AU1391888A - Retroviral expression vectors and methods for producing hbv antigens - Google Patents
Retroviral expression vectors and methods for producing hbv antigensInfo
- Publication number
- AU1391888A AU1391888A AU13918/88A AU1391888A AU1391888A AU 1391888 A AU1391888 A AU 1391888A AU 13918/88 A AU13918/88 A AU 13918/88A AU 1391888 A AU1391888 A AU 1391888A AU 1391888 A AU1391888 A AU 1391888A
- Authority
- AU
- Australia
- Prior art keywords
- gene
- protein
- cells
- pres2
- hbv
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
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Classifications
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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
Description
RETROVIRAL EXPRESSION VECTORS AND METHODS FOR PRODUCING HBV ANTIGENS
Description Technical Field
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. Background
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) . 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.
The 22-nm form of HBsAg contains structural protein antigens that are expression products of the HBV genome open reading frame (ORF) designated S. The two predominant proteins of HBsAg, P25 and GP28, have an identical 226 amino acid residue sequence encoded by the gene S of the S ORF. They differ in apparent molecular weight because GP28 is glycosylated whereas P25 is not.
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. Because the S, PreS2-S, PreSl-PreS2-S genes are all in phase, i.e., in the same reading frame, the proteins encoded by those genes all share carboxy-terminal amio acid residue sequences. See Heermann et al., J. Virol, 52:396-402 (1984) .
For instance, it has been shown that 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.
Large scale HBV vaccination programs in the United States have shown that 7-15 percent of those
persons who were vaccinated with various HBsAg preparations failed to respond with the production of antibodies. Nowicki et al., J. Infect. Pis.. 152:1245-58, (1985). Studies of the murine humoral and cellular immune response to the S protein of HBsAg identified high-responder and nonresponder phenotypes. Milich et al., J. EXP. Med.. 159:41 (1984).
Examination of the murine response to the preS2 region of the HBsAg revealed that immunization of a S protein nonresponder mouse strain with PreS2-S protein-containing HBsAg particles circumvented nonresponse to the S region by virtue of the PreS2 specific T cell help for B cell clones recognizing S region determinants. Milich et al., supra. In a similar study, Milich et al., J. Immunolo.. 137:315- 322 (1986) reported that immunization with PreSl- PreS2-S protein containing particles can induce a PreSl-specific T cell response that can bypass nonresponsiveness to the PreS2 and S regions of HBsAg. From the foregoing, it is apparent that strategies to produce recombinant HBsAg particles for vaccine development should take into account the possibility that drastic changes in the ratio of PreS to S region protein may effect antigenicity and/or immunogenicity. Thus, any recombinant process that produces HBsAg particles having PreS to S region protein ratios that mimic those found in nature would be highly advantageous.
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.
Neither the amino acid residue sequence nor the DNA sequence encoding the protein that comprises HBeAg have yet been identified. However, it is known the HBeAg is produced when core particles are denatured. For instance, the United States Patent No. 4,563,423 issued to Murray et al. describes a procedure for producing proteins displaying HBeAg antigenicity based on expressing the Core gene in bacteria. The Core protein so produced is extracted from transformed bacteria and then digested with proteases to produce proteins displaying HBeAg antigenicity. Thus, while Murray et al. teach that Core protein and HBeAg share amino acid residue sequences, it does not teach how to produce HBeAg without an ex vivo denaturation of Core protein.
Vertebrate cells have been used to produce proteins displaying either HBcAg or HBeAg antigenicity using recombinant DNA techniques. For instance, 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.
According to Gough et al. , supra. 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
(1986) reported that expression of the Core gene in Escherichia coli (E. coli) resulted in the production of nonsecreted core particles. In addition, Uy et al. reported that expression of the PreCore-Core gene in E. coli resulted in the production of a membrane bound, nonsecreted protein displaying HBeAg antigenicity. Similar results have been reported when the PreCore-Core and Core genes were expressed in the yeast Saccharomyces cerevisiae. See Miyanohara et al., J. Virol.. 59:176-180 (1986). In contrast, expression of the PreCore-Core gene in mammalian cells results in secretion of proteins that display HBeAg antigenicity. See Ou et al., Proc. Natl. Acad. Sci. USA. 83:1578-82 (1986) and Roossinck et al., Mol. Cell. Biol.. 6:1393-1400 (1986)
The introduction of cloned DNA into cells has become one of the most powerful tools of the molecular biologist. The development of expression vectors, i.e., recombinant DNA molecules (rDNA) , capable of directing the synthesis of cloned gene
products in vertebrate cells has been motivated, at least in part, by the recognition that a number of biologically and commercially important proteins require the postranslation modifications that uniquely occur in vertebrate cells in order to be biologically active.
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) . The LTRs of most retroviruses are extremely active promoters and foreign DNA cloned downstream from a viral LTR will usually be transcribed very efficiently.
The production of various mRNAs coding for different proteins is regulated at the level of RNA processing known as splicing. 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.
To be useful, 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 construction of retrovirus expression vectors has been reported by a number of laboratories. See, for example, Sorge et al., Mol Cell. Biol.. 4:1730-1737 (1984) and the references cited therein. Brief Summary of the Invention
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. In preferred embodiments 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. In preferred embodiments, the cells are NIH 3T3 cells. Also preferred are monoclonal cultures.
Further contemplated is a 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.
Also contemplated is a method of producing a composition containing a plurality of proteins displaying HBeAg 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. In preferred embodiments 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. In preferred embodiments, the vector is a retrovirus vector, preferably the vector pARV2. Also preferred
are embodiments where 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.
Further contemplated is a 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.
In a further embodiment, 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. Thus, 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.
One benefit of the present invention is that 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
In the drawings forming a portion of this disclosure:
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 ( |i-vvv.'.;i ) ; and pUC9 sequence ( BBflH ) • Arrows ("■■ ^) indicate the direction of transcription from LTR and MT promoters. Restriction endonuclease cleavage sites are indicated as follows: C = Clal; H = Hindlll; Rl = EcoRI; S = Sail; and X = Xbal. The unique restriction sites used to clone ORFs are indicated by asteriks
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. 106 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 (Δ) 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) (φ) 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.
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. Gradients were fractionated from the bottom (fraction 1) and HBsAg was determined by the AUSRIA II assay. Counts per minute (CPM) ( Q ) and corresponding sucrose density gradient fraction number are shown on the ordinate and the abscissa, respectively.
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
[Absorbance 492 nanometers (nm) , ] , and HBcAg was determined using the HBcAg-specific ELISA (Absorbance 490 n , ) , discussed hereinafter. Samples for panels A, B and D were centrifuged for 2 hours and samples for panels C and E were centrifuged
for 45 minutes as described in Example 8. Gradients were fractionated from the bottom (Fraction 1) .
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.
Portion A immunoblots were probed with a HBsAg-speσific 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) , whereas Lanes 2-4 contained 200 ug each of proteins from a cell line lysate, and 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,
University of Gottingen) . Photo G is of cell line 3T3 pARVlMTC stained with monoclonal anti-HBC 3120 (Dr. M. Mayumi) . Details of these analyses are provided in the Examples. Figure 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. The designations for the other mouse fibroblast clones were derived in a similar manner. In addition superscript "a" indicates a transfected cell line; superscript "b" indicates a helper virus infected/transfected cell line; superscript "c" indicates a clonal cell line and superscript "d" indicates a helper virus infected polyclonal cell line. Figure 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) . About 1X106 cells were plated on a 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. Cell lines from which data were obtained are: (Panel
A) 3T3 pARV2PC (■) , 3T3 pARV2PC/AMLV (•) , 3T3 ARV2PC/AMLV1 (A) , and 3T3 ARV2PC/AMLVla (♦) ; and (Panel B) 3T3 pARV2C (■) , 3T3 pARV2C/AMLV (•) , and 3T3 ARV2C/AMLVa (A) . Figure 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. Human skin fibroblasts were confluent by day 6. Cell line illustrated are: CCD- 41Sk ARV2PC/AMLVla (■) , GM2504E ARV2PC/AMLVla (•) and AG3 ARV2PC/AMLVla (A) .
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. (A) Lanes: (1) 3T3 pARV2PC DNA, (2) 3T3 ARV2PC/AMLVla DNA, and (3) CCD-41Sk
ARV2PC/AMLVla DNA. (B and C) Lanes: (1)= 3T3 pARV2C DNA, (2-4) 3T3 ARV2C/AMLV3,4 and 6 DNA, respectively, and (5) 3T3 ARV2C/AMLVa DNA. Detailed Description of the Invention A. Definitions
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 (rDNA) : 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.
B. Recombinant DNA Molecules The recombinant DNA molecules of the present invention can be produced by operatively linking a gene that encodes either a PreSl-PreS2-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 PreSι-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).
It should be understood that the 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. Thus, 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.
It should also be noted that because 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. Similarly, expression of the PreS2-S gene operatively inserted in a vector results in production of the PreS2-S and S proteins. In like manner, the DNA sequence that forms the PreCore-Core gene also contains the Core gene aligned in phase with the PreCore-Core gene. However, expression of the PreCore-Core gene does not result in expression of HBcAg. While it is advantageous to modify the DNA sequence of the PreCore-Core gene to eliminate the Core gene initiation codon so that no Core protein is expressed when the PreCore-Core gene is expressed, similar modifications of the PreS2-S and S gene initiation codons are not contemplated. This is because Core gene expression is not required in the methods of the present invention whereas expression of all in phase PreS2-S and S genes is required. Thus, the term "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. For example, 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 PreS1-PreS2-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). Of course, by chemically synthesizing the coding sequence, any desired modifications can be made simply by substituting the appropriate bases for those encoding the native amino acid residue sequence. However, in preferred embodiments, any modification to the DNA sequences of the native HBV genes do not disrupt the function of the endogenous regulatory elements those genes encode. Thus, DNA sequences exactly homologous to genes found in naturally occuring HBV subtypes are preferred.
In addition, DNA sequences that encode the PreS1-PreS2-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
Robinson, Am. J. Med. Sci.. 270:151-159 (1975) can be used to produce the entire HBV genome of any of the known subtypes in isolated form. DNA consisting essentially of genes encoding the PreS1-PreS2-S or PreCore-Core proteins can then be cloned from the isolated HBV genomic material using well known methods. See, for example, Siddiqui et al., Proc. Natl. Acad. Scif USA, 76:4664 (1979); Charmay et al., Nuc. Acids Res. f 7:335 (1979); and Sinisky et al., Nature. 279:346 (1979).
Furthermore, DNA sequences consisting essentially of genes encoding the PreSι-PreS2-S, PreS2-S or PreCore-Core proteins can be obtained from vectors containing those genes. For instance, E. coli transformed with vectors pARV2Pl/2S, pARVlMTS and
pARV2PC, which contain genes encoding the PreS^ PreS2-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.
Once obtained, DNA sequences encoding the PreS1-PreS2-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. Thus, 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
International Biotechnologies, Inc., New Haven, CN.
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.). In preferred embodiments, 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).
In preferred embodiments, 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. Such replicons are well known in the art. In addition, those embodiments that include a procaryotic replicon also include a gene whose expression confers drug resistance to a bacterial host transformed therewith.
Typical bacterial drug resistance genes are those that confer resistance to ampicillin or tetracycline.
In preferred embodiments the expression vector is a retrovirus expression vector that is preferably replication-incompetent. The construction and use of retroviral vectors has been described by Sorge et al., Mol. Cell. Biol.. 4:1730-37 (1984).
Also preferred are 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. 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, designated herein as a rRNA, is also contemplated by the present invention.
Further contemplated is a recombinant 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.
It should be noted that as used herein, the term "transfection" refers to a process of introducing a rDNa of this invention into a compatible host cell genome.
C. Transfected Cells and Cultures 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. Preferably, 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 Λ that contain a recombinant DNA molecule of the present invention and express a protein displaying either HBsAg or HBeAg antigenicity, can be identified by well known techniques. For example, 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).
In addition to directly assaying for the presence of rDNA, 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.
Thus, in addition to the transfected host vertebrate cells themselves, 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. Preferably, 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.
D. Methods for Producing HBeAg Another aspect of the present invention pertains to a method for producing proteins displaying 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.
In preferred embodiments, 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. In addition, 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. In preferred embodiments, those products display HBeAg antigenicity but no substantial HBcAg antigenicity. By 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.
As demonstrated herein, the expression products of the rDNAs containing the PreCore-Core gene secreted proteins displaying HBeAg antigenicity.
Those proteins all have amino acid residue sequences encoded by the PreCore-Core gene and have apparent molecular weights of 15, 18 and 21 kDa. Thus, the present invention contemplates compositions comprising those proteins, alone or in combination. In preferred embodiments, the proteins are affixed to a solid matrix to form a solid support useful in assembling diagnostic kits and in performing diagnostic assays. F. Proteinaceous Particle Displaying HBsAg Antigenicity
Proteinaceous particles displaying HBsAg antigenicity are particles that immunoreact with antibodies induced by native HBsAg, i.e., anti-HBs antibodies produced during HBV infection.
In one embodiment, 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. Also contemplated by the present invention is a composition 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. In preferred embodiments the particles contain a glycosylated protein. In another embodiment, 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. In preferred embodiments the particles contain a glycosylated protein.
The proteinaceous particles of the present invention do not contain any viral DNA. In addition, 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.
G. Methods for Producing Proteinaceous
Particles
The present invention also relates to a method for producing the proteinaceous particles of the present invention displaying HBsAg antigenicity. Where 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. Where 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 particle-containing medium is then recovered. In preferred embodiments the particles are isolated from the medium using well known methods.
H. Vaccines
In another embodiment, 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.
The preparation of vaccines which contain proteins as active ingredients is well understood in the art. Typically, 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. In addition, if desired, 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. For 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.
The term "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.
As used herein, the term "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.
Thus, in preferred embodiments, 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. In preferred embodiments, 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. In these embodiments, 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. Where 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
The following examples are intended to illustrate, but not limit, the present invention.
1. Recombinant DNA Molecule Constructions The construction of amphotropic retrovirus vectors pARVl, pARV2, and pARVlMT is outlined schematically in Figure 1. The various steps in the production of these vectors were performed by standard techniques [Maniatis et al., (1982) Molecular Cloning: a laboratory manual. Cold Spring Harbor Laboratory^ Press, Cold Spring Harbor, NY].
The vector CistorNeo has been described previously Sorge et al., Mol. Cell. Biol.. 4:1730- 1737 (1984) . 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.
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. From the resulting plasmid, 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. In addition, 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.
Prior to cloning the MT DNA fragment into pARVl, the StuI and Hindlll sites were modified to Xbal sites by linker insertion, and a Sail linker was cloned into the site where the Bglll/Sstll fragment was deleted. This generated a Sail cloning site for
expression of the ORFs from the MT promoter, which directs transcription in the same orientation as the retroviral LTR. The HBV DNA fragments described in Table 1 below, which were cloned into the Sail site of either PARV2 or pARVlMT, were derived from the plasmid pCPIO, Dubois et al. , Proc. Natl. Acad. Sci. USA. 77:4549-4553 (1980) by modification of appropriate restriction enzyme sites to Sail sites by linker insertion.
TABLE 1 HBV DNA Fragments (Subtype ayw) Expressed in Amphotropic Retroviruses
G418-resistant cell line
Ampho¬ established
HBV DNA tropic after trans¬
HBV gene cloned fragment Restriction retro¬ Expression fection with into retroviral coordi¬ enzyme sites*3 viral vector^ expression vector nates3 vector vector
pARV2 3T3 pARV2
PreSl-PreS2-S 2839-1990 Bglll-Bglll pARV2 PARV2P1/2S 3T3 pARV2Pl/2S
PreS2-S 3166-1411 SauI-FnuDII pARVlMT PARV2P2S 3T3 pARV2P2S
S 47- 971 Stul-Stul pARV2 pARVlMTS 3T3 pARVlMTS
PreCore-Core 1803-2804 Hinpl-Hinpl pARV2 pARV2PC 3T3 pARV2PC
Core 1890-2804c HinpI(BAL31) -Hinplc pARVlMT pARV2C 3T3 pARV2C
Core 1890-2804° HinpI(BAL31) -Hinplc pARVlMTC 3T3 pARVlMTC
a The coordinates of the 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.
^ The 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.
2. Cells and Transfections Mouse NIH/3T3 fibroblasts were grown, transfected and selected as described in Sorge et al., ■ Mol. Cell. Biol.. 4:1730-1737 (1984), except 15 micrograms (ug) of plasmid DNA without carrier DNA was used for each transfection. A cell line, derived from multiple aminoglycoside antibiotic G418 resistant colonies, was established from each transfection as shown in Table 1 above. 3. Antigen Assays
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
[polyoxyethylene (20) sorbitan monolaurate] , MacKay et al., J. Med. Virol.. 8:237-242(1981). Cell lysates were prepared by washing 5xl06 cells in PBS and resuspending the cells in 1 ml PBS containing 1 mM phenylmethylsulfonyl fluoride (PMSF) . The cell suspensions were subjected to three cycles of freezing and thawing followed by centrifugation for 30 minutes at 12,800xg. The supernatant represented the cell lysate. 4. Secretion of HBV Antigens
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.
2b) . However, inclusion of the precore in addition to the core sequence in the expression vector, i.e., the PreCore-Core gene, resulted in an approximately 10- fold higher level of HBeAg secretion as shown in Table 2 below.
TABLE 2
Estimate of Intracellular and Extracellular HBV Antigens
Rate of Rate of
HBsAg HBeAg secretion/ secretio (ng/106 (U/106 HBsAg HBeAg intra¬ intra¬ cells cells (ng/mg of (U/mg of cellular cellular
Cell Line per 24h) per 24h) protein) protein) HBsAg ratio HBeAg rat
3T3 pARV2Pl/2S 2 5 0.4 3T3 pARV2P2S 115 99 1.2 3T3 pARVlMTS 255 174 1.5 3T3 pARV2PC 1.64 0.19 8.6 3T3 pARV2C 0.15 3.16 0.05
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 5xl06 cells per confluent plate of cells. b 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)].
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5. Analysis of Soluble Intracellular
HBV Antigens
The data shown in Table 2, above, indicate that the levels of soluble intracellular HBsAg in the cell lines were approximately proportional to the rates of secretion of HBsAg from these cells.
The data shown in Table 2 also suggest that the rate of secretion may be related to the amount of soluble HBsAg inside the cell. Cell line 3T3 pARV2C had greater than 10 times more soluble intracellular HBeAg than did cell line 3T3 pARV2PC (Table 2) . This contrasts with the rate of secretion of HBeAg from these two cell lines. These observations suggest that inclusion of the precore DNA sequence in addition to the core sequence, i.e., the PreCore-Core gene, in the expression vector results in synthesis of polypeptides which are preferentially secreted compared with polypeptides lacking the precore sequence.
6. Antigenicities of Secreted and Soluble Intracellular HBV Antigens
The products of the three HBsAg-producing cell lines were analyzed by ELISA for synthesis of soluble PreS region containing polypeptides. The results of that analysis are shown in Table 3 below.
Table 3 Pre-S Polypeptide Compositions of HBsAgs
Cell Line PreSl3 % PreS2b PreSl % PreS2 3T3 pARV2Pl/2S + 5 NDC ND 3T3 pARV2P2S 0 50 0 20 3T3 pARVlMTS 0 0 0 0
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.
As expected, 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 were1 analyzed for protein displaying either HBcAg or HBeAg antigenicity. The results of that analysis are shown in Table 4 below.
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Table 4 Antigens Produced by 3T3 pARV2PC and 3T3 pARV2C Cell Lines
Soluble
Secreted Intracellular
Antigen Antigen
Cell Line HBeAg3 HBcAg HBeAg HBcAg
3T3 pARV2PC + - + - 3T3 pARV2C + _ + +
3 Determined by the Abbott HBe EIA Diagnostic Kit. b Determined by the HBcAg-speσific ELISA described in Example 3.
Since the Abbott HBe EIA detects both HBeAg and HBcAg, the 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.
Furthermore, since HBeAg was detected in the cell culture media "of the 3T3 pARV2C cell line, production of HBeAg does not necessarily require the precore sequence. 7. CsCl and Sucrose Density Gradient
Analysis of HBsAg
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)2S04, 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. This indicated that the presence of approximately 50% PreS2-S polypeptide did not significantly alter the density of the HBsAg particles. However, it appeared that 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.
The low yield of secreted HBsAg from the 3T3 PARV2P1/2S cell line did not permit detection of PreSl-PreS2-S protein containing particles by electron microscopy. 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.
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) . In addition, it is apparent that approximately half of the 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).
8. Sucrose Density Gradient Sedimentation
Analysis of HBcAg and HBeAg
The relative sizes of 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) . However, 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.
9. Immunoblotting Analysis of HBV Antigens Characterization of HBV antigens by immunoblotting was performed to determine the total intracellular antigen content and to estimate the * relative sizes of the various polypeptides.
Cell lysates were prepared by washing 5 X 106 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) .
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227:680-685 (1970). Culture medium samples were boiled with an equal volume of 2X sample buffer before electrophoresis. Transfer of the separated proteins to nitrocellulose membranes (Schleicher & Schuell, Inc., Keene, N.H. ; BA-85; 0.45-um pore size) was performed as described by Heermann et al., J. Virol.. 52:396-402 (1970). Membranes were rinsed in 25 mM Tris hydrochloride (pH 7.5)-500 mM NaCl (TN) for 10 min at 22°C and blocked in TN containing 3% (wt/vol) gelatin and 0.05% (vol/vol) Tween 20 for 8 hr at 22°C. 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 125I-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.
Analysis of intracellular HBsAg indicated that cell line 3T3 pARVlMTS contained polypeptides of 25 and 27 kDa, cell line 3T3 pARV2P2S contained polypeptides of 25, 27, and 32 kDa, and cell line 3T3 pARV2Pl/2S contained polypeptides of 25, 27, and 43 kDa, whereas the secreted CHO HBsAg particles contained polypeptides of 25, 28, and 33 kDa (Fig. 6A) . The differences in polypeptide sizes were confirmed by immunoblotting analysis of mixtures of
intracellular and secreted proteins displaying HBsAg antigenicity synthesized by these cell lines (Data not shown) . The presence of polypeptides of 27 rather than 28 kDa and 32 rather than 33 kDa suggested that the glycosylated intracellular HBsAg proteins contain only high- annose, rather than complex, oligosaccharide chains, as has been previously demonstrated for this antigen. Patzer et al., J. Virol.. 58:884-892 (1976) and Patzer et al. , O . Virol.. 51:346-353 (1986). This indicated that the intracellular HBsAg resides in a pre- or early-Golgi cellular compartment. Cell line 3T3 pARV2P2S produced the 25-kDa HBsAg in addition to the 32-kDa HBsAg, possibly because of inefficient utilization of the PreS2-S gene translation initiation codon.
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) . However, as no 32-kDa protein was apparent, it seems unlikel that inefficient translation initiation can explain this observation. 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 levels of intracellular HBsAg protein in cell lines 3T3 pARVlMTS and 3T3 pARV2P2S correlated with the amounts of soluble antigen (Table 2) . However, it was apparent that the total antigen present in cell line 3T3 pARV2Pl/2S was approximately equivalent to that in the other two cell lines. This observation, together with the relatively low level of soluble HBsAg present in the 3T3 pARV2Pl/2S cell line (Table 2) , indicated that approximately 95% of the total intracellular HBsAg was present in an aqueous,
nonextractable cellular compartment of this cell line. This change in compartmentation is attributable to a property located, at least in part, in the PreSl encoded region of the 43-kDa polypeptide since the 32- kDa protein was in the soluble intracellular compartment.
The coexistence of the 25- and 27-kDa and 43-kDa HBsAg proteins resulted in the major polypeptides residing in the aqueous nonextractable compartment, whereas when the major polypeptides were synthesized alone they were present in the soluble cellular compartment. This indicated that the 25- and 27-kDa polypeptides interact with the 43-kDa protein and are thereby trapped in the aqueous, nonextractable cellular compartment, preventing secretion of the major polypeptides.
Analysis of the product of the 3T3 pARV2C cell line indicated that, in the absence of the precore encoded sequence, the predicted 21-kDa HBcAg polypeptide was synthesized and accumulated within the cell (Fig. 6B, lanes 1 and 4) . The presence of this polypeptide and the fast-sedimenting HBeAg in lysates of these cells (Fig. 5C) is consistent with this polypeptide forming a particulate structure similar to that observed in other systems. Cohen et al., Nature (London) . 296:677-678 (1982). In addition, it suggests that this polypeptide can also form the slow- sedimenting HBeAg and subparticulate HBcAg (Fig. 5C) , although it is possible that smaller polypeptides of limited abundance account for a proportion of the observed antigens. The 3T3 pARV2C cell line secreted HBeAg (Table 3) and, from longer exposures (data not shown) of the autoradiogram shown in Fig. 6B, lane 7, this correlates with the presence of equal amounts of
core polypeptides of 15 and 21-kDa in the cell culture media.
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. Miyanohara et al. , J. Virol.. 59:176-180 (1986) . However, 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. This suggests that the 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.. 130:2903-2907 (1983), and further suggests that a larger PreCore-Core polypeptide was processed in 3T3 cells to yield secreted proteins displaying HBeAg antigenicity. In addition,' it should be noted that the relative abundance of the total intracellular Core- specific polypeptides (Fig. 6B, lanes 3 and 4) correlates with the levels of soluble intracellular antigen (Table 2) in these cell lines.- This suggests that, unlike the case of PreSl region-containing polypeptides, the PreCore sequence does not partition antigen into an aqueous, nonextractable cellular compartment but rather promotes secretion of HBeAg. 10. Cellular Localization of HBV Antigens The distribution of the HBV antigens in the
various cell lines was analyzed by immunofluorescence microscopy using the method described by Chisari et al.. Science. 230:1157-1160 (1986) (Fig. 7A to G) .
Cells were grown on microscope slides, fixed for 1 min in ice-cold acetone, and stained with appropriate antibodies. PreSl-PreS2-S polypeptides, PreS2-S polypeptides, HBsAg, HBcAg, and HBeAg 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) . In addition, 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. In addition, it appeared that the proteinaceous particles containing PreSl-PreS2-S protein have a coarser granular appearance than particles lacking this protein. As expected, the PreS-PreS2-S and PreS2-S proteins were localized to the cytoplasm in a manner similar to that of the S protein. With a HBcAg-specific monoclonal antibody, the 3T3 pARV2C and 3T3 pARVlMTC cell lines exhibited positive immunofluorescence localized predominantly to the nucleus (Fig. 7G) . Qualitatively, these two cell lines had the same distribution of immunofluorescence, but quantitatively the 3T3 pARVlMTC cell line showed a
greater signal. These results are consistent with the presence of intracellular HBcAg in these cell lines (Table 3; Fig. 5C) . 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
The ability to examine the cytotoxic T lymphocyte (CTL) response during HBV infection has been severely limited by the absence of an in vitro infection system. Most investigators have been limited to the study of antibody-dependent cell mediated cytotoxicity and natural killer cell activity present in HBV infected patients. In addition, the absence of well defined target antigens has limited the interpretation of these studies. There have been attempts to analyze the major histocompatability complex (MHC)-restricted CTL response during chronic HBV infection by utilizing autologous hepatocyte cultures derived from liver biopsies. This approach allows the analysis of only a limited number of samples and may not permit determination of CTL specificity.
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. In 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. Sorge et al., Mol. Cell. Biol.. 4:1730-1737 (1984).
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. The 3T3 ARV2PC/AMLVla, CCD-41Sk
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.
Cell culture, infection and selection conditions for mouse and human fibroblasts were as described except the antibiotic G418 concentration used was 350 ug/ml for both types of fibroblasts. The 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% C02/air. Infection was performed by co-culturing 5xl06 B cells with 1x10s 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) .
All cell lines were selected for resistance to the antibiotic, G418, and therefore were infected with recombinant retrovirus.
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12. Transmission of HBeAg and HBc/eAg Expression to Mouse Fibroblasts by Recombinant Retroviral Infection The ability of the 3T3 pARV2PC/AMLV and 3T3 pARV2C/AMLV cell lines to produce recombinant retrovirus which could transmit HBeAg and HBc/eAg expression was examined by analyzing the antigen synthesis of the 3T3 ARV2PC/AMLV1-18 and 3T3 ARV2C/AMLV1-34 clonal cell lines, respectively (Tables 5 and 6) . 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 (Table 5) 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 104 G418- resistant colony forming units per milliliter (G418R 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) .
In total, 90 clonal cell lines, 3T3 ARV2C/AMLV1.1-8.2, were examined for secreted antigen indicating that the occurrence of transmission of antigen expression by recombinant retrovirus infection from the 3T3 ARV2C/AMLV1-8 clonal cell lines was either very low or absent. In addition, 10 of these 90 clonal cell lines were examined and shown to be negative for intracellular antigen. These results demonstrated that a high titer clonal cell line, 3T3 ARV2PC/AMLV1, which transmits antigen expression by recombinant retroviral infection with high efficiency, was obtained only for one of the two antigen systems examined. Transmission of HBc/eAg expression by recombinant retrovirus derived from clonal cell lines, generated by infection with recombinant retrovirus from the infected/transfected cell line, did not occur. Therefore, transmission of HBc/eAg expression by recombinant retroviral infection was limited to the relatively inefficient virus pool produced by the 3T3 PARV2C/AMLV cell line.
TABLE 5. Transmission of HBeAg Expression by Recombinant Retrovirus Infection.
Percentage of clones expressing HBeAg3
Recombinant retrovirus (Positive clones/ Average A Mean + SD 92
Cell Line titer (G418R cfu/ml) Total clones) (Total no. of clones)
3T3 pARV2PC/AMLV 5X104 83(15/18) 0.47+0.38(18) 3T3 ARV2PC/AMLV1 2X105 90(9/10) 0.31+0.15(10) 3T3 ARV2PC/AMLV2 lXlO5 100(8/8) 0.29+0.10 (8) 3T3 ARV2PC/AMLV3 8xl04 0(0/6) 0 (0)
3T3 pARV2C/AMLV 2X104 29(10/34) 0.07+0.13(34) 3T3 ARV2C/AMLV1 5xl04 0(0/12) 0 (12) 3T3 ARV2C/AMLV2 2X104 0(0/7) 0 (7) 3T3 ARV2C/AMLV3 5X105 0(0/37) 0 (37) 3T3 ARV2C/AMLV4 2X105 0(0/13) 0 (13) 3T3 ARV2C/AMLV5 lXlO5 0(0/8) 0 (8) 3T3 ARV2C/AMLV6 1X105 0(0/7) 0 (7) 3T3 ARV2C/AMLV7 5X104 0(0/4) 0 (4) 3T3 ARV2C/AMLV8 l lO5 0(0/2) 0 (2)
3 A clone, generated by infection with a recombinant retrovirus from a cell line, was considered to be expressing HBeAg if the difference between a control medium sample and a 2
4 day medium sample from the clone was greater than A492=0.06 in the HBeAg assay. b The average A492+SD values for 2-4 day medium samples were corrected for the backgroun in the assay by subtracting the A492 value for a control medium sample.
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TABLE 6 Expression of HBeAg by Clonal Mouse NIH 3T3 Fibroblast Cell Lines Infected with Recombinant Retrovirus
Intracellular
Secreted HBeAg3 HBeAg3
Cell Line (A492) (A492>
3T3 ARV2PC/AMMLV1 1.20 ND
3T3 ARV2PC/AMLV2 1.04 ND
3T3 ARV2PC/AMLV3 0.16 ND
3T3 ARV2C/AMLV1 0.02 ND
3T3 ARV2C/AMLV2 0.08 ND
3T3 ARV2C/AMLV3 0.10 1.05
3T3 ARV2C/AMLV4 0.20 1.88
3T3 ARV2C/AMLVt 0.74 0.30
3T3 ARV2C/AMLV6 0.06 1.30
3T3 ARV2C/AMLV7 0.11 0.51
3T3 ARV2C/AMLV8 0.07 0.40
3 The HBeAg determinations were corrected for the background activity in the ELISA. ND Not determined.
13. Analysis of Secreted and Soluble
Intracellular Antigens Expressed in Mouse Fibroblast Cell Lines Generated by Recombinant Retrovirus Infection The time course of HBeAg secretion for various transfected and recombinant retrovirus infected clonal and polyclonal cell lines is shown in Figure 9. The rates of HBeAg secretion and the levels of intracellular HBeAg for these cell lines are shown in Table 7.
TABLE 7 Estimates of Intracellular and Extracellular HBeAg
Average Rate Rate of of Secretion Intracellular Secretion/ of HBeAg (U/ HBeAg (U/mg Intracellular
Cell Line ml per 24 hrs) protein)3 HBeAg ratio3
3T3 pARV2PC 0.51 0.31 1.7 3T3 pARV2PC/AMLV 0.13 0.17 0.8 3T3 ARV2PC/AMLV1 1.06 0.24 4.4 3T3 ARV2PC/AMLVla 0.58 0.022 26.4
3T3 pARV2C 0.19 18.5 0.010
3T3 pARV2C/AMLV 0.16 14.6 0.011
3T3 ARV2C/AMLVa 0.044 2.98 0.015
Determined as previously described.
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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.
In contrast, 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.
Comparison of the levels of expression indicate that the efficiency of antigen production is similar whether the retrovirus vector is introduced into the mouse fibroblasts by transfection or infection (Figure 9 and Table 7) . The 3 to 5 fold difference in antigen production between the 3T3 pARV2C/AMLV and the 3T3 ARV2C/AMLVa cell lines can e accounted for by the approximately 30% efficiency of transmission of antigen expression by the recombinant retrovirus pool produced by the 3T3 pARV2C/AMLV cell line.
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14. Expression of HBeAg in Human Skin Fibroblasts and EBV Transformed
B Lymphocytes
The secretion of HBeAg by the human skin fibroblast CCD-41Sk ARV2PC/AMLVla and GM2504E
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
NIH 3T3 fibroblasts by infection. However, due to the relatively low titer of the recombinant retroviral pool (2 X 104 G418R cfu/ml) and the observation that only about 30% of these recombinant retroviruses had the capacity to transmit the expression of HBc/eAg synthesis, it is possible that during the rather extended period of time required to select and grow the infected human skin fibroblasts a small selective advantage to the cells which were not producing antigen permitted them to outgrown the cells infected with recombinant retrovirus coding for antigen synthesis.
15. Analysis of Recombinant Retrovirus Sequences in Transfected and Infected Cell Lines
Filter hybridization analysis of 3T3 PARV2PC, 3T3 ARV2PC/AMLVla and CCD-41Sk ARV2PC/AMLVla cell line DNA digested with EcoRV demonstrated that the recombinant retroviral sequences were transmitted without gross rearrangement to both mouse and human fibroblasts (Figure 11A) . This is consistent with the secretion of HBeAg by these recombinant retrovirus infected cell lines. It should be noted that EcoRV cuts the plasmid pARV2PC in the long terminal repeat of the amphotropic murine leukemia virus 4070 producing two DNA fragments of 3.8- and 3.4-kbp. 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. However, since 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 inόreased size of these recombinant proviral genomes. Since the pattern of hybridization using an HBV probe (Figure 11A) was identical to the pattern of hybridization observed when a neomycin resistance gene probe (data not shown) , it appears that the recombinant retrovirus genomes of 6- and 9-kbp contain both HBV and neomycin resistance gene DNA sequence. Filter hybridization analysis of 3T3 pARV2C,
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 presumably reflects the integration of a single copy of the unrearranged recombinant proviral genome per cell. This proviral sequence is responsible for the HBc/eAg synthesis observed in these cells. However, when these same DNAs are probed with 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) . The nature of this recombinant proviral genome is unclear except it does not appear to contain any HBV sequences. In addition, 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.
Examination of the hybridization pattern of the 3T3 ARV2C/AMLVa polyclonal cell line DNA demonstrated that the HBV sequences present in the pARV2C expression vector had been very poorly transmitted to this cell line (Figure 11B, lane 5) . However, longer exposures of this autoradiogram did reveal a band of hybridization at 3.7-kbp which presumably corresponded to the recombinant retrovirus genome which coded for the HBc/eAg synthesized by this cell line.
The pattern of hybridization observed when 3T3 ARV2C/AMLVa polyclonal cell line DNA was probed with a neomycin resistance gene revealed, a strong band of hybridization at 9-kbp and a weak band of hybridization at 3.7-kbp. This suggests the 3T3 pARV2C/AMLV cell line is producing predominantly two types of recombinant retrovirus. Approximately 30% of mouse fibroblasts infected with this recombinant retroviral pool appear to retain the 3.7-kbp recombinant proviral genome and therefore synthesize HBc/eAg. In addition, approximately 90% (the cells not expressing HBc/eAg plus two thirds of the cells expressing antigen (Figure 11C, lanes 2-4) of mouse fibroblasts appear to retain the 9-kbp proviral genome which presumably has the capacity to code for G418 resistance. To explain the failure of all the 3T3
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. In
380
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addition, 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.
Examination of the recombinant proviral ■ structure in the CCD-41Sk ARV2PC/AMLVla polyclonal cell line revealed that in addition to the transmission of the recombinant retrovirus sequence of the expected structure, rearranged proviral sequences of a larger size than expected containing both HBV and neomycin resistance gene sequences were observed.
This indicated that the 3T3 ARV2PC/AMLV1 clonal cell line is producing aberrant recombinant retroviruses at a low frequency and cells infected with these rearranged recombinant retroviruses can preferentially grow out of a population of human skin CCD-41Sk fibroblasts.
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. However, 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.
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. However, the
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observation that the pARV2PC expression vector codes for recombinant retroviral transcripts which permit the faithful transmission of the unrearranged proviral genome indicates that small differences in the structure of a recombinant retroviral vector can dramatically alter the ability of the vector to be faithfully transmitted as a recombinant retrovirus. This illustrates one of the difficulties in constructing recombinant retroviral expression vectors which are designed to transmit expression of a non- selectable gene produce in conjunction with a selectable gene product by recombinant retrovirus infection.
Since one of the aims of this study was to examine the use of recombinant retroviral-mediated gene transfer to generate stimulator/target cells for the analysis of the CTL response to HBV antigens, it is apparent that in the case of HBeAg this is possible. For these types of studies, 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.
The foregoing specification, including the specific embodiments and examples, is intended to be illustrative of the present invention and is not to be taken as limiting. Numerous other variations and modifications can be effected without departing from the true spirit and scope of the present invention.
Claims (31)
1. A recombinant DNA molecule comprising a DNA sequence consisting essentially of the PreCore- Core gene of HBV operatively linked to a vector capable of expressing said gene in a vertebrate cell.
2. The recombinant DNA molecule of claim 1 wherein said vector is a retrovirus vector.
3. The recombinant DNA molecule of claim 2 wherein said vector is pARV2.
4. The recombinant DNA molecule of claim 2 wherein said vector is pARVlMT.
5. A recombinant DNA molecule, designated pARV2PC, characterized as containing the PreCore-Core gene containing HBV DNA Sail restriction fragment operatively inserted into the retrovirus expression vector pARV2 at the Sail restriction site of said vector.
6. A transfected host vertebrate cell culture comprising vertebrate cells in a nutrient medium, said cells containing a recombinant DNA molecule comprising a DNA sequence consisting essentially of the PreCore-Core gene of HBV operatively linked to a vector capable of expressing said gene in said cells.
7. The transfected cell culture of claim 6 wherein said cells are NIH 3T3 cells.
8. The transfected cell culture of claim 6 wherein said culture is a monoclonal culture.
9. A composition comprising an admixture of first protein having an apparent molecular weight of 15 kDa, a second protein having an apparent molecular weight of 18 kDa and a third protein having an apparent molecular weight of 21 kDa, each of said proteins having an amino acid residue sequence that corresponds to an amino acid residue sequence encoded by the HBV PreCore-Core gene, said composition displaying HBeAg antigenicity but no substantial HBcAg antigenicity.
10. A method of producing a composition containing a plurality of proteins displaying the antigenicity of HBeAg comprising the steps of:
(a) culturing, in a nutrient medium, vertebrate cells transfected with a recombinant DNA molecule comprising a DNA sequence consisting essentially of the PreCore-Core gene of HBV operatively linked to a vector capable of expressing said gene in said cells, said culturing forming a medium containing secreted proteins expressed by said gene; and (b) recovering said medium containing said secreted proteins.
11. A method of producing a protein displaying the antigenicity of HBeAg comprising the steps of: (a) initiating a culture, in a nutrient medium, of vertebrate cells transfected with a recombinant DNA molecule comprising a DNA sequence consisting essentially of the PreCore-Core gene of HBV operatively linked to a vector capable of expressing said gene in said cells;
(b) maintaining said culture for a time period sufficient for said cells to express protein from said recombinant DNA molecule and secrete said protein into said medium; and (c) recovering said protein from said medium.
12. The method of claim 11 wherein said transfected cells are NIH 3T3 cells and said protein has an apparent molecular weight selected from the group consisting of 15 kDa, 18 kDa and 21 kDa. -68-
13. A recombinant DNA molecule comprising a DNA sequence consisting essentially of the PreSl- PreS2-S gene of HBV operatively linked to a vector capable of expressing said gene in a vertebrate cell.
14. The recombinant DNA molecule of claim
13 wherein said vector is a retrovirus vector.
15. The recombinant DNA molecule of claim
14 wherein said vector is pARV2.
16. The recombinant DNA molecule of claim 13 wherein said gene is operatively linked to a mouse metallothionein promoter.
17. The recombinant DNA molecule of claim 16 wherein said vector is pARVlMT.
18. A recombinant DNA molecule, designated pARV2Pl/2S, characterized as containing the PreSl-
PreS2-S gene-containing HBV DNA restriction fragment operatively inserted into the retrovirus expression vector pARV2 at the Sail restriction site of said vector.
19. A transfected host vertebrate cell culture comprising vertebrate cells in a nutrient medium, said cells containing an expression vector capable of expressing in said cells at least one secreted protein encoded by the PreSl-PreS2-S gene.
20. The transfected cell culture of claim
19 wherein said cells are NIH 3T3 cells.
21. The transfected cell culture of claim 19 wherein said culture is monoclonal.
22. A proteinaceous particle comprising at least one protein encoded by the PreSl-PreS2-S gene of
HBV displaying HBsAg antigenicity, said particle prepared from a recombinant DNA molecule.
23. The particle of claim 22 wherein said at least one protein encoded for by the PreSl-PreS2-S gene is glycosylated. -69-
24. The particle of claim 22 wherein said particles is derived from a serum-free source.
25. A method of preparing a proteinaceous particle displaying HBsAg antigenicity wherein at least one protein is encoded by the PreSl-PreS2-S gene comprising the steps of:
(a) initiating a culture, in a nutrient medium, of vertebrate cells transfected with a recombinant DNA molecule comprising a DNA sequence consisting essentially of the PreSl-PreS2-S gene of
HBV operatively linked to vector capable of expressing said gene in said cells;
(b) maintaining said culture for a time period sufficient for said cells to express protein from said recombinant DNA molecule and secrete said protein in particle form into said medium; and
(c) recovering said particle from said medium.
26. A composition comprising proteinaceous particles that contain the PreSl-PreS2-S protein of
HBV and have a density of about 1.26 grams per milliliter wherein at least about 25 percent of said particles have a sucrose density gradient sedimentation rate that is greater than particles that contain only S protein.
27. The composition of claim 26 wherein the percent of said particles that have a sucrose density gradient sedimentation rate that is greater than particles that contain only S protein is at least about 50.
28. The composition of claim 27 wherein said particles contain a glycosylated protein.
29. A proteinaceous particle that consists essentially of the PreS2-S and S proteins, said particles having an average diameter of about 19,7 nanometers and containing at least about 35 percent PreS2-S protein as determined by immunologic methods.
30. The particle of claim 29 having at least about 50 percent PreS2-S protein.
31. The particle of claim 30 wherein said particle contains a glycosylated protein.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US1356287A | 1987-02-11 | 1987-02-11 | |
| US013562 | 1987-02-11 | ||
| US15411788A | 1988-02-08 | 1988-02-08 | |
| USNOTGIVEN | 1998-06-29 |
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| Publication Number | Publication Date |
|---|---|
| AU1391888A true AU1391888A (en) | 1988-09-14 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU13918/88A Abandoned AU1391888A (en) | 1987-02-11 | 1988-02-09 | Retroviral expression vectors and methods for producing hbv antigens |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0301083A4 (en) |
| AU (1) | AU1391888A (en) |
| NO (1) | NO885658L (en) |
| WO (1) | WO1988006185A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU616976B2 (en) * | 1988-06-03 | 1991-11-14 | Merck & Co., Inc. | Method for producing nonhyperglycosylated hepatitis b virus proteins |
| AU627465B2 (en) * | 1988-06-03 | 1992-08-27 | Smithkline Biologicals | Expression of retrovirus gag protein in eukaryotic cells |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IL93304A (en) * | 1989-02-07 | 1992-09-06 | Bio Technology General Corp | Method for production and purification of hepatitis b vaccine |
| DE4107612A1 (en) * | 1991-03-09 | 1992-09-10 | Behringwerke Ag | RECOMBINANT PROTEINS WITH THE IMMUNE REACTIVITY OF HEPATITIS B VIRUS E ANTIGENS (HBEAG), METHODS FOR THEIR PRODUCTION AND THEIR USE IN IMMUNOASSAYS AND VACCINE SUBSTANCES |
| US6297048B1 (en) | 1992-02-04 | 2001-10-02 | Chiron Corporation | Hepatitis therapeutics |
| DK0625204T3 (en) * | 1992-02-04 | 2002-07-15 | Chiron Corp | Therapeutic agents for hepatitis |
| FR2711670B1 (en) * | 1993-10-22 | 1996-01-12 | Pasteur Institut | Nucleotide vector, composition containing it and vaccine for immunization against hepatitis. |
| US6133244A (en) | 1993-10-22 | 2000-10-17 | Institut Pasteur | Method for immunization against hepatitis B |
| US7732423B2 (en) | 1994-04-27 | 2010-06-08 | Institut Pasteur | Nucleotide vector, composition containing such vector, and vaccine for immunization against hepatitis |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2559159B1 (en) * | 1984-02-02 | 1986-09-12 | Inst Nat Sante Rech Med | VIRAL VECTORS FOR THE CLONING AND EXPRESSION OF A PROTEIN IN A EUKARYOTIC CELL, COMPRISING AT LEAST ONE PART OF THE GENOME OF A RETROVIRUS; TRANSFECTED EUKARYOTIC CELLS; PROCESS USING SUCH CELLS AND PROTEINS OBTAINED |
-
1988
- 1988-02-09 AU AU13918/88A patent/AU1391888A/en not_active Abandoned
- 1988-02-09 EP EP19880902298 patent/EP0301083A4/en not_active Withdrawn
- 1988-02-09 WO PCT/US1988/000380 patent/WO1988006185A1/en not_active Application Discontinuation
- 1988-12-20 NO NO885658A patent/NO885658L/en unknown
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU616976B2 (en) * | 1988-06-03 | 1991-11-14 | Merck & Co., Inc. | Method for producing nonhyperglycosylated hepatitis b virus proteins |
| AU627465B2 (en) * | 1988-06-03 | 1992-08-27 | Smithkline Biologicals | Expression of retrovirus gag protein in eukaryotic cells |
Also Published As
| Publication number | Publication date |
|---|---|
| NO885658D0 (en) | 1988-12-20 |
| EP0301083A4 (en) | 1990-09-26 |
| NO885658L (en) | 1988-12-20 |
| WO1988006185A1 (en) | 1988-08-25 |
| EP0301083A1 (en) | 1989-02-01 |
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