séquences d'ADN, des plasmides et des lignées cellulaires codant pour des particules respectives d'antigène de surface du virus de l'hépatite B ainsi qu'un nouveau vaccin contenant ces particules sont également décrits.
PCT/EP1988/000551 1987-06-22 1988-06-22 Vaccin contenant un antigene de surface de l'hepatite b WO1988010301A1 (fr)

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KR1019890700312A KR970007153B1 (ko) 1987-06-22 1988-06-22 B형 간염 표면 항원 백신
AU19958/88A AU619753B2 (en) 1987-06-22 1988-06-22 Hepatitis b surface antigen vaccine
US08/484,408 US6117653A (en) 1987-06-22 1995-06-07 Hepatitis B surface antigen vaccine

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US6297048B1 (en) 1992-02-04 2001-10-02 Chiron Corporation Hepatitis therapeutics
EP1149917A3 (fr) * 2000-04-20 2001-11-14 Wang-Shick Ryu Vecteurs derivés du virus Hépatitis B pour la thérapie gènique
EP1288296A3 (fr) * 1992-05-11 2003-03-12 Ribozyme Pharmaceuticals, Inc. Procédé et réactif d'inhibition de la réplication virale de l'HBV
EP1997900A2 (fr) 1996-04-05 2008-12-03 Novartis Vaccines and Diagnostics, Inc. Vecteurs à base d'alpha virus recombinants avec inhibition réduite de synthèse macromoléculaire cellulaire
EP2298900A1 (fr) 1996-09-17 2011-03-23 Novartis Vaccines and Diagnostics, Inc. Compositions et procédés de traitement de maladies intracellulaires
US20150072885A1 (en) * 2013-07-16 2015-03-12 National Health Research Institutes Antibodies and method for determining deletions in hbv pre-s2 region

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NZ229260A (en) * 1988-06-03 1994-02-25 Merck & Co Inc Hepatitis b virus, expression cassette for pre-s domain, host cells and

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EP0198474A1 (fr) * 1985-04-15 1986-10-22 SMITHKLINE BEECHAM BIOLOGICALS s.a. Antigène de surface de l'hépatite B produit par des techniques d'ADN recombinant, vaccins, diagnostics, lignées cellulaires et leur procédé de préparation
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EP0156712A1 (fr) * 1984-03-07 1985-10-02 Institut Pasteur Composition utile pour la fabrication de vaccins contenant des particules portant l'antiqène de surface du virus de l'hépatite B et le récepteur de l'albumine sérique humaine polymérisée, cellules animales capables de produire de telles particules et procédé pour leur obtention
EP0180012A1 (fr) * 1984-10-27 1986-05-07 Wolfram H. Prof. Dr. Gerlich Séquence de polypeptide immunogène du virus de l'hépatite B
WO1986005198A1 (fr) * 1985-03-05 1986-09-12 Veba Oel Entwicklungs-Gesellschaft Mbh Procede d'evacution de particules de residus d'un reacteur de gaseification sous pression
EP0198474A1 (fr) * 1985-04-15 1986-10-22 SMITHKLINE BEECHAM BIOLOGICALS s.a. Antigène de surface de l'hépatite B produit par des techniques d'ADN recombinant, vaccins, diagnostics, lignées cellulaires et leur procédé de préparation
EP0244924A1 (fr) * 1986-01-31 1987-11-11 Merck & Co. Inc. Production de vaccines contre le virus de l'hépatite B
EP0248410A2 (fr) * 1986-06-03 1987-12-09 Green Cross Corporation Promoteur de levure et procédé pour préparer des protéines hétérologues
EP0250253A2 (fr) * 1986-06-20 1987-12-23 Scripps Clinic And Research Foundation Epitopes de cellules T et B de la région pré-S de l'antigène de surface de l'hépatite B

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6297048B1 (en) 1992-02-04 2001-10-02 Chiron Corporation Hepatitis therapeutics
EP1288296A3 (fr) * 1992-05-11 2003-03-12 Ribozyme Pharmaceuticals, Inc. Procédé et réactif d'inhibition de la réplication virale de l'HBV
EP1997900A2 (fr) 1996-04-05 2008-12-03 Novartis Vaccines and Diagnostics, Inc. Vecteurs à base d'alpha virus recombinants avec inhibition réduite de synthèse macromoléculaire cellulaire
EP2298900A1 (fr) 1996-09-17 2011-03-23 Novartis Vaccines and Diagnostics, Inc. Compositions et procédés de traitement de maladies intracellulaires
EP1149917A3 (fr) * 2000-04-20 2001-11-14 Wang-Shick Ryu Vecteurs derivés du virus Hépatitis B pour la thérapie gènique
US20150072885A1 (en) * 2013-07-16 2015-03-12 National Health Research Institutes Antibodies and method for determining deletions in hbv pre-s2 region
US9714284B2 (en) * 2013-07-16 2017-07-25 National Health Research Institutes Antibodies and method for determining deletions in HBV pre-S2 region

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AU619753B2 (en) 1992-02-06
KR890701743A (ko) 1989-12-21
AU1995888A (en) 1989-01-19
KR970007153B1 (ko) 1997-05-03

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