EP0301083A1 - Retrovirale expressionsvektoren und verfahren zur herstellung von hbv-antigenen - Google Patents

Retrovirale expressionsvektoren und verfahren zur herstellung von hbv-antigenen

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Publication number
EP0301083A1
EP0301083A1 EP88902298A EP88902298A EP0301083A1 EP 0301083 A1 EP0301083 A1 EP 0301083A1 EP 88902298 A EP88902298 A EP 88902298A EP 88902298 A EP88902298 A EP 88902298A EP 0301083 A1 EP0301083 A1 EP 0301083A1
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EP
European Patent Office
Prior art keywords
gene
protein
cells
pres2
hbv
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EP88902298A
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English (en)
French (fr)
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EP0301083A4 (en
Inventor
Alan Mclachlan
David R. Milich
Joseph Sorge
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Scripps Research Institute
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Scripps Clinic and Research Foundation
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Publication of EP0301083A1 publication Critical patent/EP0301083A1/de
Publication of EP0301083A4 publication Critical patent/EP0301083A4/en
Withdrawn legal-status Critical Current

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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
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • 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
    • 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

Definitions

  • the present invention relates to vertebrate cell expression vectors carrying hepatitis B virus (HBV) genes. More specifically, this invention relates to vertebrate cell expression vectors capable of expressing the HBV PreCore-Core or PreSl-PreS2-S genes and the use of those vectors to produce transfected vertebrate cell lines that secrete proteins displaying HBeAg or HBsAg antigenicity, respectively.
  • HBV hepatitis B virus
  • the classic antigens used in the diagnosis and management of hepatitis B virus infection are the surface (S) ⁇ antigen (HBsAg) , the core antigen (HBcAg) and the e antigen (HBeAg) .
  • S surface
  • HBsAg core antigen
  • HBeAg e antigen
  • the proteins that comprise those antigens and the genes that encode those proteins have been the subject of intense investigation for over two decades. However, despite those efforts, much remains unknown about the structure and expression of HBsAg, HBcAg and HBeAg in vivo.
  • Hepatitis B surface antigen (HBsAg) is synthesized in the cytoplasm of the infected hepatocyte and circulates in the plasma of chronic HBsAg carriers in a number of particulate forms.
  • One form, the Dane particle probably represents the virion of hepatitis B virus (HBV) .
  • the 22-nm spherical and filamentous forms share the antigenic HBsAg determinants with the lipoprotein coat of the virion and probably represent coat protein produced in excess by the infected hepatocytes.
  • the 22-nm form can be readily purified from carrier plasma by large- scale techniques and has been successfully used as a subviral vaccine in animal and limited human studies.
  • the recognized antigens of HBsAg appear to be specified by the HBV genome and consist of group- specific (a) and type-specific (d or y, w or r) determinants.
  • Chronic carriers of each of the four possible phenotypes (adw, adr, ayw and ayr) have been found in human populations, although the ayr phenotype is exceptionally rare and therefore not epidemiologically important.
  • Subspecificities may also exist within the group-specific a complex.
  • HBsAg contains structural protein antigens that are expression products of the HBV genome open reading frame (ORF) designated S.
  • ORF HBV genome open reading frame
  • the S gene corresponds to nucleotides 1426- 2104 of the HBV DNA sequence reported by Pasek et al., Nature. 282: 575-579 (1979).
  • the S gene has been expressed in a variety of expression systems. See, for example, Crowley et al., Mol. Cell. Biol. , 3:44-55 (1983); Davis et al. , Proc. Natl. Acad. Sci. USA, 82:7560-7564 (1985); Hsiung et al. , J. Mol. APPI. Genet. , 2:497-506 (1984) and Moriarty et al. , Proc. Natl. Acad. Sci. USA. 78:2606-2610 (1981). Synthesis of the S gene polypeptides in mammalian cells resulted in secretion of 22-nm particles with biophisical properties similar to those of subviral particles isolated from serum during HBV invection.
  • Gene S begins at the last downstream start codon in the S ORF and is therefore preceeded in phase by a 163 or 174 codon containing region, depending on the viral subtype, known as the PreS region.
  • the PreS region itself contains two functional subregions, designated PreSl and PreS2. Those regions each combine with the S gene to form the PreS2-S and PreSl- PreS2-S genes, i.e., nucleotides 1261-2104 and 947- 2104, respectively, of the HBV DNA sequence reported by Pasek et al., supra.
  • the amino acid residue sequence encoded by the PreS2-S gene i.e., protein products GP33 and GP36, consists of the amino acid residue sequence encoded by the S gene and an additional 55 amino-ter inal residues encoded by the PreS2 region of the S ORF.
  • Michel et al., Proc. Natl. Acad. Sci. USA. 81:7708-7712 (1984) reported that a vector containing expression of the PreS2-S gene in animal cells resulted in the secretion of particles containing both PreS2-S and S proteins with a relatively high proportion of PreS2-S protein.
  • the PreSl-PreS2-S gene products P43/GP46 consist of 108-119 PreSl region residues added to the amino terminus of the PreS2-S gene product.
  • Recombinant DNA molecules containing the PreSl-PreS2-S gene have been used to produce particles displaying HBsAg antigenicity but no direct evidence was provided that those particles contained the PreSl-PreS2-S protein. See European Patent Application No. 0, 198,474 by Endotronics, Inc. filed April 15, 1985.
  • the HBV core antigen (HBcAg) has not been found in the sera of HBV infected individuals.
  • the core protein is assembled into a 27 nm particle, known as the nucleocapsid, that covers the viral genome.
  • the core particle itself is enveloped by the surface layer.
  • the gene encoding the core protein is located in the C open reading frame.
  • the genetic organization of the C ORF is similar to that of the S ORF in that it contains two in phase transcription initiation codons that define two overlapping genes.
  • the Core gene starts at the second or downstream initiation codon of the C ORF.
  • the gene that starts at the 5' or upstream initiator codon is designated herein as the PreCore-Core gene and encodes the PreCore-Core protein.
  • Vertebrate cells have been used to produce proteins displaying either HBcAg or HBeAg antigenicity using recombinant DNA techniques.
  • Gough et al. J. Mol. Biol.. 162:43-67 (1982) reported transforming rat and mouse cells with a recombinant plasmid containing four tandem head-to-tail copies of the entire HBV genome.
  • Four cell lines (three mouse and one rat) secreting both HBsAg and HBeAg were isolated.
  • the rat and at least one of the mouse cell lines that synthesized HBeAg also produced small quantities of HBcAg.
  • the rat cell line producing HBeAg had many partial and 18 to 20 complete copies of the HBV genome integrated into high molecular weight DNA. Similarly, the mouse cell lines producing HBeAg were reported as having only two or three copies of the HBV genome integrated. Because not all cell lines that synthesized HBsAg also synthesized HBeAg, Gough et al. concluded that HBeAg expression is not an inevitable consequence of the acquisition of the HBV genome, and may therefore reflect the conformation or concentration of HBV sequences within a particular transfected cell. More recently, Uy et al., Virol.. 155:89-96
  • rDNA recombinant DNA molecules
  • Retrovirus vectors provide an efficient means for introducing cloned genes into mammalian and avian cells.
  • Retrovirus vectors are vectors that contain transcriptional mRNA processing and translational control elements derived from retrovirus genomes.
  • Transcriptional control elements typically include: (1) a promoter that signals the starting point of transcription; and (2) a terminator that signals the ending point of transcription.
  • Retrovirus promoters are contained in a genomic structure termed the "long terminal repeat" (LTR) .
  • LTR long terminal repeat
  • the LTRs of most retroviruses are extremely active promoters and foreign DNA cloned downstream from a viral LTR will usually be transcribed very efficiently.
  • RNA splicing is the excission of portions of the transcribed precursor mRNA that results in the production of mature mRNA.
  • the upstream and downstream splice sites in an mRNA precursor are determined by genomic structures known as the 5' (upstream) splice donor site and the 3' (downstream) splice acceptor site. During splicing, that portion of the precursor mRNA that lies between the splice acceptor and splice donor sites is excised from the transcript.
  • an expression vector should possess additional elements. For instance, it should contain one or more genes that encode a selectable marker to allow identification of transformants. It should also contain a recognition site for one or more restriction enzymes in regions of the vector that are not essential for expression but are under the control of a promoter.
  • the present invention contemplates a recombinant DNA molecule that includes a DNA sequence consisting essentially of the PreCore-Core gene of HBV operatively linked to a vector capable of expressing the gene in a vertebrate cell.
  • the vector is a retrovirus vector, preferably either pARV2 or pARVlMT. More preferably the rDNA is pARV2PC.
  • the present invention also relates to a transfected host vertebrate cell transfected with a PreCore-Core gene containing recombinant DNA molecule of the present invention.
  • the present invention also contemplates a transfected host vertebrate cell culture having the transfected host vertebrate cell in a nutrient medium.
  • the cells are NIH 3T3 cells. Also preferred are monoclonal cultures.
  • composition that includes an admixture of proteins having apparent molecular weights of 15 kDa, 18 kDa and 21 kDa wherein each of the proteins has an amino acid residue sequence corresponding to an amino acid residue sequence encoded by the HBV PreCore-Core gene and the composition displays HBeAg antigenicity but no substantial HBcAg antigenicity.
  • the method comprises the steps of culturing, in a nutrient medium, vertebrate cells transfected with a recombinant DNA molecule of the present invention that contains the PreCore-Core gene. Culturing the cells forms a medium containing secreted proteins expressed by the PreCore- Core gene. The medium containing the secreted proteins is then recovered.
  • the present invention also relates to a method of producing a protein displaying HBeAg antigenicity comprising the steps of initiating a culture, in a nutrient medium, of vertebrate cells transfected with a recombinant DNA molecule of the present invention consisting essentially of the PreCore-core gene of HBV operatively linked to a vector capable of expressing the gene in the cells.
  • the culture is maintained for a time period sufficient for the cells to express protein from the recombinant DNA and secrete the protein into the medium.
  • the protein is then recovered from the medium.
  • the cells are NIH 3T3 cells and the protein has an apparent molecular weight selected from the group consisting of 15 kDa, 18 kDa and 21 kDa.
  • the present invention also relates to a recombinant DNA molecule having a DNA sequence consisting essentially of the PreSl-PreS2-S gene of HBV operatively linked to a vector capable of expressing the gene in a vertebrate cell.
  • the vector is a retrovirus vector, preferably the vector pARV2.
  • the vector contains a mouse metallothionein promoter to which the gene is operatively linked.
  • a preferred vector containing the mouse metallothionein promoter is pARVlMT.
  • a preferred recombinant DNA molecule containing the PreSl-PreS2-S gene is pARV2Pl/2S.
  • This invention also relates to a transfected host vertebrate cell transfected with a PreSl-PreS2-S gene containing recombinant DNA molecule of the present invention.
  • transfected host vertebrate cell culture that includes in a nutrient medium, the vertebrate cells transfected with an expression vector capable of expressing in the cells at least one secreted protein encoded by the PreSl- PreS2-S gene.
  • the present also contemplates a proteinaceous particle that includes at least one protein encoded by the PreSl-PreS2-S gene of HBV displaying HBsAg antigenicity, the particle prepared from a recombinant DNA molecule of this invention.
  • Also contemplated by the present invention is a method of preparing a proteinaceous particle displaying HBsAg antigenicity wherein at least one protein is encoded by the PreSl-PreS2-S gene of HBV.
  • the method includes the steps of initiating a culture, in a nutrient medium, of vertebrate cells, preferably mammalian cells, transfected with a recombinant DNa molecule of the present invention containing the PreSl-PreS2-S gene of HBV.
  • the culture is maintained for a time period sufficient for the transfected cells to express protein from the recombinant molecule and secrete the protein in particle form into the medium.
  • the particle is then recovered from the medium.
  • the present invention contemplates a vaccine that includes an effective amount of a proteinaceous particle of the present invention that displays HBsAg antigenicity in a pharmaceutically acceptable diluent.
  • a further embodiment is a diagnostic system useful for detecting the presence of either HBeAg or antibodies to HBeAg in a body sample.
  • the system includes, in separate packages, a protein of the present invention that displays HBeAg antigenicity and a labeled specific binding agent for signaling the presence of the immunoreaction of the protein of the present invention with anti-HBe antibodies or antibody fragments.
  • the present invention provides several advantages and benefits.
  • One advantage is that the present invention provides a method of producing HBeAg in mammalian cells at a high yield.
  • Another advantage is that the present invention provides a method of producing HBsAg in mammalian cells.
  • the HBsAg particles produces by the rDNAs of this invention can be used to make a vaccine having an improved rate of nonresponsiveness.
  • Another benefit of the present invention is that it provides a method of forming transfected mammalian cells that secrete HBeAg. Brief Description of the Drawings
  • Figure 1 is a schematic representation illustrating the construction of amphotrophic retrovirus vectors pARVl, pARV2 and pARVlMT from the amphotrophic vector CistorNeo, whose construction is described by Sorge et al., Mol. Cell. Biol.. 4:1730-1737 (1984).
  • Sequence elements in the expression vectors are as follows: Ampho8 LTR and 5 ) ; Ampho8 LTR ( acceptor site ( sites ( O and / respectively) ; neomycin resistance gene ( V///////A ) ; MT gene transcription regulatory sequences (
  • Figure 2 contains two graph panels (A and B) that illustrate the time course of secretion of HBsAg (panel A) and HBeAg (panel B) into the respective culture media.
  • 10 6 Cells were plated on to 10 centimeter (cm) diameter tissue culture plates, and the media were changed the following day. Samples [0.5 milliliters (ml)] were collected 2, 4, 6 and 8 days thereafter, and were assayed for HBV antigens. Cells were confluent by day 2.
  • Cell lines studied were the transfected NIH/3T3 mouse fibroblast cells, and are identified as follows: 3T3 pARV2Pl/2S (A) »
  • 3T3 pARV2P2S ( ⁇ ) ,* 3T3 pARVlMTS ( ); 3T3 pARV2C ( ⁇ ) 3T3 pARV2PC (El); and 3T3 pARV2 (O)- Tne ordinates are in micrograms per milliliter (ug/ml) of HBSAg or HBeAg, whereas the abscissas are in days on which samples were collected.
  • Figure 3 contains three graph panels that illustrate CsCl density gradient analyses of secreted HBsAg from cell lines 3T3 pARVlMTS (panel A) ; 3T3 pARV2P2S (panel B) ; and 3T3 pARV2Pl/2S. Gradients were fractionated from the bottom (Fraction 1) and HBsAg was determined by the AUSRIA II assay. Counts per minute (CPM) ( ⁇ ) and densities in grams per illiliter (g/ml) (A) are shown on the ordinates. The inserts to panels A and B show electron microscopic analyses of the negatively stained HBsAg particles isolated from the CsCl gradients.
  • CPM Counts per minute
  • g/ml grams per illiliter
  • Figure 4 contains three graph panels that illustrate sucrose density gradient analyses of secreted HBsAg from cell line 3T3 pARVlMTS CsCl gradient fractions 7 to 9 (Fig. 3A) ; cell line 3T3 pARV2P2S CsCl gradient fractions 6 to 8 (Fig. 3B) ; and cell line 3T3 pARV2Pl/2S CsCl gradient fractions 4 and 5 (Fig. 3C) .
  • the pooled CsCl gradient fractions were dialized against PBS containing 1 mM phenylmethylsulfonyl flouride before sucrose gradient analysis. Dialyzed fractions were then centrifuged as described in Example 7.
  • Figure 5 contains five graph panels (A-E) that illustrate results from sucrose density gradient analyses of HBeAg and HBcAg.
  • HBeAg and HBcAg were obtained from: medium of 3T3 pARV2C cells (panel A) ; medium of 3T3 pARV2PC cells (panel B) ; cell lysate of 3T3 pARV2C cells (panel C) ; cell lysate of 3T3 pARV2PC cells (panel D) ; and recombinant HBcAg (Biogen; panel E) .
  • HBeAg plus HBcAg was determined by HBe EIA
  • Figure 6 contains two portions (A and B) that illustrate autoradiographic immunoblotting analyses of HBV antigens. Visualization of the immunoblots was as discussed in Example 9. Markers GP33, GP28, P25, P21 and P15 indicate positions of specific glycoproteins (GP) and proteins (P) in the blots.
  • HBsAg-spe ⁇ ific anti-polypeptide antiserum a HBsAg-spe ⁇ ific anti-polypeptide antiserum.
  • Lane 1 contained 60 nanogra s (ng) of CHO (Chinese hamster ovary) cell-derived, secreted HBsAg prepared as described in Michel et al. , Proc. Natl. Acad. Sci. USA. 81:7708-7712 (1984); Lanes 2-5 contained 200 micrograms (ug) each of protein from a cell line lysate.
  • the cell lines of those lysates were as follows: Lane 2, 3T3 pARV2; Lane 3, 3T3 pARV2Pl/2S; Lane 4, 3T3 pARV2P2S; and Lane 5, 3T3 pARVlMTS.
  • Portion B immunoblots were probed with an HBcAg-specific anti-polypeptide antiserum.
  • Lane 1 contained 60 ng of recombinant HBcAg (Biogen)
  • Lanes 2-4 contained 200 ug each of proteins from a cell line lysate
  • Lanes 5-7 contained 50 microliters- (ul) of a cell line culture medium.
  • the cell lines from which the lysates and media were obtained were as follows: Lanes 2 and 5, 3T3 pARV2; Lanes 3 and 6, 3T3 pARV2PC; and Lanes 4 and 7, 3T3 pARV2C.
  • Figure 7 contains seven photomicrographs (A-G) that illustrate results of immunofluorescence analyses of HBV antigen-expressing cell lines.
  • Photos A, B and C are of cell lines 3T3 pARVlMTS, 3T3 pARV2P2S and 3T3 pARV2Pl/2S, respectively, stained with monoclonal anti-HBs antibodies (Dr. D. P. Kaplan, Ortho Laboratories) .
  • Photos D and E are of cell lines 3T3 pARV2P2S and 3T3 pARV2Pl/2S, respectively, stained with pre-S(2)-specific monoclonal antibody 5520 (Dr. M. Mayumi, Jichi Medical School) .
  • Photo F is of cell line 3T3 pARV2Pl/2S stained with pre-S(1)-specific monoclonal antibody MA 18/7 (Dr. . H. Gerlich,
  • FIG. 8 illustrates two lineages (A and B) of retrovirus infected cell lines.
  • the parental cell lines in lineages A and B were 3T3 pARV2PC and 3T3 pARV2C, respectively, both produced as described in Examples 11 and 12.
  • the recombinant plus helper retrovirus from the 3T3 ARV2PC/AMLV1 clonal cell line was used to generate each of the 3T3 ARV2PC/AMLV1.1- 1.10 clonal cell lines.
  • FIG. 9 contains two panels (A and B) illustrating the line course of the secretion of HBeAg from transfected and infected cell lines derived from 3T3 pARV2PC (panel A) and 3T3 pARV2C (panel B) .
  • tissue culture plate (10 cm in diameter) , and the culture medium was changed the following day. Tissue culture medium samples (0.5 ml) were collected 2, 4, 6 and 8 days later and assayed for HBeAg as described in Example 3. Data are expressed as units of HBeAg per ml of culture medium.
  • FIG. 10 illustrates the time courses of the secretion of HBeAg from three retrovirus transfected human cell lines. Cells were plated, media samples were collected and HBeAg was assayed as described in Figure 9.
  • Figure 11 contains 3 panels (A, B and C) illustrating filter hybridization analysis of transfected and infected mouse and human fibroblast
  • DNA DNAs (15 ug per lane) were digested with ECoRV.
  • the 3 P-labeled probes used were (Panels A and B) the HBV precore ORF DNA fragment cloned into pCU13 and (Panel C) pBRneo DNA.
  • Structural Gene is a DNA sequence that is expressed as a polypeptide, i.e., an amino acid residue sequence.
  • Expression is the combination of intracellular processes, including transcription and translation, undergone by a structural gene to produce a polypeptide.
  • Recombinant DNA Molecule is a hybrid DNA sequence comprising at least two nucleotide sequences not normally found together in nature.
  • Expression Vector a DNA sequence that forms control elements that regulate expression of structural genes when operatively linked to those genes.
  • Operatively Linked or Inserted means that a structural gene is joined to an expression vector so that it is under the control of the expression vector.
  • Promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur.
  • Retrovirus Expression Vector is an expression vector that includes a promoter derived from the long terminal repeat (LTR) region of a retrovirus genome.
  • Restriction Fragment is a linear segment of double-stranded DNA that is the product of a specific restriction endonuclease reaction.
  • Secreted Protein is a protein that is formed intracellularly and released by the cell into its surrounding medium.
  • the recombinant DNA molecules of the present invention can be produced by operatively linking a gene that encodes either a PreSl-PreS 2 -S, PreS2-S or a PreCore-Core HBV protein to a vector capable of expressing those genes in a vertebrate cell.
  • the DNA sequence of genes that encode the PreS ⁇ -PreS2-S and PreCore-Core HBV proteins are well known in the art. See, for example, Galibert et al.. Nature. 281:646- 650 (1979); Pasek et al.. Nature. 282:575-579 (1979); and Valenzuela et al.. Nature, 280:815-819 (1979).
  • nucleotide sequence or gene fragment inserted at the selected restriction site of the expression vector can include nucleotides that are not part of the actual structural gene for the desired protein.
  • functional endogenous HBV regulatory elements can be included in the rDNAs of the present invention. Such regulatory elements include promoters, splice acceptor and donor sites and transcription initiation and termination sequences.
  • the gene that encodes the PreSl-PreS2-S protein also includes the genes encoding the PreS2-S and S proteins aligned in phase
  • expression of the PreSl-PreS2-S gene also results in expression of the PreS2-S and S genes.
  • expression of the PreS2-S gene operatively inserted in a vector results in production of the PreS2-S and S proteins.
  • the DNA sequence that forms the PreCore-Core gene also contains the Core gene aligned in phase with the PreCore-Core gene.
  • expression of the PreCore-Core gene does not result in expression of HBcAg.
  • the DNA sequence consisting essentially of when used in reference to a gene contained in a DNA sequence refers to the designated gene and any gene that has a smaller number of nucleotides but is in phase with the designated gene.
  • the DNA sequence consisting essentially of the PreSl-PreS2-S gene contemplates the three in phase genes: PreSl-PreS2-S, PreS2-S and S.
  • DNA sequences that encode the PreS 1 -PreS 2 -S, PreS2-S and PreCore-Core proteins can easily be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci et al. , J. Am. Chem. Soc.. 103:3185 (1981).
  • any desired modifications can be made simply by substituting the appropriate bases for those encoding the native amino acid residue sequence.
  • any modification to the DNA sequences of the native HBV genes do not disrupt the function of the endogenous regulatory elements those genes encode.
  • DNA sequences exactly homologous to genes found in naturally occuring HBV subtypes are preferred.
  • DNA sequences that encode the PreS 1 -PreS 2 -S, PreS2-S and PreCore-Core proteins can be obtained, as is known in the art, from Dane particle DNA. For instance, the procedure of
  • DNA sequences consisting essentially of genes encoding the PreS ⁇ -PreS 2 -S, PreS2-S or PreCore-Core proteins can be obtained from vectors containing those genes.
  • E. coli transformed with vectors pARV2Pl/2S, pARVlMTS and pARV2PC, which contain genes encoding the PreS ⁇ PreS 2 -S, PreS2-S and PreCore-Core HBV proteins, respectively, have been deposited pursuant to Budapest Treaty requirements with the American Type Culture Collection, (ATCC) 12301 Parklawn Drive, Rockville, MD 20852.
  • DNA sequences encoding the PreS 1 -PreS 2 -S, PreS2-S and PreCore-Core HBV proteins can then be provided with appropriate linkers and operatively ligated into vertebrate cell expression vectors.
  • a variety of methods have been developed to link double-stranded cDNA to expression vectors. For instance, complementary homopolymer tracts are added to the double-stranded cDNA of the structural gene to be inserted and to the vector DNA. The vector and double-stranded cDNA are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.
  • Synthetic linkers containing one or more restriction sites provide an alternative method to join double-stranded cDNA to expression vectors.
  • Double-stranded cDNA is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3', single-stranded termini with their 3'-5' exonucleolytic activities and fill in recessed 3' ends with their polymerizing activities. The combination of these activities therefore generates blunt-ended cDNA molecules, which are then incubated with a large molar excess of linker molecules in the presence of bacteriophage T4 DNA ligase, an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules.
  • the products of the reaction are cDNA molecules carrying polymeric linker sequences at their ends. These molecules are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with a compatible enzyme.
  • Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including
  • Expression vectors compatible with vertebrate cells are well known in the art and are available from several commercial sources. Typically such vector are provided containing convenient restriction sites for insertion of the desired DNA coding sequence. Typical of such vectors are pSVL and pKSV-10 (Pharmacia, Piscataway, NJ) and pBPV-l/pML2d (International Biotechnologies, Inc.).
  • the expression vectors used to construct the recombinant DNA molecules of the present invention contain a selection marker that is effective in a vertebrate cell, preferably a drug resistance selection marker.
  • a preferred drug resistance marker is the gene whose expression results in neomycine-resistance, i.e., the neomycin phosphotransferase (neo) gene. Southern et al., J. Mol. APPI. Genet.. 1:327-341 (1982).
  • the recombinant DNA molecules of the present invention include a procaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a procaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a procaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a procaryotic host cell, such as a bacterial host cell, transformed therewith.
  • procaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a procaryotic host cell, such as a bacterial host cell, transformed therewith.
  • procaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recomb
  • the expression vector is a retrovirus expression vector that is preferably replication-incompetent.
  • retroviral vectors The construction and use of retroviral vectors has been described by Sorge et al., Mol. Cell. Biol.. 4:1730-37 (1984).
  • recombinant DNA molecules having the PreSl-PreS2-S, PreS2-S or PreCore-Core genes operatively linked to the mouse metallothionein (MT) gene promoter so that expression is regulated, at least in part, by the level of heavy metals, such as zinc and cadmium, in the transfected vertebrate host cell.
  • MT mouse metallothionein
  • the use of the MT gene promoter in the construction of expression vectors is well known in the art. See, for instance, Searle et al., Mol. Cell. Biol.. 5:1480-1490 (1985).
  • a ribonucleic acid equivalent of a rDNA of the present invention is also contemplated by the present invention.
  • retrovirus containing a rRNA of this invention, wherein the retrovirus is capable of transfecting a mammalian cell to produce a cell that contains an rDNA of this invention and secretes HBeAg or a proteinaceous particle displaying HBsAg of this invention.
  • transfection refers to a process of introducing a rDNa of this invention into a compatible host cell genome.
  • the present invention also relates to a transfected host vertebrate cell, preferably a mammalian cell, transformed with a recombinant DNA molecule of the present invention.
  • the host is a mouse, rat or avian fibroblastic cell line, a Chinese hamster ovary (CHO) cell line, an African Green Monkey kidney cell line or a human liver cell line.
  • Preferred cell lines include the African green monkey kidney cell line COS-7 available from the ATCC as CRL 1651 and the NIH Swiss mouse embryo cell line NIH/3T3 available from the ATCC as CRL 1658.
  • Transfection of appropriate vertebrate cell hosts with a recombinant DNA molecule of the present invention is accomplished by well known methods. See, for example, Sorge et al., Mol. Cell. Biol. , 4:1730-37 (1984); Graham et al., Virol.. 52:456 (1973); and Wigler et al., Proc. Natl. Acad. Sci. USA, 76:1373-76 (1979) .
  • Successfully transfected cells i.e., cells ⁇ that contain a recombinant DNA molecule of the present invention and express a protein displaying either HBsAg or HBeAg antigenicity
  • cells resulting from the attempted introduction of an rDNA of the present invention can be cloned to produce monoclonal colonies. Cells from those colonies can be harvested, lysed and their DNA content examined for the presence of the rDNA using a method such as that described by Southern, J. Mol. Biol.. 98:503 (1975).
  • successful transfection can be confirmed by well known immunological methods. For example, cells successfully transfected secrete proteins displaying HBsAg or HBeAg antigenicity into their culture medium. Samples of culture medium from cells suspected of being transfected are harvested and assayed for HBsAg or HBeAg using antibodies specific for those antigens, such as those contained in the AUSRIA II or HBe EIA Diagnostic kits available from Abbott Laboratories, Chicago, IL.
  • the present invention also contemplates a culture of those cells, preferably a monoclonal culture, or a culture derived from a monoclonal culture, in a nutrient medium.
  • the culture also contains a secreted protein displaying HBsAg or HBeAg antigenicity.
  • Nutrient media useful for culturing transfected host cells are well known in the art and may be obtained from several commercial sources. In preferred embodiments, a "serum-free" medium is used.
  • HBeAg antigenicity proteins that display HBeAg antigenicity are proteins that immunoreact with antibodies induced by native HBeAg, i.e., anti-HBe antibodies produced during HBV infection.
  • the present method entails culturing, in a nutrient medium, host vertebrate cells transfected with a PreCore-Core gene-containing rDNA of the present invention. During culturing, the transfected cells secrete into the nutrient medium proteins that display HBeAg antigenicity. The medium containing the secreted proteins is then recovered.
  • one or more of the secreted proteins displaying HBeAg antigenicity is isolated from the medium.
  • These preferred methods entail fractionation of the secreted protein- containing medium using well known biochemical techniques. For instance, the various methods of gel filtration, gel chromatography, ultrafiltration, electrophoresis, ion exchange and the like, such as are known for protein fractionations, can be used to isolate the various secreted proteins found in the culture medium.
  • immunochemical methods such as immunoaffinity, immunoadsorption and the like can be performed using well known methods.
  • E. HBeAg Expression Products Also contemplated by the present invention are the secreted protein expression products of the rDNAs of the present invention that contain the PreCore-Core gene.
  • those products display HBeAg antigenicity but no substantial HBcAg antigenicity.
  • no substantial HBcAg antigenicity is meant that, of the total HBV related antigenicity present, at least no more than about 10 percent, preferably no more than about 5 percent and more preferably no more than about 1 percent, is attributable to HBcAg.
  • the expression products of the rDNAs containing the PreCore-Core gene secreted proteins displaying HBeAg antigenicity.
  • proteins all have amino acid residue sequences encoded by the PreCore-Core gene and have apparent molecular weights of 15, 18 and 21 kDa.
  • the present invention contemplates compositions comprising those proteins, alone or in combination.
  • the proteins are affixed to a solid matrix to form a solid support useful in assembling diagnostic kits and in performing diagnostic assays.
  • Proteinaceous particles displaying HBsAg antigenicity are particles that immunoreact with antibodies induced by native HBsAg, i.e., anti-HBs antibodies produced during HBV infection.
  • a proteinaceous particle of the present invention comprises a protein encoded by the PreSl-PreS2-S gene of HBV that is prepared from an rDNA of the present invention that contains the PreSl-PreS2-S gene. That is, the present invention contemplates a proteinaceous particle containing the PreSl-PreS2-S protein.
  • the PreSl-PreS2-S containing particles of the present invention have a density of about 1.26 g/ml.
  • compositions comprising proteinaceous particles that include the PreSl-PreS2-S protein of HBV and have a density of about 1.26 g/ml wherein at least about 25 percent, preferably about 50 percent and more preferably about 75 percent of the particles have a sucrose density gradient sedimentation rate that is greater than particles that contain on S protein.
  • the particles contain a glycosylated protein.
  • the present invention contemplates a proteinaceous particle that consists essentially of the PreS2-S and S proteins, has an average diameter of about 19.7 nm and contains at least about 35 percent, preferably at least about 50 percent and more preferably at least about 65 percent PreS2-S protein as determined by immunologic methods.
  • the particles contain a glycosylated protein.
  • the proteinaceous particles of the present invention do not contain any viral DNA.
  • the proteinaceous particles of the present invention are characterized by their increased immunogenicity and their ability to overcome nonresponsiveness to HBV vaccines that do not include the PreSl-PreS2-S or PreS2-S proteins.
  • the present invention also relates to a method for producing the proteinaceous particles of the present invention displaying HBsAg antigenicity.
  • a method for producing the proteinaceous particles of the present invention displaying HBsAg antigenicity.
  • at least one protein subunit of the produced particle is the PreSl-PreS2-S protein
  • a rDNA of the present invention that contains the PreSl-PreS2-S gene is used.
  • the particle produced contains the PreS2-S and S proteins but does not contain the PreSl- PreS2-S protein
  • a rDNA of the present invention that contains the PreS2-S gene is used.
  • the method is performed by culturing, in a nutrient medium, host vertebrate cells transfected with a PreSl-PreS2-S gene-containing or a PreS2-S gene-containing rDNA of the present invention. Culturing of the. transfected cells results in the proteinaceous particles of the present invention being secreted into the nutrient medium.
  • the proteinaceous particles of the present invention are used in a pharmaceutically acceptable composition that, when administered in an effective amount, is capable of inducing protective immunity against HBV infection.
  • vaccines which contain proteins as active ingredients are well understood in the art.
  • such vaccines are prepared -as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • the preparation may also be emulsified.
  • the active immunogenic ingredient is often mixed with excipient which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents or adjuvants or immunopotentiators which enhance the effectiveness of the vaccine.
  • the vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly.
  • Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • suppositories traditional binders and carriers may include, for example, polyalkalene glucose or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%.
  • Oral formulations include such normally employed excipients as-, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like.
  • the compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25-70%.
  • the proteinaceous particles can be formulated into the vaccine as. * neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • unit dose refers to physically discrete units suitable as unitary dosages for humans, each unit containing a predetermined-quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic.
  • the quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to synthesize antibodies, and. degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges are of the order of one to several hundred micrograms active ingredient per individual. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed in one or two week intervals by a subsequent injection or other administration. I. Diagnostic Systems
  • a diagnostic system preferably in kit form, useful for detecting the presence of HBeAg or antibodies to HBeAg in a body sample, includes, in separate packages, (a) a protein of the present invention that displays HBeAg antigenicity, and (b) a labeled specific binding agent for signaling the presence of the immunoreaction of the protein of the present invention with anti-HBe antibodies.
  • a “specific binding agent” is a molecular entity capable of selectively binding a ligand such as a protein of the present invention or an antibody that immunoreacts with a protein of the present invention.
  • exemplary specific binding agents are antibodies or antibody fragments such as Fab' and F(ab') 2 , complement fragments, protein A and the like.
  • label in its various gramatical forms refers to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a immunoreactant. Any labeling means can be linked to or incorporated in a specific binding agent or used separately, and those atoms or molecules can be used alone or in conjunction with additional reagents. Such labels are themselves well-known in immunochemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel proteins methods and/or systems.
  • the diagnostic kits of thepresent invention are typically used in an "ELISA" format to detect the presence or quantity of anti-HBe antibodies in a body sample such as serum or plasma.
  • ELISA refers to an enzyme-linked immunosorbent assay that employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and quantify the amount of antigen or antibody present in a sample.
  • a description of the ELISA technique is found in Chapter 22 of the 4th Edition of Basic and Clinical Immunology by D.P. Sites et al., published by Lange Medical Publications of Los Altos, CA in 1982 and in U.S. Patents No. 3,654,090; No. 3,850,752; and No. 4,016,043, which are all incorporated herein by reference.
  • the protein displaying HBeAg antigenicity is affixed to a solid matrix to form a solid support.
  • the protein is typically affixed to the solid matrix by adsorption from an aqueous medium although several modes of adsorption from an aqueous medium although several modes of adsorption, as well as other modes of affixation, well known to those skilled in the art can be used.
  • Exemplary of such modes are the reaction of the receptor or antigen with the reactive carboxyl functionality produced by the reaction of cyanogen bromide with glucose-containing matrices such as cross-linked dextrose or cellulose, glutaraldehyde linking as discussed hereinafter in conjunction with latex particles and the like.
  • Useful solid matrices are well known in the art. Such materials include the cross-linked dextran available under the trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, NJ) ; agorse; beads of polystyrene beads about 1 micron to about 5 millimeters in diameter available from Abbott Laboratories of North Chicago, IL; polyvinyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose of nylon-based webs such as sheets, strips or paddles; or tubes, plates or the wells of a microtiter plate such as those made from polystyrene or polyvinylchloride.
  • the kit further includes, in a separate package, an amplifying reagent such as complement, like guinea pig complement, anti- immunoglobulin antibodies or S. aureus cowan strain protein A that reacts with the antigen or antibodies being detected.
  • an amplifying reagent such as complement, like guinea pig complement, anti- immunoglobulin antibodies or S. aureus cowan strain protein A that reacts with the antigen or antibodies being detected.
  • the labeled specific binding agent is capable of specifically binding the amplifying means when the amplifying means is bound to HBeAg or an anti-HBe antibody.
  • the labeled specific binding agent of any diagnostic system described herein, as well as the above-described amplifying reagent, may be provided in solution, as a liquid dispersion or as a substantially dry power, e.g., in lyophilized form.
  • the indicating means is an enzyme
  • the enzyme's substrate can also be provided in a separate package of the system.
  • a solid support such as the before-described microtiter plate and one or more buffers can also be included as separately packaged elements in this diagnostic assay system.
  • the packages discussed herein in relation to diagnostic systems are those customarily utilized in diagnostic system. Such packages include glass and plastic (e.g., polyethylene, polypropylene and polycarbonate) bottles, vials, plastic and plastic- foil laminated envelopes and the like. Examples
  • Vector pARVl was derived from CistorNeo by cloning the Moloney murine leukemia virus (Mo-MLV) Xbal/Smal fragment (coordinates 5325-5750) , Shinnick et al.. Nature 293:543-548 (1981), from clone no. 48, Bacheler et al., J. Virol.. 37:181-190 (1981), into the Clal site 5' to the neomycin phosphotransferase (neo) gene. This was achieved by converting the Clal site 5' to the neo gene in CistorNeo to an Xbal site by linker insertion.
  • Mo-MLV Moloney murine leukemia virus
  • the Smal site of the Mo-MLV fragment was converted to a Clal site by linker insertion. This generated a Mo-MLV Xbal/Clal DNA fragment that was used to replace the Xbal/Clal neo fragment of the modified CistorNeo.
  • pARVl was produced by cloning the Clal neo DNA fragment of CistorNeo into the unique Clal site. These cloning steps resulted in the introduction of the Mo-MLV 3' splice acceptor site into CistorNeo such that splicing between this site and the 5'splice donor site produced a transcript coding for the neo gene product.
  • a unique Xbal cloning site was generated for expression of ORFs from the LTR promoter in the absence of transcript splicing.
  • Vector pARV2 was derived by insertion of a Sail linker into the Xbal site of pARVl.
  • Vector pARVlMT was derived by cloning the Stul/Hindlll mouse metallothionein-I(MT) gene fragment, deleted for the internal Bglll/Sstll fragment, (coordinates -700 to +64 and +930 to +1241) Glanville et al.. Nature, 292:267-269 (1981) and Searle et al., Mol. Cell. Biol.. 4:1221-1230 (1984), into the Xbal site of pARVl.
  • HBV fragments are derived from the DNA sequence as reported in the GenBank genetic sequence data bank.
  • k Restriction enzyme sites defining the HBV fragments. These sites were modified to Sa sites before cloning into the Sail sites of the retroviral vectors.
  • c This 5' coordinate is an estimate from detailed restriction enzyme analysis of this fragment, which represents a BAL31 5' deletion of the Hinpl fragment with coordinates 1803 to 2804.
  • HBV DNA fragments were cloned into the amphotropic retroviral vectors such that t antigen ORG was oriented in the same direction as transcription from the LTR arid MT promoter.
  • HBsAg was measured by a solid phase radioimmunoassay (AUSRIA II, Abbott Laboratories, North Chicago, IL) , using the HBsAg positive control (20 ng/ml) as a standard.
  • HBeAg was measured by ELISA (HBe EIA, Diagnostic Kit, Abbott Laboratories) , using the HBeAg positive control (arbitrarily defined as 1 unit/ml undiluted) as a standard. For all studies, a single batch of control HBeAg was used as standard.
  • PreSl and PreS2 antigenic determinants were measured by an ELISA as previously described by Milich et al., J. Immunol.. 137:315-322 (1986). Briefly, PreSl and PreSl-specific monoclonal antibodies MA18/7, (Heermann et al. J. Virol.. 52:396-402 (198,4()) and 4408 (Machida et al. Gastroenterology 86:910-918 (1984)), respectively, were used bound on the solid phase, and horseradish peroxidase-labelled, monoclonal anti-HBs, provided by Dr. P. Kaplan (Ortho Laboratories, Raritan, NJ) , was used as the probe.
  • HBcAg was measured by ELISA, using monoclonal anti-HBc 3120 bound on the solid phase, and horseradish peroxidase-labelled monoclonal anti-HBc 3105 as the probe. This assay is HBcAg specific and does not detect HBeAg. Takahashi et al., J. Immunol.. 130:2903-2907 (1983).
  • Antigen assays were performed on cell culture media or cell lysates after suitable dilutions in PBS (10 mM sodium phosphate, pH 7.4, 145 mM NaCl) containing 1% (w/v) bovine serum albumin (BSA) , 5% (v/v) fetal calf serum, and 0.005% (v/v) Tween 20
  • Fig. 2 After transfection of the expression vectors into NIH 3T3 cells, G418-resistant cell lines were established and examined for expression of HBV antigens. Secretion of HBsAg and HBeAg into the culture media was examined (Fig. 2) . Cell lines 3T3 pARVlMTS and 3T3 pARV2P2S secreted relatively large amounts of HBsAg, whereas cell line 3T3 pARV2Pl/2S secreted HBsAg at approximately 1% of the level of these two cells lines (Fig. 2A; Table 2) . Cell lines 3T3 pARV2PC and 3T3 pARV2C both secreted HBeAg (Fig.
  • a Rate of secretion was calculated from the following formula: (HBV antigen concentratio 10)/(5x8), based on the HBV antigen concentration on day 8, a tissue culture medium volume 10 ml, and 5xl0 6 cells per confluent plate of cells.
  • Soluble xntracellular HBV antigen was determined in cell lysates prepared by freezing thawing as described in Example 3. Protein concentrations were determined by the Bradford assay [Bradford, Anal. Biochem.. 72:248-254 (1976)].
  • a The symbol + means that the protein displaying HBsAg antigenicity contained detectable levels of PreSl encoded sequences. Estimation of the percentage of polypeptides containing PreSl sequences was not possible because of lack of a suitable standard. b The amount of PreS2-containing polypeptides was estimated by comparison with recombinant HBsAg having 35% PreS2-containing polypeptides Michel et al., Proc. Natl. Acad. Sci. USA. 81:7709-7712 (1984). c ND, Not detectable.
  • cell line 3T3 pARVlMTS produced
  • HBsAg containing no PreS sequences.
  • the PreS2 content of the HBsAg produced by cell line 3T3 pARV2P2S was estimated at approximately 20% for soluble intracellular antigen and 50% for secreted antigen. This is consistent with inclusion of the PreS2 DNA sequence in the expression vector used to generate this cell line. Because of the low level of soluble intracellular antigen produced by the 3T3 pARV2Pl/2S cell line, the presence of PreS sequences was not detectable on this antigen. However, a low level (5%) of PreS2-containing polypeptides was detected on the HBsAg secreted from this cell line. In addition, PreSl sequences were detected on the secreted HBsAg. As each large HBsAg polypeptide contains the PreS2 sequence in addition to the PreSl sequence, it is likely that this secreted HBsAg contains no more than 5% PreSl-containing molecules.
  • the antigens produced by the 3T3 pARV2PC and 3T3 pARV2C cell lines were 1 analyzed for protein displaying either HBcAg or HBeAg antigenicity. The results of that analysis are shown in Table 4 below. -41-
  • HBcAg-specific assay was required to differentiate between these two antigens.
  • HBcAg was detected only in the soluble intracellular compartment of the 3T3 pARV2C cell line. In combination with the data in Table 2, this indicates that the presence of the PreCore-Core gene results in the synthesis of HBeAg, which is readily secreted. In the absence of the precore sequence, i.e. the Core gene only, HBcAg is synthesized and accumulates inside the cell.
  • Proteins displaying HBsAg antigenicity secreted from cell lines 3T3 pARVlMTS, 3T3 pARV2P2S, and 3T3 pARV2Pl/2S were precipitated from cell culture media ith 45% (NH4) 2 S0 4 , pH7.5, and analyzed by CsCl isopycnic density gradient centrifugation according to the method of Michel et al., Proc. Natl. Acad. Sci. USA. 81:7708-7712 (1984) (Fig. 3A, 3B and 3C) .
  • the densities of the HBsAg particles from these cell lines were estimated to be 1.21, 1.22, and 1.26 g/ml, respectively.
  • PreS2-S polypeptide did not significantly alter the density of the HBsAg particles.
  • a relatively small amount of PreSl-PreS2-S polypeptide increased the density of the HBsAg particles.
  • the PreSl-PreS2-S protein containing particles also showed a broad range of densities, suggesting a spectrum of HBsAg particles containing larger proportions of PreSl-PreS2-S protein as the density of the particles increased (Fig. 3C) .
  • Electron microscopy of the CsCl gradient fractions containing HBsAg revealed particles having estimated diameters of 23 nm for those particles containing only S protein and 21 nm for those containing both PreS2-S and S proteins. (Fig. 3A and B) . This suggests that the synthesis of a high percentage of PreS2 polypeptides can affect subunit packaging and, therefore, the nature of the assembled particle, including its antigenicity.
  • HBsAg particles from the CsCl gradients were further characterized by sucrose gradient sedimentation analysis (Fig. 4A to C) .
  • HBsAg particles from CsCl gradient peak fractions were sedimented through 4.2 ml of 5 to 20% (wt/vol) sucrose gradients prepared in PBS containing 1 mM phenylmethylsulfonyl fluoride. Samples were centrifuged for 45 min at 50,000 rpm in an SW 60 rotor at 20°C.
  • Fig. 4 The results, shown in Fig. 4 demonstrate that the PreS2 protein-containing particles (Fig. 4B) sedimented at approximately 70% of the rate of HBsAg particles lacking PreSl-PreS2-S or PreS2-S proteins (Fig. 4A) . This has been confirmed in similar experiments in which the HBsAg particles were sedimented for longer times. The difference in the rates of sedimentation of these particles presumably reflects a difference in their relative masses. A 30% difference in mass would predict a spherical particle diameter of 19.7 nm for particles assembled from both PreS2-S and S proteins assuming a diameter of 22.0 nm for native HBsAg particles. This is consistent with the observed electron microscopy data (Fig. 3A and B) .
  • PreSl-PreS2-S protein-containing particles sedimented at the same rate as HBsAg lacking PreSl- PreS2-S and PreS2-S proteins, and the remainder represented larger particles (Fig. 4C) .
  • These larger particles may represent filaments that preferentially contain PreSl-PreS2-S polypeptides. Heermann et al., J. Virol.. 52:396-402 (1984).
  • HBeAg and HBcAg were investigated by sucrose gradient analysis.
  • Cell culture media and cell lysates (0.4 ml), prepared as described in the Example 3, were sedimented through 4 ml of 5 to 25% (wt/vol) sucrose gradients prepared in 10 mM Tris hydrochloride (pH 7.6)-l mM phenylmethylsulfonyl fluoride. Samples were centrifuged for various times at 50,000 rpm in a SW60 rotor (Beck an Instruments) at 20°C.
  • HBeAg synthesized in each of the various cell lines was slow sedimenting and therefore nonparticulate in nature (Fig. 5A to D) .
  • a significant proportion of the HBcAg activity measured in 3T3 pARV2C cell lysate was fast sedimenting, suggesting that this HBcAg has a particulate nature (Fig. 5C and E) .
  • the rate of sedimentation of the fast-sedimenting HBcAg in this cell lysate was similar to that observed for purified recombinant HBcAg (Biogen S.A., Geneva, Switzerland), indicating that it may be similar in nature to the 27 nm HBcAg particles synthesized in Escherichia coli or isolated from HBV- infected human liver. Cohen et al., Nature (London) . 296:677-678 (1982). In addition, some of the HBcAg activity was slower sedimenting, suggesting the presence of subparticulate HBcAg in this cell line. The relative amounts of HBcAg- and HBeAg-reactive material in the various gradient fractions also suggested that the lysate of 3T3 pARV2C cells contained significant amounts of slow-sedimenting HBeAg.
  • Cell lysates were prepared by washing 5 X 10 6 cells in PBS and homogenizing them in 0.3 ml of sample buffer. Laemmli, Nature (London) r 227:680-685 (1970) . Samples were boiled for 10 min and then centrifuged for 15 min at 12,800 X g. The supernatants were separated on 12.5% polyacrylamide gels as previously described Laemmli, Nature (London) . -45-
  • the membranes were probed with rabbit anti-peptide antisera specific for S protein (anti-49a) , Gerin et al., Proc. Natl. Acad. Sci. USA. 80:2365-2369 (1983), and HBcAg (antibodies to a synthetic peptide corresponding to amino acid residue positions 73-87 of the Core protein of HBV subtype ayw) in 20 ml of TN containing 1% (wt/vol) gelatin and 0.05% (vol/vol) Tween 20 for 16 hr at 22°C.
  • the membranes were washed four times in 100 ml TN containing 0.05% (vol/vol) Tween 20 for 15 min at 22°C and developed with 5 uCi of 125 I-protein A (Amersham Corp., Arlington Heights, 111.) in 50 ml of TN containing 1% (wt/vol) gelatin and 0.05% (vol/vol) Tween 20 for 1 hr at 22°C. After washing the membranes four times in TN containing 0.05% (vol/vol) Tween 20 for 15 min at 22°C, the immunoblot was dried and subjected to autoradiography.
  • Cell line 3T3 pARV2Pl/2S also produced the 25-, 27-kDa HBsAg proteins in addition to the expected 43-kDa HBsAg protein (PreSl-PreS2-S) .
  • PreSl-PreS2-S the expected 43-kDa HBsAg protein
  • the 25- and 27-kDa proteins were probably produced from transcripts initiating at the endogenous HBV promoter located in the PreSl region of the viral genome. Siddiqui et al., Proc. Natl. Acad. Sci. USA. 83:566-570 (1986).
  • the inclusion of the precore sequence, i.e., the PreCore-Core gene, in the 3T3 pARV2PC cell line was predicted to result in synthesis of a 24-kDa polypeptide.
  • the observed intracellular antigen was a polypeptide of approximately 21 kDa (Fig. 6B, lane 3) .
  • This antigen displayed no HBcAg antigenicity (Table 3) and therefore is probably different from the 21-kDa polypeptide synthesized by the pARV2C cell line.
  • 21-kDa polypeptide synthesized in the 3T3 pARV2PC cell line is a processed product of the predicted 24-kDA polypeptide " encoded by the PreCore-Core gene.
  • the secreted products of this cell line were a major polypeptide of approximately 15-kDa and two minor polypeptides of approximately 18- and 21-kDa (Fig. 6B, lane 6) .
  • the size of the major secreted polypeptide is similar to that found in HBeAg from serum, Takahashi et al., J. Immunol..
  • HBV Antigens were analyzed by immunofluorescence microscopy using the method described by Chisari et al.. Science. 230:1157-1160 (1986) (Fig. 7A to G) .
  • PreSl-PreS2-S polypeptides were assayed indirectly by probing with specific mouse monoclonal antibodies and staining with a fluorescein- conjugated, affinity-purified goat antibody to mouse immunoglobulin (Bochringer Mannheim Biochemicals, Indianapolis, Ind.). The specificity of the various antibodies was assessed by examining the staining of the 3T3 pARV2 cell line. S protein was observed in the cytoplasm of the three cell lines producing this antigen (Fig. 7A to C) .
  • PreS2-S proteins were detected in the 3T3 pARV2P2S and 3T3 pARV2Pl/2S cell lines (Fig. 7D and E) , whereas PreSl-PreS2-S proteins were detected only in the 3T3 pARV2Pl/2S cell line (Fig. 7F) .
  • These results are in agreement with the immunoblotting analysis performed in Example 9 and shown in Fig. 6A.
  • the proteinaceous particles containing PreSl-PreS2-S protein have a coarser granular appearance than particles lacking this protein.
  • the PreS-PreS2-S and PreS2-S proteins were localized to the cytoplasm in a manner similar to that of the S protein.
  • the 3T3 pARV2C and 3T3 pARVlMTC cell lines exhibited positive immunofluorescence localized predominantly to the nucleus (Fig. 7G) .
  • these two cell lines had the same distribution of immunofluorescence, but quantitatively the 3T3 pARVlMTC cell line showed a greater signal.
  • HBcAg The distribution of HBcAg in these cell lines indicates that, at least in this heterologous expression system where HBV replication is absent, HBcAg can be transported to the nucleus, presumably by a mechanism analogous to that occurring during HBV liver infection.
  • the 3T3 pARV2PC cell line was negative for HBeAg.
  • Immunofluorescence analysis of the 3T3 pARV2PC, 3T3 pARV2C, and 3T3 pARVlMTC cell lines for HBcAg was negative with three different monoclonal antibodies, suggesting that this assay was not sensitive enough to detect the intracellular antigen. 11. Generation of Cell Lines Infected with a Recombinant Retrovirus
  • CTL cytotoxic T lymphocyte
  • Retroviral-mediated transfer and expression of non-selectable gene products in conjunction with a selectable gene product in human cells has been described. It is anticipated that this approach will be useful in generating autologous stimulator/target cells for the analysis of antigen-specific CTL populations present in the PBL of HBV patients.
  • Examples 11-15 the use of recombinant retroviral- mediated gene transfer to express HBeAg and HBc/eAg in human skin fibroblasts and EBV transformed B lymphocytes was examined.
  • the lineages of the cell lines infected with the various recombinant retroviruses is outlined in Figure 8.
  • the 3T3 pARV2PC and 3T3 pARV2C cell lines were produced as described in Example 2.
  • the 3T3 pARV2PC/AMLV and 3T3 pARV2C/AMLV cell lines were generated by infecting the 3T3 pARV2PC and 3T3 pARV2C cell lines, respectively, with the amphotropic murine leukemia virus 4070 produced by the mouse NIH 3T3 fibroblast prA8 cell line.
  • the 3T3 ARV2PC/AMLV1-18 and 3T3 ARV2C/AMLV1- 34 clonal cell lines were generated by infecting mouse NIH 3T3 fibroblasts with recombinant plus helper retrovirus produced by the 3T3 pARV2PC/AMLV and 3T3 pARV2C/AMLV cell lines, respectively.
  • the 3T3 ARV2PC/AMLV1.1-3.6 and 3T3 ARV2C/AMLV1.1-8.2 clonal cell lines were generated by infecting mouse NIH 3T3 fibroblasts with the recombinant plus helper retrovirus produced by the 3T3 ARV2PC/AMLV1-3 and 3T3 ARV2C/AMLV1-8 clonal cell lines.
  • ARV2PC/AMLVla, GM2504E ARV2PC/AMLVla and AG3 ARV2PC/AMLVla polyclonal cell lines were generated by infecting mouse NIH 3T3 fibroblasts (ATCC #CRL 1505) , human skin CCD-41Sk fibroblasts, human skin GM2504E fibroblasts and EBV transformed AG3 B lymphocytes, respectively, with recombinant plus helper retrovirus produced by the 3T3 ARV2PC/AMLV1 clonal cell line.
  • the 3T3 ARV2C/AMLVa and CCD-41Sk ARV2C/AMLVa polyclonal cell lines were generated by infecting mouse NIH 3T3 fibroblasts and human skin CCD-41Sk fibroblasts, respectively, with recombinant plus helper virus produced by the 3T3 pARV2C/AMLV cell line.
  • EBV transformed B lymphocytes were grown in RPMI 1640 medium containing 10% fetal calf serum and 50 ug/ml gentamicin at 37°C in 5% C0 2 /air. Infection was performed by co-culturing 5xl0 6 B cells with 1x10 s recombinant retroviral producing mouse fibroblasts for 48"hours in Dulbecco's modified Eagle medium containing the B cells, was then removed and centrifuged at 200 xg for 10 minutes.
  • the cells were subsequently resuspended in RPMI 1640 medium containing 10% fetal calf serum, 50 ug/ml gentamicin and 350 ug/ml G418.
  • the G418-resis ant B cells were separated from contaminating mouse fibroblasts by serial passage to fresh culture flasks.
  • HBeAg synthesis was determined as described in Example 3 and filter hybridization analysis was performed by standard techniques as described in Maniatis et al. , Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982) .
  • the 3T3 pARV2PC/AMLV cell line transmits HBeAg expression at high efficiency (approximately 80%, Table 5) whereas the 3T3 pARV2C/AMLV transmits HBc/eAg expression at a lower efficiency (approximately 30%, Table 5) .
  • As an aim of this study was to generate high titer clonal cell lines which efficiently transmit antigen expression by recombinant retroviral infection, several clonal cell lines generated from the infected/transfected cell lines were characterized further (Tables 5 and 6) .
  • the clonal cell lines producing the highest titers of recombinant retrovirus were examined for their ability to synthesize HBeAg (Table 6) .
  • the cell lines synthesizing HBc/eAg were examined for intracellular as well as secreted antigen.
  • the 3T3 ARV2PC/AMLV1-3 and 3T3 ARV2C/AMLV1-8 clonal cell lines had titers of recombinant retrovirus greater than 2 X 10 4 G418- resistant colony forming units per milliliter (G418 R cfu/ l) and produced HBeAg and HBc/eAg, respectively.
  • These clonal cell lines were examined for their - ability to transmit antigen expression by recombinant retrovirus infection (Table 5) .
  • the 3T3 ARV2PC/AMLV1 and 2 clonal cell lines transmitted antigen expression with high efficiency (approximately 90-100%) , whereas, the 3T3 ARV2PC/AMLV3 clonal cell line failed to transmit antigen expression by recombinant retrovirus infection although this cell line synthesized HBeAg.
  • the 3T3 ARV2C/AMLV1-8 clonal cell lines also failed to transmit antigen expression by recombinant retroviral infection despite the fact that these cell lines synthesized high levels of HBc/eAg (Tables 5 and 6) .
  • the data in Table 7 indicate that the cell lines derived from the transfected 3T3 pARV2PC cell line have an approximately 100 times greater ratio of secreted to intracellular HBeAg as compared to the cell lines derived from the transfected 3T3 pARV2C cell line. This is consistent with the 3T3 pARV2PC cell line, and the cell lines derived from it by recombinant retrovirus infection, synthesizing a polypeptide containing the precore signal sequence which results in the secretion of a 15-kDa HBeAg polypeptide.
  • the 3T3 pARV2C cell line, and the cell lines derived from it by recombinant retroviral infection preferentially retain the HBc/eAg inside the cell which is consistent with the synthesis of a 21-kDa nucleocapsid polypeptide lacking a signal sequence.
  • the observation that the cell lines generated by recombinant retrovirus infection show a similar intracellular to extracellular distribution to the parental transfected cell line from which they were derived is consistent with the faithful transmission of the recombinant retrovirus sequences by infection.
  • ARV2PC/AMLVla cell lines and the EBV transformed B lymphocyte AG3 ARV2PC/AMLVla cell line is shown in Figure 10.
  • the intracellular HBeAg levels in these cell lines was below the detection level of the assay, indicating HBeAg was efficiently secreted from both types of human cells. This suggests the expression vector has been faithfully transmitted to these human cells since the antigen was preferentially secreted, as would be expected for a polypeptide which contains the precore signal sequence prior to processing.
  • the human skin fibroblast CCD-41Sk ARV2C/AMLVa cell line did not secrete HBeAg or synthesize intracellular HBc/eAg. This was unexpected as the recombinant retrovirus pool produced by the 3T3 pARV2C/AMLV did transmit antigen synthesis to mouse
  • the 3.8-kbp fragment represents the recombinant retrovirus sequences and the 3.4-kbp fragment represents the pUC9 and flanking sequences.
  • the transfected 3T3 pARV2PC cell line contained several copies of the integrated recombinant retrovirus DNA per cell as indicated by the prominent bands of hybridization at 3.8- and 3.4-kbp.
  • the infected 3T3 ARV2PC/AMLVla cell line DNA only showed a single band of hybridization at 3.8-kbp indicating each cell contained a single integrated copy of the recombinant retrovirus genome. This is consistent with the high frequency of antigen expression observed in the clonal cell lines derived from the recombinant retrovirus produced by the 3T3 ARV2PC/AMLV1 cell line (Table 5) .
  • the infected CCD-41Sk ARV2PC/AMLVla cell line DNA showed three bands of hybridization at 3.8-, 6- and 9-kbp.
  • the 3.8-kbp band of hybridization was predominant and presumably represents an unrearranged recombinant retrovirus genome which is responsible for the antigen secretion observed from this cell line.
  • the bands of hybridization observed at 6- and 9-kbp must represent recombinant viral genomes which contain HBV sequences, it is possible that they might also code for HBeAg. This would depend on the nature of the rearrangement responsible for the in ⁇ reased size of these recombinant proviral genomes.
  • 3T3 ARV2C/AMLV3, 4 and 6 and 3T3 ARV2C/AMLVa cell line DNA digested with EcoRV was performed to examine the structure of the recombinant retrovirus genomes generated as a consequence of a single round of retroviral replication ( Figure 11B-C) .
  • the 3T3 pARV2C cell line DNA showed two prominent bands, of hybridization at 3.5- and 3.7-kbp. These represent several copies per cell of the pUC9 and flanking sequences and the recombinant retrovirus genome, respectively.
  • the 3T3 ARV2C/AMLV3, 4 and 6 clonal cell lines show a single band of hybridization at 3.7- kbp when an HBV prove was used for the analysis ( Figure 11B, lanes 2-4) .
  • This proviral sequence is responsible for the HBc/eAg synthesis observed in these cells.
  • a neomycin resistance gene probe an additional band of hybridization at 9-kpb is observed in the 3T3 ARV2C/AMLV3 and 4 cell line DNAs ( Figure 4C, lanes 2-4) .
  • this recombinant proviral genome is unclear except it does not appear to contain any HBV sequences.
  • the 9-kbp recombinant retrovirus genome appears to be present at less than one copy per cell unless the difference in the intensities between the 3.7- and 9- kbp bands of hybridization is due to their differential transfer to the hybridization filter.
  • ARV2C/AMLV1-8 clonal cell lines to produce recombinant retrovirus capable of transmitting antigen expression it must be assumed that the efficiency of transmission of the 3.7 kbp versus the 9 kbp recombinant retrovirus genome greatly favors the 9 kbp proviral genome.
  • clonal cell lines such as 3T3 ARV2C/AMLV6 ( Figure 11C, lane 4) , which do not contain a 9 kbp recombinant proviral genome, must also efficiently produce a rearranged retrovirus genome during the next round of replication. This rearranged genome must code for G418 resistance but not HBc/eAg to explain its failure to transmit antigen expression by recombinant retrovirus infection (Table 5) . 16. Summary of Examples 11-15 The expression of HBeAg in the various cell types examined was achieved.
  • Retroviral-mediated HBeAg expression occurred in approximately 90% of the mouse NIH 3T3 fibroblasts and the structure of the recombinant retrovirus showed no signs of gross rearrangements after two cycles of retroviral replication. Therefore, it appears that the recombinant retrovirus genome was transmitted faithfully from the transfected 3T3 pARV2PC cell line to the 3T3 ARV2PC/AMLV1 clonal cell line and subsequently to the 3T3 ARV2PC/AMLVla polyclonal cell line.
  • the generation of the high titer 3T3 ARV2PC/AMLV1 clonal cell line permitted the examination of the ability to transmit antigen expression into human skin fibroblasts and EBV transformed B lymphocytes. These cell types did express HBeAg after infection with the recombinant retrovirus and subsequent selection for G418 resistance.
  • the efficiency of retroviral-mediated transmission of HBc/eAg expression was not sufficient to obtain expression of this antigen in human fibroblasts.
  • the 3T3 pARV2C/AMLV cell line transmitted antigen synthesis to 35% of the clonal mouse fibroblast cell lines generated by recombinant retrovirus infection.
  • none of the high titer clones produced recombinant retrovirus with the capacity to transmit antigen synthesis by infection to mouse fibroblasts.
  • the failure to transmit antigen synthesis by recombinant retrovirus infection appeared, in part, to correlate with the generation of an aberrant recombinant proviral genome of 9-kbp.
  • the production of altered recombinant proviral genomes has been reported in other systems.
  • the retroviral expression vector, pARV2C Since the recombinant retrovirus pool derived from the 3T3 pARV2C/AMLV cell line failed to transmit any detectable level of antigen synthesis to the human skin fibroblast CCD-41Sk cell line, it is apparent that the retroviral expression vector, pARV2C will not be useful in generating stimulator/target cells for the analysis of the CTL response to HBV infection.
  • the reason the pARV2C expression vector which lacks approximately 90 nucleotides of HBV DNA sequence compared to the pARV2PC expression vector, codes for retroviral transcripts that result in the frequent production of rearranged recombinant proviral genomes that fail to synthesize antigen is unclear.
  • recombinant retroviral-mediated gene transfer to generate stimulator/target cells for the analysis of the CTL response to HBV antigens
  • the use of recombinant retroviral vectors may offer certain advantages over other infectious recombinant viral vector systems such as adenovirus, herpes and vaccinia virus.
  • a major advantage would appear to be the ability to generate stable cell lines. This differs from infection using the other viral systems where lysis of human cells occurs.

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WO1990010058A2 (en) * 1989-02-07 1990-09-07 Bio-Technology General Corp. Method for production and purification of hepatitis b vaccine
DE4107612A1 (de) * 1991-03-09 1992-09-10 Behringwerke Ag Rekombinante proteine mit der immunreaktivitaet des hepatitis b virus e antigens (hbeag), verfahren zu ihrer herstellung und ihre verwendung in immunoassays und impfstoffen
US6297048B1 (en) 1992-02-04 2001-10-02 Chiron Corporation Hepatitis therapeutics
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US7732423B2 (en) 1994-04-27 2010-06-08 Institut Pasteur Nucleotide vector, composition containing such vector, and vaccine for immunization against hepatitis

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