CA1340934C - Hepatitis b surface antigen formed by recombinant dna techniques, vaccines, diagnostics, cell lines and method of forming same - Google Patents
Hepatitis b surface antigen formed by recombinant dna techniques, vaccines, diagnostics, cell lines and method of forming sameInfo
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- CA1340934C CA1340934C CA000506686A CA506686A CA1340934C CA 1340934 C CA1340934 C CA 1340934C CA 000506686 A CA000506686 A CA 000506686A CA 506686 A CA506686 A CA 506686A CA 1340934 C CA1340934 C CA 1340934C
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
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Abstract
Bacterial plasmids carrying the PreS1-PreS2-S protein coding region, but lacking sequences encoding the hepatitis B core antigen, are used for transfection of eukaryotic cell lines for production of particles containing polypeptides encoded by the PreS1-PreS2-S protein coding region.
Description
HEPATITI~; B SURFACE ANTIGEN FORMED BY RECOMBINANT
DNA TE;CHNIQIJES, VACCINES, DIAGNOSTICS, CELL
L,ILdES AND PMETHODS OF FORMING SAME
BACFCGROUND OF THE INVENTION
05 1. Field of the Invention.
The present invention relates to bacterial Plasmids carrying the PreSl-PreS2-S protein coding region, but lacking sequences encoding the hepatitis B core antigen, which are used for transfection of eukaryotic cell lines for production of particles containing polypeptides encoded by the PreSl-PreS2-S protein coding region.
:?. Background of the Invention.
Recent advances in genetic engineering have made it possible for a number of peptides and proteins to be expressed in bacteria, yeast as well as mammalian cells, in increased amounts. Though many proteins and peptides have been expressed in considerable amounts in bacteria, it is a special desire to expres:~ large amounts of those proteins and polypeptides which are to be used in human medicine in mammalian cells in order to avoid drawbacks asso ciated with any contaminating products resulting from the bacterial cell, or from expression in bacterial cells.
Of special interest is the expression of hepatitis B surface antigen proteins in increased amounts in mammalian cells. The expression rate of these polypeptides in mammalian cells has so far not been high enough to allow an economic production thereof.
40934 ' Hepatitis B virus is transmitted among humans and causes a chronic and debilitating infection that results in severe liver damage, primary carcinoma and death. In most cases, complete 05 recovery from hepatitis B infection can be expected.
However, i.n ma.ny African and Asian countries, hepatitis :3 virus infection is endemic. A large number of individuals in the populations of these countries a.re chronic carriers of hepatitis B virus and thereby have the dangerous potential of further transmitting the disease.
Vac cination is the only known protection against hepatitis B virus. Recently several companies (for example, Dterck, Sharp and Dohme, U.S.A., and the Pasteur Institute, France) have introduced ~~ pla~~ma-derived vaccine against hepatitis B virus. The vaccine contains, as the antigen, hepatitis B viral proteins which are normally located on the envelope of the viral particle. Such antigen 2C is commonl~,~ referred to as the hepatitis B virus surface antigen (HBsAg), and the viral gene encoding such antigen is called the HBsAg gene. The above-referenced vaccines contain surface antigen isolated from t:he plasma of hepatitis B virus infected humans. Although the surface antigen itself is not pathogenic, it stimulates the production of antibodies that are directed against hepatitis B
virus particles i:n human beings.
The' only commercially available source of hepatitis B surface antigen is the blood of hepatitis B virus infected humans. The isolation and purification of tl:~e surface antigen from human serum 134Q9~4 is a cumbersome and time-consuming process and is, therefore, relatively expensive. In addition, as long as the human serum is the only commercially available source for surface antigen, the amount of 05 surface antigen available for use in vaccines, will be limited.
For large-scale vaccination, especially in areas where hepatitis B virus is endemic, it is of great importance to have a cheap and virtually unlimited source for surface antigen of hepatitis B
virus. Because of the potential danger of contamination with pathogenic agents when surface antigen is taken from human serum, it is desirable to avoid the use of human serum as a source for surface antigen. ~"here is also no biological system known besides human beings and chimpanzees in which hepatitis B virus can grow and be propagated.
Recent advances in molecular biology have made it X~ossible to clone the sequence of the complete genome of hepatitis B virus (Siddiqui, A. et al, Proc. Dfatl. ,~lca. Sci. , U.S.A. , Vol. 76, p. 4664, 1979; Sninsky, J.J. et al, Nature, Vol. 279, p. 346, 1979; and c~harnay, P. et al, Nucl. Acids Res., Vol.
7, p. 335, 1979). In addition, the coding regions for the different. viral proteins of hepatitis B virus have been identified (see, e.g., European Patent Application Publication Nos. 13828, 20251 and 38765;
Galibert et al, Nature, Vol. 281, pp. 646-650, 1979;
Pasel~ et al, Nature, Vol. 282, pp. 575-579, 1979; and Valenzuela et al, Nature, Vol. 280, pp. 815-819, 1979).
1340934 '~
DNA TE;CHNIQIJES, VACCINES, DIAGNOSTICS, CELL
L,ILdES AND PMETHODS OF FORMING SAME
BACFCGROUND OF THE INVENTION
05 1. Field of the Invention.
The present invention relates to bacterial Plasmids carrying the PreSl-PreS2-S protein coding region, but lacking sequences encoding the hepatitis B core antigen, which are used for transfection of eukaryotic cell lines for production of particles containing polypeptides encoded by the PreSl-PreS2-S protein coding region.
:?. Background of the Invention.
Recent advances in genetic engineering have made it possible for a number of peptides and proteins to be expressed in bacteria, yeast as well as mammalian cells, in increased amounts. Though many proteins and peptides have been expressed in considerable amounts in bacteria, it is a special desire to expres:~ large amounts of those proteins and polypeptides which are to be used in human medicine in mammalian cells in order to avoid drawbacks asso ciated with any contaminating products resulting from the bacterial cell, or from expression in bacterial cells.
Of special interest is the expression of hepatitis B surface antigen proteins in increased amounts in mammalian cells. The expression rate of these polypeptides in mammalian cells has so far not been high enough to allow an economic production thereof.
40934 ' Hepatitis B virus is transmitted among humans and causes a chronic and debilitating infection that results in severe liver damage, primary carcinoma and death. In most cases, complete 05 recovery from hepatitis B infection can be expected.
However, i.n ma.ny African and Asian countries, hepatitis :3 virus infection is endemic. A large number of individuals in the populations of these countries a.re chronic carriers of hepatitis B virus and thereby have the dangerous potential of further transmitting the disease.
Vac cination is the only known protection against hepatitis B virus. Recently several companies (for example, Dterck, Sharp and Dohme, U.S.A., and the Pasteur Institute, France) have introduced ~~ pla~~ma-derived vaccine against hepatitis B virus. The vaccine contains, as the antigen, hepatitis B viral proteins which are normally located on the envelope of the viral particle. Such antigen 2C is commonl~,~ referred to as the hepatitis B virus surface antigen (HBsAg), and the viral gene encoding such antigen is called the HBsAg gene. The above-referenced vaccines contain surface antigen isolated from t:he plasma of hepatitis B virus infected humans. Although the surface antigen itself is not pathogenic, it stimulates the production of antibodies that are directed against hepatitis B
virus particles i:n human beings.
The' only commercially available source of hepatitis B surface antigen is the blood of hepatitis B virus infected humans. The isolation and purification of tl:~e surface antigen from human serum 134Q9~4 is a cumbersome and time-consuming process and is, therefore, relatively expensive. In addition, as long as the human serum is the only commercially available source for surface antigen, the amount of 05 surface antigen available for use in vaccines, will be limited.
For large-scale vaccination, especially in areas where hepatitis B virus is endemic, it is of great importance to have a cheap and virtually unlimited source for surface antigen of hepatitis B
virus. Because of the potential danger of contamination with pathogenic agents when surface antigen is taken from human serum, it is desirable to avoid the use of human serum as a source for surface antigen. ~"here is also no biological system known besides human beings and chimpanzees in which hepatitis B virus can grow and be propagated.
Recent advances in molecular biology have made it X~ossible to clone the sequence of the complete genome of hepatitis B virus (Siddiqui, A. et al, Proc. Dfatl. ,~lca. Sci. , U.S.A. , Vol. 76, p. 4664, 1979; Sninsky, J.J. et al, Nature, Vol. 279, p. 346, 1979; and c~harnay, P. et al, Nucl. Acids Res., Vol.
7, p. 335, 1979). In addition, the coding regions for the different. viral proteins of hepatitis B virus have been identified (see, e.g., European Patent Application Publication Nos. 13828, 20251 and 38765;
Galibert et al, Nature, Vol. 281, pp. 646-650, 1979;
Pasel~ et al, Nature, Vol. 282, pp. 575-579, 1979; and Valenzuela et al, Nature, Vol. 280, pp. 815-819, 1979).
1340934 '~
Following these advances, several host systems have been tested for the expression of that portion of the viral genome encoding the surface antigen proteins of hepatitis B virus.
05 It has been demonstrated in many cases that bacteria ca.n provide a cheap production system for certain eukaryotic products. However, many difficulties are observed when eukaryotic products are synthesized in bacteria. For example:
1) the eukaryotic structural gene may not be efficiently transcribed in bacteria because codon usage in bactex-ia differs from codon usage in eukaryotes;
2) the eukaryotic gene product can be toxic to the host bacteria cell;
3) the structure and function of the eukaryotic gene product may be dependent on certain post-translational processes, such as glycosylation or special linkage of disulfide bonds, neither of which can be accomplished by the bacterial host;
4) only rarely will the gene product be secreted from the bacterial host cell;
05 It has been demonstrated in many cases that bacteria ca.n provide a cheap production system for certain eukaryotic products. However, many difficulties are observed when eukaryotic products are synthesized in bacteria. For example:
1) the eukaryotic structural gene may not be efficiently transcribed in bacteria because codon usage in bactex-ia differs from codon usage in eukaryotes;
2) the eukaryotic gene product can be toxic to the host bacteria cell;
3) the structure and function of the eukaryotic gene product may be dependent on certain post-translational processes, such as glycosylation or special linkage of disulfide bonds, neither of which can be accomplished by the bacterial host;
4) only rarely will the gene product be secreted from the bacterial host cell;
5) euk~aryotic promoters usually do not work in bacteria and must be substituted by a bacterial promoter, and such substitution can result in modificat=ion of the eukaryotic gene product, i.e., the N-terminal part of the product is of bacterial origin and includes the N-formyl methionine as the initial amino acid which does not occur in eukaryotes and which might present a new immunogenic determinant for the mammalian immune system; and 1340934:, 6) during the purification process, bacterial cell wall components may co-purify with the gene product and cause serious allergic reactions or lead to anaphylactic shock in mammalian recipients of 05 the gene product.
The=_re have been problems with hepatitis B
surface antigen made in bacteria. First of all, the production rate in bacteria is very low and, in addition to this,. the purification has to start from a bacterial lysate because the product is not secreted from the bacterial cell. Another problem arises from the fact that certain post-translational processes coo not exist in bacteria, for example glycosylation of the surface antigen which has an influence o:n the level and specificity of the immune response. For any of these reasons it is desirable to avoid bacteria as production hosts.
Yeast cells transformed with an appropriate recombinant vector containing the hepatitis B surface 2G antigen coding sequence synthesize and accumulate said surface antigen in considerable amounts in yeast culture. Lut there are some serious drawbacks in this host system,. too, i.e., 1) the surface antigen is not secreted from the yeast cells and has to be isolated from a cell lysate of such cells, and 2) the surface antigen monomers made in yeast cells form no covalently-:joined dimers, unlike the surface antigen secreted from mammalian cells.
There 7zave been several attempts to establish mammalian cell lines that produce surface antigen. These cell lines have been derived from human hepatocellu:Lar carcinomas (Alexander, J.J. et 1 3 40 93 4' al, Afr. D~E~d. J. , Vol. 50, p. 2124, 1976) as well as from cells transfected with cloned hepatitis B virus DNA. In ,sddition, monkey kidney cells have been infected with simian virus 40 (SV40)-based 05 recombinant DNA vectors carrying 40% of the hepatitis B virus genome (Laub, O. et al, J. of Virol., Vol.
48, p. 271, 198~~). Also, co-transfection approaches have been used to select cell lines expressing the surface antigen I;Standring, D.N. et al, J. of Virol., Vol. 50, p. 563, 1984). Amplification of the dihydrofolote reductase (DHFR) gene has been used to establish mamma7~~ian cell lines producing greater quantities of antigen (Michel et al, Proc. Natl. Aca.
Sci., Vol. 81, p. 7708, 1984). Also, eukaryotic viral vectors that utilize the transformed phenotype as the selectable marker (Hsiung, N, et al, Molec.
and Applied Genetics, Vol. 2, p. 497, 1984) have been chosen for transferring the surface antigen gene into mammalian cells. In these cases, DNA constructions containing oncogE~nic DNA sequences, or DNA sequences from oncogenic viruses, were used in the selection of cell lines produ~~ing surface antigen. The use of an oncogene sequence as a selection marker poses new safety problems when surface antigen made by these cells is used for vaccination.
A number of references discuss the expression of polypeptides synthesized by the hepatitis B virus. coding sequence.
European Patent Application Publication PJo.
013,828-A1 (EPA 013,828), published August 8, 1980, states that nucleotide sequences encoding a 163 amino _7_ acid stretch (the PreS coding region) immediately preceding the S protein coding region in the HBV
genome, ma.y also be included in the fragments employed to produce useful recombinant DNA molecules 05 for production o:E polypeptides displaying hepatitis B
virus surface antigen antigenicity. In addition, it is described that gene fragments including both the precursor sequence and the structural gene for HBsAg may be excised by the use of one or more of a variety of restriction endonucleases prior to or during construction of a~ cloning vector. Furthermore, it is described that the 163 codon precursor preceding the structural gene coding for HBsAg should be excised from a cl«ning vehicle containing the S protein coding sequence prior to using the vector for transformation.
European Patent Application Publication No.
072,318-82 (EPA 072,318), published February 16, 1983, describes a method of making HBsAg by growing yeast cell; transformed with a vector including a yeast replicon, a yeast promoter and a DNA segment coding for the 6. protein and specifically excluding the 163 codon precursor preceding the structural gene coding for HBsAg. A vaccine including an HBsAg antigen and a method of obtaining an HBsAg antibody by purifying the antibody from the serum of animals immunized against HBsAg is also described. A 14 to 18 nm antigen particle capable of forming an immune complex with HBsAg antibody is also described.
In Feitelson et al, Virology, Vol. 130, pp.
75-90, 1983,, it was observed that a number of surface antigen-associated polypeptides may be partially 1 :~ 4~J 93 4 _8_ encoded within the PreS coding sequence, including 24,000, 28,000, 32,000, 43,000, and 50,000 dalton species.
Laub et al, J. Virol., Vol. 48, No. 1, pp.
05 271-280, 1°.'83, describe the construction of a simian virus 40 early replacement vector that has a structural gene coding for HBsAg including the 163 codon precursor sequence immediately preceding the structural gene, and expression of the coding sequence b~~ SV40~-transformed CV-1 cells (COS cells) transformed by such a vector. Laub et al also report that more HBsAg i.s expressed by COS cells transformed by a vector containing only the S protein coding sequence as compared to those transformed with a vector containing the 163 codon precursor immediately preceding the structural gene coding for HBsAg.
Ta:~eda Chemical Ind., Japanese Patent Application No. J5-8194-897-A, published November 12, 1983, (Takeda I)" describes an HBV DNA (adw subtype) coding for the entire PreS-S protein polypeptide and a vector containing such DNA and a host transformed with the DL~A. Takeda Chemical Ind., Japanese Patent Application No. J5-9080-615-A, published Play 10, 1984, (Takeda II ) , describes an HBV DNA (adw subtype ) coding for the entire PreS-S protein polypeptide, as well as the polypeptide produced by such DNA and its use in a vaccine. Takeda Chemical Ind., Japanese Patent Application No. J5-9074-985-A, published April 27, 1984 ;Takeda III), describes a DrdA fragment containing one or more of the entire adw type PreS-S
protein coding sequence, the S protein coding sequence or the Hepatitis B Virus Core Antigen 1340934 ~~
(HBcAg) coding sequence, a vector containing such DNA, and hosts transformed with such DNA. Takeda III
states that. the 43,000 dalton polypeptide coded for by the PreS-S protein coding sequence "may be used as 05 vaccines for prevention of HBV infection." Cloning of the Pre:-S protein coding sequence in E. coli and identification of the polypeptide produced by such sequence is described in Takeda III.
Gerlich, in a presentation at the European Association of Clinical Microbiology in Bologna, October 18, 1983,, reported that four potential genes of HBV have been deduced from the DNA sequence of cloned HBV-DNA; that the gene for the surface proteins (S-gene) of HBV consists of one uninterrupted coding sequence which is translated into at least three polypeptides; that the sequence of tile major protein, P24, and its glycosylated form, GP27, begins with the third conserved translational start signal of the S-gene; that the minor surface proteins, CfP3:s or GP36, begin at the second start signal, that only the P41 polypeptide of HBV probably uses the full length coding sequence of the S-gene;
that P41 carries a major antigenic determinant of IiBV; and that it is present only in the complete viral particles but not in the 20 nm particles of surplus surface antigen from which the current hepatitis E. vaccines derive. There is no report in Gerlich specifically regarding the PreSl region of P41 or that the PreSl region specifically contains an antigenic determinant which would be useful in an HBV vaccine.
Stibbe eat al, Develop. Biol. Standard, Vol.
54, pp. 33-43, 1983, reported at the second WHO/IABS
1344934 '~
- to -Symposium i.n Athens on Viral Hepatitis: Standardiza-tion in Immunoprophylaxis of Infections by Hepatitis Viruses, P.thens,, Greece, 1982, that the 20 nm particles c~f HBsAg isolated from serum of humans 05 infected with HBV contain at least three minor proteins, CTP33, GP36 and P41 as ascertained by SDS
gel electrophoresis. Stibbe et al, conclude that GP33 and G:?36 are probably not essential components of HBV vaccines. There is no report in Stibbe et al specifically regarding the PreSl region of P41, or that such region specifically contains an antigenic determinant that would be useful in an HBV vaccine.
Stibbe et al, J. Virol., Vol. 46, No. 2, pp.
626-628, 1983, disclose that minor glycoproteins from the HBsAg codina~ region, named GP33 and GP36, are coded for by the PreS2-S protein coding sequence.
The PreS2 region is a portion of the PreS coding region in the hepatitis B virus genome and codes for the first 55 amino acid immediately preceding the S
protein.
LTeurath et al, Science, 224, pp. 392-394, 1984, disclose that a polypeptide having a sequence identical t~~ the amino-terminal 26 amino acids of the PreS2 region acts as a highly efficient immunogen, and suggest that the antibodies elicited by the immunogen can be utilized for diagnostic tests.
Hec:rman et al, J. Virol., Vol. 52, No. 2, pp. 396-402, 1984, describe the entire 389 amino acid coding sequence comprising both the S protein coding sequence and the PreS coding sequence, which codes for a polypeptide, P39, found in naturally-derived HBV particl<~s and in viral surface antigen filaments, ~340934'~
along with its glycosylated form, GP42, as well as the other FfBV surface antigen-associated polypeptides P24, GP27, GP33 and GP36. Heerman et al also disclose that the unique portion of the P39/P42 05 protein, i.e., its PreSl region, bound monoclonal antibodies which had been induced by immunization with HBV F~artic7Les . They suggest that such PreSl region is, probably, highly immunogenic. The PreSl region is that portion of the PreS coding region, immediately preceding the PreS2 region and comprises the coding sequences for amino acids 1-108 (or 1-122 depending on virus subtype), which is the stretch of amino acids immediately preceding the PreS2 coding sequence. Heerman et al also state that in the search for immunogenic and protective poly- or oligopeptides as an alternate vaccine against HBV, sequences of the PreS region may be of interest.
fdi~~hel et al, Proc. Natl . Acad. Sci . U. S.A. , Vol. 81, p~~. 7708-7712, 1984, describe the synthesis of HBsl~,g carrying human serum polyalbumin receptors in Chinese hamster ovary (CHO) cells transfected with a plasmid carrying the PreS2-S protein coding sequence.
Cavtaneo et al, Nature, Vol. 305, pp.
335-338, 1985, disclose that the S gene of the HBV
genome initiates translation at the beginning of the PreS region (i.e., at 489 nucleotides or 163 codons upstream of the S gene) and the mRNA is processed/polyadenylated at a site within the core gene.
Persing et al, Proc. Natl. Acad. Sci.
U.S.A., Vol. 82, pp. 3440-3444, 1985, disclose that 134~tg34.
mouse L cells transformed with a PreS2-S protein coding sequence produce three HBsAg related polypeptides of molecular weight 24,000, 27,000 and 35,000 daltons, all of which are organized into 05 complex immunorE:active HBsAg particles of 22 nm diameter v~hich bind to polymerized human serum albumin (HSA); while mouse L cells transformed with the PreS2--S protein coding sequence bearing a frame-shift mutation near the 3' end of the PreS2 region produce only the 24,000 and 27,000 dalton polypeptides organized into 22 nm diameter immunoreactive HBsAg particles which are unable to bind HSA. Persing et al conclude that the PreS2-S
protein coding sequence encodes the 35,000 dalton species, that the PreS2 coding portion accounts for the HSA-binding activity of HBsAg, but is not required for assembly and secretion of the HBsAg particles; and that the predominant polypeptide of HBsAg (i.e., th.e 24,000 dalton species) is not derived primarily by cleavage of larger presursors (i.e., the 27,00() and 35,000 dalton species) encoded by the PreS.~-S protein coding sequence.
Mi:Lich et al, Science, Vol. 228, pp.
1195-1199, 1985, disclose that vaccines that contain HBsAg particles with amino acids of both PreS2 and HBsAg, wherein such particles were secreted by Chinese hamster ovary (CHO) cells transfected with a plasmid containing the PreS2-S protein coding sequence, can circumvent nonresponsiveness to vaccines which just contain HBsAg since the immunogenic response to HBsAg is independent of the immunogenic response to PreS2; and that the 26 1340834 .
amino acid residues at the amino-terminus of the 33,000 dali:on polypeptide coded for by the PreS2-S
protein coding sequence represent a dominant antibody binding site on t:he PreS2 region.
05 Neurath et al, Nature, Vol. 315, pp. 154-156, 1985, disclose that the PreS2 region codes for a protein on the H13V envelope with domains specifically recognized by liver cells; the PreS2-S protein coding sequence codes for a protein present in HBV
particles; synth~~thic peptides corresponding to the gene encoding I?reS2 are highly immunogenic; and conclude that HBV vaccines should contain PreS2.
Valenzuela disclosed, at the Dlay 15, 1985 Bio-Expo-5 Meeting in Boston, expression of the entire PreS2 protein coding sequence in yeast; that such yeast cells do synthesize a particle containing both HBsAg and I?reS2 peptide which is very similar in electron microscopy and sedimentation properties to particles containing only HBsAg, and that the nreS2 region does not interfere with the ability to form the 22 nm HBsAg particles. Valenzuela also states that it has been hypothesized that HBV gets into the liver by binding polyalbumin to its polyalbumin receptor which in turn binds the polyalbumin receptor in the liver cell and thus, the virus gets internalized. The polyalbumin receptor of HEV is encoded by the PreS2 region. Valenzuela concludes that a vaccine containing PreS2 will elicit antibodies which, in addition to inactivating HBV through the normal mechanism, might interfere with the way the virus enters liver cells. See, also, Valenzuela et al, Biotechnology, Vol. 3, pp.
317-320, 1985.
1340934 ' Valenzue~la et al, Biotechnology, Vol. 3, pp.
323-327, 1.985, disclose the use of the HBsAg polyalbumin receptor coded for by the PreS2 coding sequence a~: a me~a~s to prepare polyvalent vaccines.
05 Valenzuela prepared a hybrid HBsAg-Herpes simplex 1 virus glycoprote~.n D (HSVIgD) particle by expressing the entire HSVIgD-PreS2S protein coding sequence in yeast.
Ne~arath et al European Patent Application Publication No. 0,154,902-A2, published September 18, 1985, describes a hepatitis B vaccine containing a peptide with an amino acid chain of at least six consecutive amino acids within the PreS gene coding region of the envelope of hepatitis B virus. The vaccine is free of an amino acid sequence corre-sponding to the naturally occuring envelope proteins of hepatitis E! virus and the physiologically acceptable diluant. The peptide is free or can be linked to a carrier.
Kent et al, Pept. Chem., Vol. 22, pp. 167-70, 1984, disclose that a chemically synthesized peptide comprising the N-terminal 26 amino acids of the PreS2 region is a very good antigen and is a good candidate for a synthetic vaccine.
Lo et al, Biochem. Biophys. Res. Comm., Vol.
129, No. 3, pp. 797-803, 1985, disclose the characterization of the restriction endonuclease map of full length HBV DNA for different subtypes (adw, adr, a~w an~i adyw) of HBV, including characterization of the re=~trict:ion endonuclease map of the entire PreS region. Lo et al also disclose cloning and expression of the full length HBV DNA in E. coli using a pUc~8 expression vector, and state that they 05 intend to use such HBV gene product, expressed in either prokaryotic or eukaryotic cells, as diagnostic agents and as vaccination sources.
along et al, J. Virology, Vol. 55, No. 1, pp.
223-231, 1985, disclose the identification of two HBV
polypeptides encoded by the entire PreS open reading frame, i.e., 42 and 46 kd glycosylated polypeptides, containing determinants of both HBsAg and the PreS
region. Wc>ng et. al also disclose expression of a tribrid fusion protein containing 108 amino acids correspondi:zg to the N-terminal 27 through 133 amino acids of the li'4 amino acids of the PreS region (adw2 subtype), as well as ~ -galactosidase and chloramphen.icol acetyltransferase sequences. This peptide contains 15 amino acids from the PreS2 reuion and 93 from the PreSl region. Wang et al also disclose that (a) polyclonal antiserum generated to the tribrid fusion protein was capable of detecting 42 and 46 kd polypeptides in partially purified virus particle preparations, and (b) the glycosylate<3 42 and 46 kd polypeptides appear to correspond to the 39 and 42 kd non-glycosylated polypeptide:~ described in Heerman et al, cited above.
Of:Eensperger et al, Proc. Natl. Acad. Sci.
USA, Vol. 82, pp. 7540-7544, 1985, disclose expression of a cloned DNA sequence encoding the PreS2 peptide-~~-galactosidase fusion protein by E-coli.
134~93~ ~
A number of references discuss plasmids containing they promoter of a mouse metallothionein Pavlakis et al,, Proc. Natl. Acad. Sci USA, Vol. 80, p.
397, 1983, disc: loses recombinant bovine papilloma virus plasrnids containing the gene for human growth hormone (hGH) and a rnet:allothionein gene (MMT) promoter.
Hofschneider et al, European Patent Appln.
No. EP 0,105,141A2, published April 11, 1984, disclose recombinant plasmids containing the S
protein coding sequence and either bovine papilloma virus type 1 DI:JA or Maloney mouse sarcoma virus DNA.
The plasmids disclosed by Hofschneider et al preferably employ the natural promoter associated with the S protein coding sequence, but the Hofschneic3er reference does state (but does not exemplify) that "[a] further example for a preferred natural eukaryotic expression signal is the metalloth:ionein signal from mouse cells."
hogel et al, European Patent Appln. Publica-tion No. ~~P 0, 096, 491A2, disclose the use of plasmids containincr an entire metallothionein gene sequence, including the promoter region, as a means for obtaining strong expression of genes downstream of the MMT gene in mammalian hosts transformed with such plasmid.
Lniveraity Patent, Inc., PCT Patent Appln.
Publication No. WO 83/01783, published May 26, 1983, and Palmiter et al, Cell, Vol. 29, p. 701, 1982, disclose a plasmid comprising the mouse MT-1 gene 1 340 g3 4 '~
promoter fused to a structural gene (preferably the gene for thymidine kinase from herpes simplex virus), and microi.nject:ion of mouse embryos with such plasmids, .and state that gene expression of the 05 resulting fused polypeptide product in differentiated cells of adult :mice resulting from the embryos is subsequentl:~ regulable by administration of heavy metal ions. Palmiter et al also disclose plasmids containing the mouse MT-1 promoter fused to rat growth hormone, microinjection of such plasmids into mouse embryo, and regulation of gene expression of the resulting fused polypeptide product by heavy metal admin:~stration.
SUriMARY OF THE INVENTION
The present invention relates to a particle prepared by recombinant DNA techniques which comprises a.t least one protein coded for by the entire PreS~_-PreS,2-S protein coding region.
ThE~ present invention also relates to a 2t, recombinant DLTA vector comprising the PreSl PreS2-S protein coding region and a mouse metallothior~ein (MP4T) promoter. Such vector preferably additionally comprises a transcription termination site <3nd a selection marker.
The pre:~ent invention also relates to a transfected host eukaryotic cell transfected with a recombinant DNA vector comprising the PreSl-PreS2-S protein coding region and an MMT-promoter.
Such vector preferably additionally comprises a a transcription termination site and a selection marker. Such host is preferably a mammalian cell.
Additionally, the invention relates to a method of preparing such t:ransfected host cell which comprises transfectin~~ a eukaryotic cell with the recombinant DNA vector ~~f the present invention.
Th~~ present invention also relates to a 05 method for preparing particles comprising at least one protein coded for by the entire PreSl-PreS2-S
protein coding :region which comprises: a) culti vating the transfected host eukaryotic cell of this invention under culture medium conditions which enable such host: to express such protein; and b) isolating such particles. Preferably, such transfected host is able to secrete such proteins assembled into such particles into the medium.
Preferably, such method additionally comprises the addition of heav5r metal ions or steroid hormones to such culture medium to enhance the expression of the proteins. 'rhe present invention also relates to the particles prepared by such method.
The' present invention also relates to a vaccine comprising an immunoprotective amount of the particles oi: this invention.
The: present invention also relates to a method for detecting the presence of antibodies to proteins coded for by the PreSl-PreS2-S protein coding region in a sample of mammalian blood sera which comprises:
a) contacting the sample with a solid substrate coated with non-labeled particles of this invention;
b) incubating and washing said contacted sample;
c) contacting said contacted sample with labeled particlea> of this invention thereby producing a labeled c~~ntacted sample;
d) incubating and washing said labeled 05 contacted sample; and e) determining the extent of labeled particle in the labeled contacted sample.
The present invention also relates to a method for detecting the presence of proteins coded for by the PreSl-PreS2-S protein coding region in a sample of mammalian blood sera which comprises:
a) producing a composition containing an antibody produced by an immunogen comprised by the particles o~_ this invention;
b) contacting the sample with a first portion of the composition and the, immunogen which has been libeled, incubating and washing the first portion;
c) contacting an antigen-free control with a second portion of the composition and the immunogen which has been labeled, incubating and washing the second portion;
d ) add:ing the same amount of staphylococci bearing protein A to the composition of steps b) and c) above, incubating both compositions and separating liquid from solids; and e) determining the extent of labeled immunogen in each of the resultant compositions from step d) above.
'I'he present invention also relates to a diagnostic kit for detecting the presence of antibodies to prot:eins coded for by the PreSl-PreS2-S protein coding region in a sample of mammalian b:Lood sera which comprises:
a) unlabeled proteins comprising the particle of this invention attached to a solid 05 support; and b) labeled antibodies to human IgG or IgM, for example,.
The present invention also relates to a diagnostic l;it for detecting the presence of proteins coded for by the PreSl-PreS2-S protein coding region in a sample of mammalian blood sera which comprises:
a) antibodies, produced by the particles of this invention, attached to a solid support; and b) labeled antibodies, produced by the particles of this invention.
The preaent invention also relates to a co-transfect.ed eukaryotic host cell co-transfected with a recombinant DNA vector of this invention and a second recombinant DNA vector comprising the PreS2-S protein coding region, or only the S
protein coding region, and an L,4MT-promoter.
Preferably, the second recombinant vector additionally comprises a transcription termination site and, opi_ionally, a selection marker.
Optionally, the co-transfected host is co-transfected with the recombinant DNA vector of this invention, the second recombinant DNA vector and a third recombinant DNA vector comprising an rZP~T-promoter and a selectic>n marker. Preferably, the third recombinant vector additionally comprises a transcription termination site. Additionally, this 1 3 40 9'~ 4 invention :elates to a method of preparing such co-transfeci:ed host cell which comprises co-transfec~_ing a eukaryotic cell with the recombinant DNA vector of this invention, the second 05 recombinant DNA vector described above, and, optionally, the third recombinant DNA vector described above.
Thc~ present invention also relates to a method for preparing particles comprising at least one protein coded for by the entire PreSl-PreS2-S
protein coding region which comprises: a) cultivating the co-transfected host eukaryotic cell of this ir,.vention under culture medium conditions which enable such host to express such protein; and b) isolating such particles. Preferably, such co-transfec~:ed host is able to secrete such proteins assembled into such particles in the medium.
Preferably, such method additionally comprises the addition of heavy metal ions or steroid hormones to such culture medium to enhance the expression of such proteins. This invention also relates to the particles prepared by such method.
This invention relates to a recombinant DNA
concatamer comprising: a) a vector comprising the PreSl-PreS2-S protein coding region and at least one vector comprising one of the following coding regions: PreSl-PreS2-S protein coding region or PreS2-S protein coding region or S protein coding region; and b) the MMT promoter. Such concatamer preferably additionally comprises a selection marker and a transcription termination site. Such concatamers can be prepared by techniques known in the art.
This invention also relates to a host ~ukaryotic cell transfected with the concatamer of 1340934 .
this invention. Such host is preferably a mammalian cell. Additionally, the invention relates to a method of preparing such transfected host cell with the concatamer of this invention which comprises 05 transfectin<3 a eukaryotic cell with the concatamer of this invention.
This invention also relates to a method for preparing a particle comprising at least one protein coded for by the entire PreSl-PreS2-S protein coding region which comprises: a) cultivating the transfected host of this invention, which comprises the concatamer of this invention, under culture medium conditions which enable such host to express such protein; .and b) isolating such particle.
Preferably, such transfected host is able to secrete such proteins as~cembled into such particles into the medium. :Preferably, such method additionally comprises the addition of heavy metal ions or steroid hormones to such culture medium to enhance the 2C expression of t-he proteins. This invention also relates to t:he particles prepared by such method.
BRIEF DESCRIPTION OF THE DP,AWINGS
Figure 1 includes a partial restriction endonucleasEa map of plasmid pBPV342-12. Figure 1 also illustrates the location of plasmid pML2-derived DNA, bovine papilloma virus (BPV) DNA, the MMT-promoter and the sv-PAS-t transcription termination region on plasmid pBPV342-7.2. Figure 1 also illustrates the splitting of plasmid pBPV342-12 with the restriction endonuclease BamHI.
Figure 2 includes a partial restriction endonuclease map of plasmid pA01 which contains the complete hepatitis B virus genome in linear form.
Figure 2 a:Lso illustrates the splitting of pA01 with the restri~=tion endonuclease EcoRI, ligation of the EcoRI linear HBV product with T4 DNA-ligase to form 05 concatamers and splitting of the concatamer product obtained w:th the restriction endonuclease BglII to generate a specific DNA fragment containing the intact DNA coding sequence for the PreSl-PreS2-S
protein coding region.
Figure 3 includes a partial restriction endonuclease map of plasmid pDMl and illustrates its construction by ligation of a BamHI DNA fragment comprising plasmid pMMT-neo with a BglII genomic fragment «f hepatitis E virus encoding the PreSl-PreS2~-S protein coding sequence.
Fi<3ures 4A and 4B include a partial restriction endonuclease map of plasmid pDM2 and illustrates its construction from ligation of a BamHI
restriction endonuclease fragment and a BamliI-BglII
restriction endonuclease fragment, both from plasmid pDMl.
Figure 5 includes a partial restriction endonuclease map of plasmid pDM3 and illustrates its construction from pDMl and pMMT-neo.
Figure 6 is a graphical representation illus-trating the elution pattern of the particles of this invention from a BioRad (A5m) column.
Figure 7 is a graphical representation of a typical isopycn~lc CsCl centrifugation of the particles o:E this; invention from about 30 fractions from the first peak resulting from the BioRad (A5m) column.
1 340934 '~
Figures 8A, 8B and 8C are graphs that illustrate seropositive conversion induced in mice by different dilutions of purified particles of the present invention.
05 Figures 9A and 9B include a partial restriction endonuclease map of plasmid pENDO-1 and illustrates its multi-step construction.
Fi~3ure 10 includes a partial restriction endonucleas~e map of plasmid pENDO-2 and illustrates its construction from ligation of restriction endonuclease DNA fragments of plasmid pENDO-1 and plasmid pDPM:Z .
Fi~~ure 11 includes a partial restriction endonucleas~~ map of plasmid pENDO-0 and illustrates its constru~~tion.
DET~~ILED DESCRIPTION OF THE INVENTION
By the term "PreSl-PreS2-S protein coding region" is meant the region of the HBV DNA
genome that encodes the entire polypeptide known as the PreSl-P:reS2-S polypeptide, including the codon for the initial methionine through to the final termination codon (TAA) that specifies insertion of no amino acid and directs that translation terminate, or any functional derivative of such coding region.
Proteins coded for by such region include the PreSl-PreS2--S polypeptide (389 amino acid residues i:z HBV subtype adw, for example), the PreS2-S polypeptide (281 amino acid residues in HBV
subtype adw, for example), and the S polypeptide (226 amino acid residues in HBV subtype adw, for example).
Preferred PreSl-PreS2-S protein coding regions are derived from the following subtypes: adr, ayw, adyw and adw. By the term "functional derivative" is meant any derivative of the PreSl-PreS2-S protein coding region which codes for polypeptides which, when assembled into a particle, function in 05 substantially the same way as the particle resulting from the a:~sembl:y of the polypeptides coded for the native PrE~Sl-PreS2-S protein coding region in terms of i.mmunoc3enic capability. Such derivatives include pe~rtial sequences of the PreSl-PreS2-S
10 protein cc>ding region, as well as derivatives produced by modification of such coding region.
Techniques for modifying such coding region are known in the art and include, for example, treatment with chemical cr biological mutagens, irradiation or 15 direct genetic engineering, such as by inserting, deleting or substituting nucleic acids by the use of enzymes or other recombinant and molecular biological techniques.
By the term "PreS2-S protein coding 20 sequence" is meant the region of the HBV DNA genome that encodE~s the entire polypeptide known as the PreS2-S polypept:ide including the colon for the initial met:hionine through to the final termination colon (TAA) that specifies insertion of no amino 25 acid, as specified above, or any functional derivative of such coding region. Proteins coded for by such region include the PreS2-S polypeptide (281 amino acid residues in HBV subtype adw) and the S
polypeptide (226 amino acid residues in HBV subtype ad~w, for example) . Preferred PreS2-S protein coding regions are derived from the following HBV
subtypes: _adr, a.yw, a- dyw and adw. By the term 1340834 ' "functional derivative" is meant any derivative of the PreS2-~> prot:ein coding region which codes for polypeptide,s which, when assembled into a particle, function in substantially the same way as the 05 particle resulting from the assembly of polypeptides coded for by the native PreS2-S protein coding region in terms of immunogenic capability. Such derivatives include partial sequences of the PreS2-~ p.rotein coding region, as well as derivatives produced by modification of such coding region. Techniques which may be employed for modifying such coding region are known in the art and some have been outlined above.
By the term "S protein coding sequence" is meant the region of the HBV DNA genome that encodes the entire polypeptide known as the S polypeptide including the codon for the initial methionine through to the final termination codon (TAA) that specifies insertion of no amino acid, as specified above, or a3ny functional derivative of such coding region. Proteins coded for by such region include the S peptide (226 amino acid residues in HBV subtype adw, for examp:le). Preferred S protein coding regions are derived from the following HBV subtypes:
adr, ayw, adyw and adw. By the term "functional derivative" is meant any derivative of the S protein coding region which codes for polypeptides which, when assembled into a particle, function in substantially the same way as the particle resulting from the a:asembly of polypeptides coded for by the native S protein coding region in terms of immunogenic capability. Such derivatives include 1 340 g34 ~
partial sequence~~ of the S protein coding region, as well as derivatives produced by modification of such coding region. ~('echniques which may be employed for modifying sv~ch coding region are known in the art and 05 some have bf~en outlined above.
By the term "promoter" is meant that DiJA
sequence within a DNA molecule upstream of a gene that direct, the appropriate host cell P,NA polymerase complex to attach to the DNA molecule at a specific site on said DN'A molecule and to become properly positioned to begin transcription of the gene at a specific point on the DNA to result in the synthesis of an P.NA copy of one of the DNA strands.
By the term "transcription" is meant that process carried out by the host cell biosynthetic machinery, including RNA polymerase complexes, that, using one of the two complementary DNA strands as a template, polymerizes ribonucleotides in a sequential fashion, 5' to 3' , ( 3' to 5' with respect to the DNA
template strand) to yield an exact copy of one of tree DNA strands, but containing ribonucleotides instead of deoxyribonucleotides.
By the term "transcription termination sequence" is meant that region within the DNA
molecule, ~iesign~ated t, that signals to the RNA
polymerase compl'_ex engaged in the process of transcription to terminate progress of such transcription to yield an RNA molecule that can be processed .properly, including polyadenylation and transport for such RNA molecules that are destined to be used as messenger RNA molecules in the host cell cytoplasm.
134934 '~
By the term "polyadenylation" is meant the process by which the host cell biosynthetic machinery recognizes a specific sequence, usually, 5'-AATAAA-3', designated PAS, for polyadenylation signal, within 05 the RNA molecule and that directs the addition to the 3' end of said molecule some 15-20 nucleotides downstream (in t:he direction of the 3' end) of a non-template determined polyriboadenylate moiety, specifically added by the enzyme poly-A polymerase.
By the term "selection marker" is meant a gene determinant that, when expressed in the cell, confers a specific set of characteristics upon the cell that allows such a cell to be distinguished, or selected ovat, from other cells not carrying or expressing raid gene determinant.
PrE~ferred selection markers include drug resistance markers. By the term "drug-resistance marker" is meant a special class of selection markers that confer upon a cell expressing such a marker, resistance to th.e lethal effects of a drug or an antibiotic that ordinarily blocks growth, or kills, cells not carrying or expressing said drug-resistance marker.
Preferred drug resistance markers include the coding sequences for the neomycine-resistance gene, neo; i=he Eco-gpt gene (Mulligan, R.C. and Berg, P., Science, Vol. 209, p. 1422, 1980), the dihydrofolat:e reduction (DHFR) gene (Ringold, G. et al, J. of N.olec. and Appld. Genet., Vol. 1, p. 165, 1981).
By the term "particles comprising at least one protein coded for by the entire PreSl-PreS2-S
protein coding region" is meant a hepatitis B
particle, devoid of any nucleic acid, formed by the assembly within or from a culture lysate of a euraryotic cell transfected with the entire PreSl, PreS2 and S region wherein such particle contains 05 subunits composed predominantly of the S polypeptide (dimers), but also composed of smaller amounts of the entire PreSl-PreS2-S, optionally, the PreS2-S
polypeptide;a.
This invention relates to a recombinant DNA
vector comprising the PreSl-PreS2-S protein coding region and a MMT-promoter. The MN1T-promoter may be incorporated into the DNA vector either in addition to the natural promoter for the DNA sequence (hepatitis B promoter) or in place of the hepatitis B
promoter. Preferably, the MMT-promoter is located in the DNA vector immediately upstream of the PreSl-PreS2-S protein coding region. Preferably, such vector also comprises a transcription termination sequence and a selection marker. Preferably, such selection marker is a drug-resistance marker, such as the neomyci.ne gene. Preferably, such transcription termination sequence is an SV40 termination site (sv-PAS-t) or more preferably the DEF region (mg-PAS-t) of t:he mouse globin gene. The SV40 termination sequence is well-known in the art and is described by various references such as Mulligan and Berg, Science, Vol. 209, p. 1422, 1980. The DEF
region of the mouse globin gene is described by Falck-Pederson et al, Cell, Vol. 40, p. 897, 1985.
Such vect~~r may be prepared by conventional recombinant DNA and other molecular biological techniques. In the most preferable case, using the mg-PAS-t region, the vector does not contain any viral DNA segments and is not oncogenic, i.e., it will not 134A9~4 transform (make cancerous) any host cell into which it is introduced. Such vector, upon transfection into a host, if it does not contain an autonomous replication sequence (replicon) capable of functioning 05 in a host t:ransfected therewith, integrates into the host chromosome and replicates passively with the host genome.
Preferably, the recombinant DNA vector of this invention should comprise the following characteristics:
1} The PreSl-PreS2-S protein coding region.
2) A MMT-promoter located immediately upstream of the PreSl-PreSZ-S protein coding region.
3) The vector should be able to replicate in bacteria, or other procaryotic host into which it is transformed, for growth, amplification and preparation of large quantities of the recombinant vector. Thus, such vector should include a bacterial or other procaryotic replicon, i.e., a DNA segment bearing al:L the functions required for autonomous replication and maintenance of the vector extrachromosomally in a procaryotic host cell, such as a bacterial host cell, transformed therewith.
Such replicons are well known in the art.
4) The vector replicon should be small (i.e., smal_ler than 6-8 kilobase pairs) to enable easy genetic an<9 molecular biological manipulation thereof .
5) The vector should carry a selection marker, preferably a drug-resistance marker such as 134Q9:i4 s ampicillin, for use in bacterial host cells transformed therewith.
6) T'he vector should carry a second selection marker, preferably a drug-resistance marker 05 such as neomycine, for use as such in eukaryotic host cells trans;_ected therewith.
The=_re have been problems with hepatitis B
surface antigen made in bacteria. First of all, the production rate in bacteria is very low and, in addition to this,. the purification has to start from a bacterial lysate because the product is not secreted from the bacterial cell. Another problem arises from the fact that certain post-translational processes coo not exist in bacteria, for example glycosylation of the surface antigen which has an influence o:n the level and specificity of the immune response. For any of these reasons it is desirable to avoid bacteria as production hosts.
Yeast cells transformed with an appropriate recombinant vector containing the hepatitis B surface 2G antigen coding sequence synthesize and accumulate said surface antigen in considerable amounts in yeast culture. Lut there are some serious drawbacks in this host system,. too, i.e., 1) the surface antigen is not secreted from the yeast cells and has to be isolated from a cell lysate of such cells, and 2) the surface antigen monomers made in yeast cells form no covalently-:joined dimers, unlike the surface antigen secreted from mammalian cells.
There 7zave been several attempts to establish mammalian cell lines that produce surface antigen. These cell lines have been derived from human hepatocellu:Lar carcinomas (Alexander, J.J. et 1 3 40 93 4' al, Afr. D~E~d. J. , Vol. 50, p. 2124, 1976) as well as from cells transfected with cloned hepatitis B virus DNA. In ,sddition, monkey kidney cells have been infected with simian virus 40 (SV40)-based 05 recombinant DNA vectors carrying 40% of the hepatitis B virus genome (Laub, O. et al, J. of Virol., Vol.
48, p. 271, 198~~). Also, co-transfection approaches have been used to select cell lines expressing the surface antigen I;Standring, D.N. et al, J. of Virol., Vol. 50, p. 563, 1984). Amplification of the dihydrofolote reductase (DHFR) gene has been used to establish mamma7~~ian cell lines producing greater quantities of antigen (Michel et al, Proc. Natl. Aca.
Sci., Vol. 81, p. 7708, 1984). Also, eukaryotic viral vectors that utilize the transformed phenotype as the selectable marker (Hsiung, N, et al, Molec.
and Applied Genetics, Vol. 2, p. 497, 1984) have been chosen for transferring the surface antigen gene into mammalian cells. In these cases, DNA constructions containing oncogE~nic DNA sequences, or DNA sequences from oncogenic viruses, were used in the selection of cell lines produ~~ing surface antigen. The use of an oncogene sequence as a selection marker poses new safety problems when surface antigen made by these cells is used for vaccination.
A number of references discuss the expression of polypeptides synthesized by the hepatitis B virus. coding sequence.
European Patent Application Publication PJo.
013,828-A1 (EPA 013,828), published August 8, 1980, states that nucleotide sequences encoding a 163 amino _7_ acid stretch (the PreS coding region) immediately preceding the S protein coding region in the HBV
genome, ma.y also be included in the fragments employed to produce useful recombinant DNA molecules 05 for production o:E polypeptides displaying hepatitis B
virus surface antigen antigenicity. In addition, it is described that gene fragments including both the precursor sequence and the structural gene for HBsAg may be excised by the use of one or more of a variety of restriction endonucleases prior to or during construction of a~ cloning vector. Furthermore, it is described that the 163 codon precursor preceding the structural gene coding for HBsAg should be excised from a cl«ning vehicle containing the S protein coding sequence prior to using the vector for transformation.
European Patent Application Publication No.
072,318-82 (EPA 072,318), published February 16, 1983, describes a method of making HBsAg by growing yeast cell; transformed with a vector including a yeast replicon, a yeast promoter and a DNA segment coding for the 6. protein and specifically excluding the 163 codon precursor preceding the structural gene coding for HBsAg. A vaccine including an HBsAg antigen and a method of obtaining an HBsAg antibody by purifying the antibody from the serum of animals immunized against HBsAg is also described. A 14 to 18 nm antigen particle capable of forming an immune complex with HBsAg antibody is also described.
In Feitelson et al, Virology, Vol. 130, pp.
75-90, 1983,, it was observed that a number of surface antigen-associated polypeptides may be partially 1 :~ 4~J 93 4 _8_ encoded within the PreS coding sequence, including 24,000, 28,000, 32,000, 43,000, and 50,000 dalton species.
Laub et al, J. Virol., Vol. 48, No. 1, pp.
05 271-280, 1°.'83, describe the construction of a simian virus 40 early replacement vector that has a structural gene coding for HBsAg including the 163 codon precursor sequence immediately preceding the structural gene, and expression of the coding sequence b~~ SV40~-transformed CV-1 cells (COS cells) transformed by such a vector. Laub et al also report that more HBsAg i.s expressed by COS cells transformed by a vector containing only the S protein coding sequence as compared to those transformed with a vector containing the 163 codon precursor immediately preceding the structural gene coding for HBsAg.
Ta:~eda Chemical Ind., Japanese Patent Application No. J5-8194-897-A, published November 12, 1983, (Takeda I)" describes an HBV DNA (adw subtype) coding for the entire PreS-S protein polypeptide and a vector containing such DNA and a host transformed with the DL~A. Takeda Chemical Ind., Japanese Patent Application No. J5-9080-615-A, published Play 10, 1984, (Takeda II ) , describes an HBV DNA (adw subtype ) coding for the entire PreS-S protein polypeptide, as well as the polypeptide produced by such DNA and its use in a vaccine. Takeda Chemical Ind., Japanese Patent Application No. J5-9074-985-A, published April 27, 1984 ;Takeda III), describes a DrdA fragment containing one or more of the entire adw type PreS-S
protein coding sequence, the S protein coding sequence or the Hepatitis B Virus Core Antigen 1340934 ~~
(HBcAg) coding sequence, a vector containing such DNA, and hosts transformed with such DNA. Takeda III
states that. the 43,000 dalton polypeptide coded for by the PreS-S protein coding sequence "may be used as 05 vaccines for prevention of HBV infection." Cloning of the Pre:-S protein coding sequence in E. coli and identification of the polypeptide produced by such sequence is described in Takeda III.
Gerlich, in a presentation at the European Association of Clinical Microbiology in Bologna, October 18, 1983,, reported that four potential genes of HBV have been deduced from the DNA sequence of cloned HBV-DNA; that the gene for the surface proteins (S-gene) of HBV consists of one uninterrupted coding sequence which is translated into at least three polypeptides; that the sequence of tile major protein, P24, and its glycosylated form, GP27, begins with the third conserved translational start signal of the S-gene; that the minor surface proteins, CfP3:s or GP36, begin at the second start signal, that only the P41 polypeptide of HBV probably uses the full length coding sequence of the S-gene;
that P41 carries a major antigenic determinant of IiBV; and that it is present only in the complete viral particles but not in the 20 nm particles of surplus surface antigen from which the current hepatitis E. vaccines derive. There is no report in Gerlich specifically regarding the PreSl region of P41 or that the PreSl region specifically contains an antigenic determinant which would be useful in an HBV vaccine.
Stibbe eat al, Develop. Biol. Standard, Vol.
54, pp. 33-43, 1983, reported at the second WHO/IABS
1344934 '~
- to -Symposium i.n Athens on Viral Hepatitis: Standardiza-tion in Immunoprophylaxis of Infections by Hepatitis Viruses, P.thens,, Greece, 1982, that the 20 nm particles c~f HBsAg isolated from serum of humans 05 infected with HBV contain at least three minor proteins, CTP33, GP36 and P41 as ascertained by SDS
gel electrophoresis. Stibbe et al, conclude that GP33 and G:?36 are probably not essential components of HBV vaccines. There is no report in Stibbe et al specifically regarding the PreSl region of P41, or that such region specifically contains an antigenic determinant that would be useful in an HBV vaccine.
Stibbe et al, J. Virol., Vol. 46, No. 2, pp.
626-628, 1983, disclose that minor glycoproteins from the HBsAg codina~ region, named GP33 and GP36, are coded for by the PreS2-S protein coding sequence.
The PreS2 region is a portion of the PreS coding region in the hepatitis B virus genome and codes for the first 55 amino acid immediately preceding the S
protein.
LTeurath et al, Science, 224, pp. 392-394, 1984, disclose that a polypeptide having a sequence identical t~~ the amino-terminal 26 amino acids of the PreS2 region acts as a highly efficient immunogen, and suggest that the antibodies elicited by the immunogen can be utilized for diagnostic tests.
Hec:rman et al, J. Virol., Vol. 52, No. 2, pp. 396-402, 1984, describe the entire 389 amino acid coding sequence comprising both the S protein coding sequence and the PreS coding sequence, which codes for a polypeptide, P39, found in naturally-derived HBV particl<~s and in viral surface antigen filaments, ~340934'~
along with its glycosylated form, GP42, as well as the other FfBV surface antigen-associated polypeptides P24, GP27, GP33 and GP36. Heerman et al also disclose that the unique portion of the P39/P42 05 protein, i.e., its PreSl region, bound monoclonal antibodies which had been induced by immunization with HBV F~artic7Les . They suggest that such PreSl region is, probably, highly immunogenic. The PreSl region is that portion of the PreS coding region, immediately preceding the PreS2 region and comprises the coding sequences for amino acids 1-108 (or 1-122 depending on virus subtype), which is the stretch of amino acids immediately preceding the PreS2 coding sequence. Heerman et al also state that in the search for immunogenic and protective poly- or oligopeptides as an alternate vaccine against HBV, sequences of the PreS region may be of interest.
fdi~~hel et al, Proc. Natl . Acad. Sci . U. S.A. , Vol. 81, p~~. 7708-7712, 1984, describe the synthesis of HBsl~,g carrying human serum polyalbumin receptors in Chinese hamster ovary (CHO) cells transfected with a plasmid carrying the PreS2-S protein coding sequence.
Cavtaneo et al, Nature, Vol. 305, pp.
335-338, 1985, disclose that the S gene of the HBV
genome initiates translation at the beginning of the PreS region (i.e., at 489 nucleotides or 163 codons upstream of the S gene) and the mRNA is processed/polyadenylated at a site within the core gene.
Persing et al, Proc. Natl. Acad. Sci.
U.S.A., Vol. 82, pp. 3440-3444, 1985, disclose that 134~tg34.
mouse L cells transformed with a PreS2-S protein coding sequence produce three HBsAg related polypeptides of molecular weight 24,000, 27,000 and 35,000 daltons, all of which are organized into 05 complex immunorE:active HBsAg particles of 22 nm diameter v~hich bind to polymerized human serum albumin (HSA); while mouse L cells transformed with the PreS2--S protein coding sequence bearing a frame-shift mutation near the 3' end of the PreS2 region produce only the 24,000 and 27,000 dalton polypeptides organized into 22 nm diameter immunoreactive HBsAg particles which are unable to bind HSA. Persing et al conclude that the PreS2-S
protein coding sequence encodes the 35,000 dalton species, that the PreS2 coding portion accounts for the HSA-binding activity of HBsAg, but is not required for assembly and secretion of the HBsAg particles; and that the predominant polypeptide of HBsAg (i.e., th.e 24,000 dalton species) is not derived primarily by cleavage of larger presursors (i.e., the 27,00() and 35,000 dalton species) encoded by the PreS.~-S protein coding sequence.
Mi:Lich et al, Science, Vol. 228, pp.
1195-1199, 1985, disclose that vaccines that contain HBsAg particles with amino acids of both PreS2 and HBsAg, wherein such particles were secreted by Chinese hamster ovary (CHO) cells transfected with a plasmid containing the PreS2-S protein coding sequence, can circumvent nonresponsiveness to vaccines which just contain HBsAg since the immunogenic response to HBsAg is independent of the immunogenic response to PreS2; and that the 26 1340834 .
amino acid residues at the amino-terminus of the 33,000 dali:on polypeptide coded for by the PreS2-S
protein coding sequence represent a dominant antibody binding site on t:he PreS2 region.
05 Neurath et al, Nature, Vol. 315, pp. 154-156, 1985, disclose that the PreS2 region codes for a protein on the H13V envelope with domains specifically recognized by liver cells; the PreS2-S protein coding sequence codes for a protein present in HBV
particles; synth~~thic peptides corresponding to the gene encoding I?reS2 are highly immunogenic; and conclude that HBV vaccines should contain PreS2.
Valenzuela disclosed, at the Dlay 15, 1985 Bio-Expo-5 Meeting in Boston, expression of the entire PreS2 protein coding sequence in yeast; that such yeast cells do synthesize a particle containing both HBsAg and I?reS2 peptide which is very similar in electron microscopy and sedimentation properties to particles containing only HBsAg, and that the nreS2 region does not interfere with the ability to form the 22 nm HBsAg particles. Valenzuela also states that it has been hypothesized that HBV gets into the liver by binding polyalbumin to its polyalbumin receptor which in turn binds the polyalbumin receptor in the liver cell and thus, the virus gets internalized. The polyalbumin receptor of HEV is encoded by the PreS2 region. Valenzuela concludes that a vaccine containing PreS2 will elicit antibodies which, in addition to inactivating HBV through the normal mechanism, might interfere with the way the virus enters liver cells. See, also, Valenzuela et al, Biotechnology, Vol. 3, pp.
317-320, 1985.
1340934 ' Valenzue~la et al, Biotechnology, Vol. 3, pp.
323-327, 1.985, disclose the use of the HBsAg polyalbumin receptor coded for by the PreS2 coding sequence a~: a me~a~s to prepare polyvalent vaccines.
05 Valenzuela prepared a hybrid HBsAg-Herpes simplex 1 virus glycoprote~.n D (HSVIgD) particle by expressing the entire HSVIgD-PreS2S protein coding sequence in yeast.
Ne~arath et al European Patent Application Publication No. 0,154,902-A2, published September 18, 1985, describes a hepatitis B vaccine containing a peptide with an amino acid chain of at least six consecutive amino acids within the PreS gene coding region of the envelope of hepatitis B virus. The vaccine is free of an amino acid sequence corre-sponding to the naturally occuring envelope proteins of hepatitis E! virus and the physiologically acceptable diluant. The peptide is free or can be linked to a carrier.
Kent et al, Pept. Chem., Vol. 22, pp. 167-70, 1984, disclose that a chemically synthesized peptide comprising the N-terminal 26 amino acids of the PreS2 region is a very good antigen and is a good candidate for a synthetic vaccine.
Lo et al, Biochem. Biophys. Res. Comm., Vol.
129, No. 3, pp. 797-803, 1985, disclose the characterization of the restriction endonuclease map of full length HBV DNA for different subtypes (adw, adr, a~w an~i adyw) of HBV, including characterization of the re=~trict:ion endonuclease map of the entire PreS region. Lo et al also disclose cloning and expression of the full length HBV DNA in E. coli using a pUc~8 expression vector, and state that they 05 intend to use such HBV gene product, expressed in either prokaryotic or eukaryotic cells, as diagnostic agents and as vaccination sources.
along et al, J. Virology, Vol. 55, No. 1, pp.
223-231, 1985, disclose the identification of two HBV
polypeptides encoded by the entire PreS open reading frame, i.e., 42 and 46 kd glycosylated polypeptides, containing determinants of both HBsAg and the PreS
region. Wc>ng et. al also disclose expression of a tribrid fusion protein containing 108 amino acids correspondi:zg to the N-terminal 27 through 133 amino acids of the li'4 amino acids of the PreS region (adw2 subtype), as well as ~ -galactosidase and chloramphen.icol acetyltransferase sequences. This peptide contains 15 amino acids from the PreS2 reuion and 93 from the PreSl region. Wang et al also disclose that (a) polyclonal antiserum generated to the tribrid fusion protein was capable of detecting 42 and 46 kd polypeptides in partially purified virus particle preparations, and (b) the glycosylate<3 42 and 46 kd polypeptides appear to correspond to the 39 and 42 kd non-glycosylated polypeptide:~ described in Heerman et al, cited above.
Of:Eensperger et al, Proc. Natl. Acad. Sci.
USA, Vol. 82, pp. 7540-7544, 1985, disclose expression of a cloned DNA sequence encoding the PreS2 peptide-~~-galactosidase fusion protein by E-coli.
134~93~ ~
A number of references discuss plasmids containing they promoter of a mouse metallothionein Pavlakis et al,, Proc. Natl. Acad. Sci USA, Vol. 80, p.
397, 1983, disc: loses recombinant bovine papilloma virus plasrnids containing the gene for human growth hormone (hGH) and a rnet:allothionein gene (MMT) promoter.
Hofschneider et al, European Patent Appln.
No. EP 0,105,141A2, published April 11, 1984, disclose recombinant plasmids containing the S
protein coding sequence and either bovine papilloma virus type 1 DI:JA or Maloney mouse sarcoma virus DNA.
The plasmids disclosed by Hofschneider et al preferably employ the natural promoter associated with the S protein coding sequence, but the Hofschneic3er reference does state (but does not exemplify) that "[a] further example for a preferred natural eukaryotic expression signal is the metalloth:ionein signal from mouse cells."
hogel et al, European Patent Appln. Publica-tion No. ~~P 0, 096, 491A2, disclose the use of plasmids containincr an entire metallothionein gene sequence, including the promoter region, as a means for obtaining strong expression of genes downstream of the MMT gene in mammalian hosts transformed with such plasmid.
Lniveraity Patent, Inc., PCT Patent Appln.
Publication No. WO 83/01783, published May 26, 1983, and Palmiter et al, Cell, Vol. 29, p. 701, 1982, disclose a plasmid comprising the mouse MT-1 gene 1 340 g3 4 '~
promoter fused to a structural gene (preferably the gene for thymidine kinase from herpes simplex virus), and microi.nject:ion of mouse embryos with such plasmids, .and state that gene expression of the 05 resulting fused polypeptide product in differentiated cells of adult :mice resulting from the embryos is subsequentl:~ regulable by administration of heavy metal ions. Palmiter et al also disclose plasmids containing the mouse MT-1 promoter fused to rat growth hormone, microinjection of such plasmids into mouse embryo, and regulation of gene expression of the resulting fused polypeptide product by heavy metal admin:~stration.
SUriMARY OF THE INVENTION
The present invention relates to a particle prepared by recombinant DNA techniques which comprises a.t least one protein coded for by the entire PreS~_-PreS,2-S protein coding region.
ThE~ present invention also relates to a 2t, recombinant DLTA vector comprising the PreSl PreS2-S protein coding region and a mouse metallothior~ein (MP4T) promoter. Such vector preferably additionally comprises a transcription termination site <3nd a selection marker.
The pre:~ent invention also relates to a transfected host eukaryotic cell transfected with a recombinant DNA vector comprising the PreSl-PreS2-S protein coding region and an MMT-promoter.
Such vector preferably additionally comprises a a transcription termination site and a selection marker. Such host is preferably a mammalian cell.
Additionally, the invention relates to a method of preparing such t:ransfected host cell which comprises transfectin~~ a eukaryotic cell with the recombinant DNA vector ~~f the present invention.
Th~~ present invention also relates to a 05 method for preparing particles comprising at least one protein coded for by the entire PreSl-PreS2-S
protein coding :region which comprises: a) culti vating the transfected host eukaryotic cell of this invention under culture medium conditions which enable such host: to express such protein; and b) isolating such particles. Preferably, such transfected host is able to secrete such proteins assembled into such particles into the medium.
Preferably, such method additionally comprises the addition of heav5r metal ions or steroid hormones to such culture medium to enhance the expression of the proteins. 'rhe present invention also relates to the particles prepared by such method.
The' present invention also relates to a vaccine comprising an immunoprotective amount of the particles oi: this invention.
The: present invention also relates to a method for detecting the presence of antibodies to proteins coded for by the PreSl-PreS2-S protein coding region in a sample of mammalian blood sera which comprises:
a) contacting the sample with a solid substrate coated with non-labeled particles of this invention;
b) incubating and washing said contacted sample;
c) contacting said contacted sample with labeled particlea> of this invention thereby producing a labeled c~~ntacted sample;
d) incubating and washing said labeled 05 contacted sample; and e) determining the extent of labeled particle in the labeled contacted sample.
The present invention also relates to a method for detecting the presence of proteins coded for by the PreSl-PreS2-S protein coding region in a sample of mammalian blood sera which comprises:
a) producing a composition containing an antibody produced by an immunogen comprised by the particles o~_ this invention;
b) contacting the sample with a first portion of the composition and the, immunogen which has been libeled, incubating and washing the first portion;
c) contacting an antigen-free control with a second portion of the composition and the immunogen which has been labeled, incubating and washing the second portion;
d ) add:ing the same amount of staphylococci bearing protein A to the composition of steps b) and c) above, incubating both compositions and separating liquid from solids; and e) determining the extent of labeled immunogen in each of the resultant compositions from step d) above.
'I'he present invention also relates to a diagnostic kit for detecting the presence of antibodies to prot:eins coded for by the PreSl-PreS2-S protein coding region in a sample of mammalian b:Lood sera which comprises:
a) unlabeled proteins comprising the particle of this invention attached to a solid 05 support; and b) labeled antibodies to human IgG or IgM, for example,.
The present invention also relates to a diagnostic l;it for detecting the presence of proteins coded for by the PreSl-PreS2-S protein coding region in a sample of mammalian blood sera which comprises:
a) antibodies, produced by the particles of this invention, attached to a solid support; and b) labeled antibodies, produced by the particles of this invention.
The preaent invention also relates to a co-transfect.ed eukaryotic host cell co-transfected with a recombinant DNA vector of this invention and a second recombinant DNA vector comprising the PreS2-S protein coding region, or only the S
protein coding region, and an L,4MT-promoter.
Preferably, the second recombinant vector additionally comprises a transcription termination site and, opi_ionally, a selection marker.
Optionally, the co-transfected host is co-transfected with the recombinant DNA vector of this invention, the second recombinant DNA vector and a third recombinant DNA vector comprising an rZP~T-promoter and a selectic>n marker. Preferably, the third recombinant vector additionally comprises a transcription termination site. Additionally, this 1 3 40 9'~ 4 invention :elates to a method of preparing such co-transfeci:ed host cell which comprises co-transfec~_ing a eukaryotic cell with the recombinant DNA vector of this invention, the second 05 recombinant DNA vector described above, and, optionally, the third recombinant DNA vector described above.
Thc~ present invention also relates to a method for preparing particles comprising at least one protein coded for by the entire PreSl-PreS2-S
protein coding region which comprises: a) cultivating the co-transfected host eukaryotic cell of this ir,.vention under culture medium conditions which enable such host to express such protein; and b) isolating such particles. Preferably, such co-transfec~:ed host is able to secrete such proteins assembled into such particles in the medium.
Preferably, such method additionally comprises the addition of heavy metal ions or steroid hormones to such culture medium to enhance the expression of such proteins. This invention also relates to the particles prepared by such method.
This invention relates to a recombinant DNA
concatamer comprising: a) a vector comprising the PreSl-PreS2-S protein coding region and at least one vector comprising one of the following coding regions: PreSl-PreS2-S protein coding region or PreS2-S protein coding region or S protein coding region; and b) the MMT promoter. Such concatamer preferably additionally comprises a selection marker and a transcription termination site. Such concatamers can be prepared by techniques known in the art.
This invention also relates to a host ~ukaryotic cell transfected with the concatamer of 1340934 .
this invention. Such host is preferably a mammalian cell. Additionally, the invention relates to a method of preparing such transfected host cell with the concatamer of this invention which comprises 05 transfectin<3 a eukaryotic cell with the concatamer of this invention.
This invention also relates to a method for preparing a particle comprising at least one protein coded for by the entire PreSl-PreS2-S protein coding region which comprises: a) cultivating the transfected host of this invention, which comprises the concatamer of this invention, under culture medium conditions which enable such host to express such protein; .and b) isolating such particle.
Preferably, such transfected host is able to secrete such proteins as~cembled into such particles into the medium. :Preferably, such method additionally comprises the addition of heavy metal ions or steroid hormones to such culture medium to enhance the 2C expression of t-he proteins. This invention also relates to t:he particles prepared by such method.
BRIEF DESCRIPTION OF THE DP,AWINGS
Figure 1 includes a partial restriction endonucleasEa map of plasmid pBPV342-12. Figure 1 also illustrates the location of plasmid pML2-derived DNA, bovine papilloma virus (BPV) DNA, the MMT-promoter and the sv-PAS-t transcription termination region on plasmid pBPV342-7.2. Figure 1 also illustrates the splitting of plasmid pBPV342-12 with the restriction endonuclease BamHI.
Figure 2 includes a partial restriction endonuclease map of plasmid pA01 which contains the complete hepatitis B virus genome in linear form.
Figure 2 a:Lso illustrates the splitting of pA01 with the restri~=tion endonuclease EcoRI, ligation of the EcoRI linear HBV product with T4 DNA-ligase to form 05 concatamers and splitting of the concatamer product obtained w:th the restriction endonuclease BglII to generate a specific DNA fragment containing the intact DNA coding sequence for the PreSl-PreS2-S
protein coding region.
Figure 3 includes a partial restriction endonuclease map of plasmid pDMl and illustrates its construction by ligation of a BamHI DNA fragment comprising plasmid pMMT-neo with a BglII genomic fragment «f hepatitis E virus encoding the PreSl-PreS2~-S protein coding sequence.
Fi<3ures 4A and 4B include a partial restriction endonuclease map of plasmid pDM2 and illustrates its construction from ligation of a BamHI
restriction endonuclease fragment and a BamliI-BglII
restriction endonuclease fragment, both from plasmid pDMl.
Figure 5 includes a partial restriction endonuclease map of plasmid pDM3 and illustrates its construction from pDMl and pMMT-neo.
Figure 6 is a graphical representation illus-trating the elution pattern of the particles of this invention from a BioRad (A5m) column.
Figure 7 is a graphical representation of a typical isopycn~lc CsCl centrifugation of the particles o:E this; invention from about 30 fractions from the first peak resulting from the BioRad (A5m) column.
1 340934 '~
Figures 8A, 8B and 8C are graphs that illustrate seropositive conversion induced in mice by different dilutions of purified particles of the present invention.
05 Figures 9A and 9B include a partial restriction endonuclease map of plasmid pENDO-1 and illustrates its multi-step construction.
Fi~3ure 10 includes a partial restriction endonucleas~e map of plasmid pENDO-2 and illustrates its construction from ligation of restriction endonuclease DNA fragments of plasmid pENDO-1 and plasmid pDPM:Z .
Fi~~ure 11 includes a partial restriction endonucleas~~ map of plasmid pENDO-0 and illustrates its constru~~tion.
DET~~ILED DESCRIPTION OF THE INVENTION
By the term "PreSl-PreS2-S protein coding region" is meant the region of the HBV DNA
genome that encodes the entire polypeptide known as the PreSl-P:reS2-S polypeptide, including the codon for the initial methionine through to the final termination codon (TAA) that specifies insertion of no amino acid and directs that translation terminate, or any functional derivative of such coding region.
Proteins coded for by such region include the PreSl-PreS2--S polypeptide (389 amino acid residues i:z HBV subtype adw, for example), the PreS2-S polypeptide (281 amino acid residues in HBV
subtype adw, for example), and the S polypeptide (226 amino acid residues in HBV subtype adw, for example).
Preferred PreSl-PreS2-S protein coding regions are derived from the following subtypes: adr, ayw, adyw and adw. By the term "functional derivative" is meant any derivative of the PreSl-PreS2-S protein coding region which codes for polypeptides which, when assembled into a particle, function in 05 substantially the same way as the particle resulting from the a:~sembl:y of the polypeptides coded for the native PrE~Sl-PreS2-S protein coding region in terms of i.mmunoc3enic capability. Such derivatives include pe~rtial sequences of the PreSl-PreS2-S
10 protein cc>ding region, as well as derivatives produced by modification of such coding region.
Techniques for modifying such coding region are known in the art and include, for example, treatment with chemical cr biological mutagens, irradiation or 15 direct genetic engineering, such as by inserting, deleting or substituting nucleic acids by the use of enzymes or other recombinant and molecular biological techniques.
By the term "PreS2-S protein coding 20 sequence" is meant the region of the HBV DNA genome that encodE~s the entire polypeptide known as the PreS2-S polypept:ide including the colon for the initial met:hionine through to the final termination colon (TAA) that specifies insertion of no amino 25 acid, as specified above, or any functional derivative of such coding region. Proteins coded for by such region include the PreS2-S polypeptide (281 amino acid residues in HBV subtype adw) and the S
polypeptide (226 amino acid residues in HBV subtype ad~w, for example) . Preferred PreS2-S protein coding regions are derived from the following HBV
subtypes: _adr, a.yw, a- dyw and adw. By the term 1340834 ' "functional derivative" is meant any derivative of the PreS2-~> prot:ein coding region which codes for polypeptide,s which, when assembled into a particle, function in substantially the same way as the 05 particle resulting from the assembly of polypeptides coded for by the native PreS2-S protein coding region in terms of immunogenic capability. Such derivatives include partial sequences of the PreS2-~ p.rotein coding region, as well as derivatives produced by modification of such coding region. Techniques which may be employed for modifying such coding region are known in the art and some have been outlined above.
By the term "S protein coding sequence" is meant the region of the HBV DNA genome that encodes the entire polypeptide known as the S polypeptide including the codon for the initial methionine through to the final termination codon (TAA) that specifies insertion of no amino acid, as specified above, or a3ny functional derivative of such coding region. Proteins coded for by such region include the S peptide (226 amino acid residues in HBV subtype adw, for examp:le). Preferred S protein coding regions are derived from the following HBV subtypes:
adr, ayw, adyw and adw. By the term "functional derivative" is meant any derivative of the S protein coding region which codes for polypeptides which, when assembled into a particle, function in substantially the same way as the particle resulting from the a:asembly of polypeptides coded for by the native S protein coding region in terms of immunogenic capability. Such derivatives include 1 340 g34 ~
partial sequence~~ of the S protein coding region, as well as derivatives produced by modification of such coding region. ~('echniques which may be employed for modifying sv~ch coding region are known in the art and 05 some have bf~en outlined above.
By the term "promoter" is meant that DiJA
sequence within a DNA molecule upstream of a gene that direct, the appropriate host cell P,NA polymerase complex to attach to the DNA molecule at a specific site on said DN'A molecule and to become properly positioned to begin transcription of the gene at a specific point on the DNA to result in the synthesis of an P.NA copy of one of the DNA strands.
By the term "transcription" is meant that process carried out by the host cell biosynthetic machinery, including RNA polymerase complexes, that, using one of the two complementary DNA strands as a template, polymerizes ribonucleotides in a sequential fashion, 5' to 3' , ( 3' to 5' with respect to the DNA
template strand) to yield an exact copy of one of tree DNA strands, but containing ribonucleotides instead of deoxyribonucleotides.
By the term "transcription termination sequence" is meant that region within the DNA
molecule, ~iesign~ated t, that signals to the RNA
polymerase compl'_ex engaged in the process of transcription to terminate progress of such transcription to yield an RNA molecule that can be processed .properly, including polyadenylation and transport for such RNA molecules that are destined to be used as messenger RNA molecules in the host cell cytoplasm.
134934 '~
By the term "polyadenylation" is meant the process by which the host cell biosynthetic machinery recognizes a specific sequence, usually, 5'-AATAAA-3', designated PAS, for polyadenylation signal, within 05 the RNA molecule and that directs the addition to the 3' end of said molecule some 15-20 nucleotides downstream (in t:he direction of the 3' end) of a non-template determined polyriboadenylate moiety, specifically added by the enzyme poly-A polymerase.
By the term "selection marker" is meant a gene determinant that, when expressed in the cell, confers a specific set of characteristics upon the cell that allows such a cell to be distinguished, or selected ovat, from other cells not carrying or expressing raid gene determinant.
PrE~ferred selection markers include drug resistance markers. By the term "drug-resistance marker" is meant a special class of selection markers that confer upon a cell expressing such a marker, resistance to th.e lethal effects of a drug or an antibiotic that ordinarily blocks growth, or kills, cells not carrying or expressing said drug-resistance marker.
Preferred drug resistance markers include the coding sequences for the neomycine-resistance gene, neo; i=he Eco-gpt gene (Mulligan, R.C. and Berg, P., Science, Vol. 209, p. 1422, 1980), the dihydrofolat:e reduction (DHFR) gene (Ringold, G. et al, J. of N.olec. and Appld. Genet., Vol. 1, p. 165, 1981).
By the term "particles comprising at least one protein coded for by the entire PreSl-PreS2-S
protein coding region" is meant a hepatitis B
particle, devoid of any nucleic acid, formed by the assembly within or from a culture lysate of a euraryotic cell transfected with the entire PreSl, PreS2 and S region wherein such particle contains 05 subunits composed predominantly of the S polypeptide (dimers), but also composed of smaller amounts of the entire PreSl-PreS2-S, optionally, the PreS2-S
polypeptide;a.
This invention relates to a recombinant DNA
vector comprising the PreSl-PreS2-S protein coding region and a MMT-promoter. The MN1T-promoter may be incorporated into the DNA vector either in addition to the natural promoter for the DNA sequence (hepatitis B promoter) or in place of the hepatitis B
promoter. Preferably, the MMT-promoter is located in the DNA vector immediately upstream of the PreSl-PreS2-S protein coding region. Preferably, such vector also comprises a transcription termination sequence and a selection marker. Preferably, such selection marker is a drug-resistance marker, such as the neomyci.ne gene. Preferably, such transcription termination sequence is an SV40 termination site (sv-PAS-t) or more preferably the DEF region (mg-PAS-t) of t:he mouse globin gene. The SV40 termination sequence is well-known in the art and is described by various references such as Mulligan and Berg, Science, Vol. 209, p. 1422, 1980. The DEF
region of the mouse globin gene is described by Falck-Pederson et al, Cell, Vol. 40, p. 897, 1985.
Such vect~~r may be prepared by conventional recombinant DNA and other molecular biological techniques. In the most preferable case, using the mg-PAS-t region, the vector does not contain any viral DNA segments and is not oncogenic, i.e., it will not 134A9~4 transform (make cancerous) any host cell into which it is introduced. Such vector, upon transfection into a host, if it does not contain an autonomous replication sequence (replicon) capable of functioning 05 in a host t:ransfected therewith, integrates into the host chromosome and replicates passively with the host genome.
Preferably, the recombinant DNA vector of this invention should comprise the following characteristics:
1} The PreSl-PreS2-S protein coding region.
2) A MMT-promoter located immediately upstream of the PreSl-PreSZ-S protein coding region.
3) The vector should be able to replicate in bacteria, or other procaryotic host into which it is transformed, for growth, amplification and preparation of large quantities of the recombinant vector. Thus, such vector should include a bacterial or other procaryotic replicon, i.e., a DNA segment bearing al:L the functions required for autonomous replication and maintenance of the vector extrachromosomally in a procaryotic host cell, such as a bacterial host cell, transformed therewith.
Such replicons are well known in the art.
4) The vector replicon should be small (i.e., smal_ler than 6-8 kilobase pairs) to enable easy genetic an<9 molecular biological manipulation thereof .
5) The vector should carry a selection marker, preferably a drug-resistance marker such as 134Q9:i4 s ampicillin, for use in bacterial host cells transformed therewith.
6) T'he vector should carry a second selection marker, preferably a drug-resistance marker 05 such as neomycine, for use as such in eukaryotic host cells trans;_ected therewith.
7) The vector should contain convenient endonuclease restriction sites for cloning.
8) The vector should contain a tran-scription termination and polyadenylation sequence.
Most preferably, the vector of this invention does not comprise an autonomous replicating sequence (~_eplicon) capable of functioning in a eulcaryotic host cell transfected therewith. A
primary reason i:or using a non-replicating vector system in eaukaryotic host cells is that all vector systems capable of autonomous and extrachromosomal replication in a mammalian host eukaryotic cell transfected therewith comprise replicons which are derived from oncogenic viruses. It is desirable to employ vectors comprising DNA not derived from oncogenic viruses for expressing DNA sequences encoding polypept:ides of pharmaceutical importance, such as the px-oteins coded for by the PreSl-PreS2-S protein coding region.
They vector of this invention comprises the MMT-promoter. The MMT-promoter is a non-viral, strong transcriptional promoter. The coding sequence of the MMT-promoter is described by Pavlakis and Hamer, Proc. Nat.l. Acad. Sci., Vol. 11, p. 397, 1983. The MMT-promoter is regulable by heavy metal ions, such a.s zinc. and cadmium, and by steroid ~ 34O 9,3,~ v hormones, such as dexamethasone. Such regulation is well known (see, e.g.. Yagle and Palmiter, Molecular and Cellular Biol" Vol. 5, p. 291, 1985).
This invention also relates to a transfected C5 host eukary«tic cell transformed with a recombinant DNA vector «f this invention. Preferably, such host is a mamm~ilian cell, most preferably a Chinese hamster ovary (CHO) cell line, a vero cell line, an L-cell line, or a mouse or rat fibroblastic cell line. Such host may be prepared by transfecting a eukaryotic cell with a recombinant DNA vector of this invention b~~ conventional techniques, such as by the method of Graham and van der Eb, Virology. Vol. 52, p. 456, 1973, or ~~~igler, h:. , Cell, Vol. 14, p. 725, 1978.
This invention also relates to a method for preparing a particle comprising proteins coded for by the PreSl-PreS2-S protein coding region wherein at least one of such proteins corresponds to a polypeptide coded for by the entire PreSl-PreS2-S protein coding region which comprises: a) cultivating the transfected host of this invention under culture medium conditions which enable such host to ex~~ress such proteins, and b) isolating such particle. Preferably, such co-transfected host is able to secrete such proteins assembled into such particles into the medium. Preferably, heavy metal ions or steroid hormones, such as dexamethasone, are added to such culture medium to induce the MMT-promoter and thereby enhance expression of such coding region. Heavy metal ions such as cadmium or zinc are most preferred. The optimal concentration 134934 ' of heavy metal ions or steroid hormone contained in the medium can be determined by conventional techniques.
This invention also relates to the particles 05 prepared b~~ such method. If the transfected host cell of this invention secretes such particles directly into the' culture medium, such particles can then be isolated from the culture medium of the transfected host of this invention by conventional protein isolation techniques. If the transfected host cell of this invention does not secrete such particles, 1=hey are obtained from a culture lysate of such host by conventional culture lysate techniques.
Such particles are obtained in glycosylated form and are compo=>ed of proteins coded for by the PreSl-PreS2--S protein coding region, including at least one protein coded for by the entire PreSl-PreS2--S protein coding region.
Th:~s invention also relates to a vaccine comprising an immunoprotective amount of the particle of this invention. By "immunoprotective amount" is meant that quantity (preferably 1-20 ug) of the particles of this invention required to induce and mantain a lc=vel of antibody in the host sufficient to neutralize the infectious agent and prevent proliferation and subsequent disease by said infectious agent.
The particle of the subject invention contains no viral DNA components and therefore is free from undesirable side effects commonly found in naturally-derived vaccines, such as unintentional infection with the virus, allergic reactions, fevers, and the like. The vaccine of the present invention 1340934 .
comprising an immunoprotective amount of the particles c~f th:is invention can be used to improve the HBV immune response and to overcome non-respon-siveness to hepatitis B virus vaccines which do not 05 include proteins coded for by the entire PreSl-PreS2-S protein coding sequence.
A vaccine of this invention can be prepared by combining a.n immunoprotective amount of the particles of this invention in an appropriate buffer, such as phosphate buffered saline. The vaccine may additionall comprise an adjuvant, such as aluminum hydroxide a.nd the' like.
Alternat=ively, the polypeptides comprised by the particles of the present invention can be disassociated from said particles and reassociated in a lipid ve~~icle. A vaccine against hepatitis B virus can be pre;~ared using said lipid vesicles comprising the polypeptides of the present invention. The lipid vesicles in the appropriate concentration can be used as a vaccine with or without adjuvant, such as aluminum hydroxide.
Th.e particles of the vaccine of this invention can be employed with a physiologically acceptable diluant (medium), such as phosphate buffered saline. ' The vaccine of this invention, comprising an immunoprotective amount of the particles of this invention, can be prepared and used in the same general mariner as disclosed in U.S. Patent 4,118,479.
1340934 ?
Thc~ vaccine can be administered by subcu-taneous, intradermal or intramuscular injection.
V~Thile the preferred route would depend on the particular 'vaccine, it is believed that intramuscular 05 injection is generally more suitable. Frequency of administrat_~.on will vary depending upon the vaccine.
This invention also relates to a method for detecting t=he presence of antibodies to proteins coded for by the PreSl-PreS2-S protein coding region in a sample of mammalian blood sera which comprises:
a) contacting the sample with a solid substrate coated with non-labeled particles of this invention;
b) incubating and washing said contacted sample;
c) convtacting said contacted sample with labeled part=icles of this invention thereby producing a labeled cc>ntacted sample;
d) incubating and washing said labeled contacted sample; and e) determining the extent of labeled particle in the labeled contacted sample.
This invention also relates to a method for detecting the presence of proteins coded for by the PreSl-PreS2-~S protein coding region in a sample of mammalian: blood sera which comprises:
a) producing a composition containing an antibody produced by an immunogen comprised by the particles of this invention;
b) cons=acting the sample with a first portion of the composition and the immunogen which has been l~3beled, incubating and washing the first portion;
c) contacting an antigen-free control with a second portion of the composition and the immunogen 05 which has been :Labeled, incubating and washing the second portion;
d) adding the same amount of staphylococci bearing protein P, to the composition of steps b) and c) above, incubating both compositions and separating liquid from solids; and e) determining the extent of labeled immunogen in each of the resultant compositions from step d) above.
Th_Ls invention also relates to a diagnostic kit for dE~tecting the presence of antibodies to proteins coded for by the PreSl-PreS2-S protein coding region in a sample of mammalian blood sera which compr:~ses:
a) unlabeled proteins comprising the ?0 particle o:~ this invention attached to a solid support ; an~i b) labeled antibodies to human IgG or IgM, for example..
This invention also relates to a diagnostic kit for detecting the presence of proteins coded for by the PreSl-Pr~~S2-S protein coding region in a sample of mammalian blood sera which comprises:
a) antibodies, produced by the particles of this invention, attached to a solid support; and b) labeled antibodies, produced by the particles of. this invention.
1340934 "~
Thc~ present invention also includes diagnostic tests for direct detection of hepatitis B
surface antigens and antibodies, raised to such antigens. Radioimmunoassay (RIA) or enzyme-linked 05 immunosorbant a w ay (ELISA) are employed to detect HBV antigens containing proteins coded for the PreSl-PreS2--S coding regions in sera of HBV
infected animals, such as humans. One diagnostic test includes the following:
1. A solid substrate containing binding sites thereon, such as polystyrene beads, is coated with antibad.ies to particles containing amino acid sequences corresponding to those of the PreSl-PreS2--S containing polypeptides;
2. The coated beads are then washed with, for example, Tris buffered saline to remove excess antibodies;
3. The beads are then contacted with a protein-cone=aining solution, such as bovine serum albuMin (FSA) or gelatin, to saturate protein binding sites on the beads (to reduce non-specific binding).
A convenient concentration of such protein-containing solution can be employed, such as 1 mg/ml to 50 mg/ml;
4. The beads are then washed to remove excess BSA or gelatin;
5. The beads are then incubated with serum samples suspected to contain HBV or HBV surface antigens (normal serum is utilized as a control);
6. The beads are then washed with a solution, such as Tris buffered saline solution, and mixed with a radio-labeled antibody, such as 125I
labeled antibody to HBV surface antigens;
1344934 _.
7. The beads are then incubated; and 8. The beads are then washed and counted in a gamma counter.
Particles of the present invention coded for 05 by the Pre~51-PreS2-S protein coding region can be employed as a diagnostic tool to detect antibodies to the PreSl-F'reS2-S regions of HBV surface antigen proteins in a given sample. The PreSl-PreS2-S
containing particles are adsorbed on a solid substrate containing binding sites thereon, for example, polystyrene beads. The substrate is thereafter contacted with a substance, for example, gelatin, BSA or powdered milk, to saturate the non-specific. binding sites thereon. Thereafter, the substrate i.s wa:~hed with a buffered solution and thereafter the buffer is removed. A specimen, such as human serum, diluted with animal serum, is added to the substrate. The resulting mass is then incubated and washed. Thereafter, radio-labeled, such as iodinated (125I), antibodies to human IgG
or IgM is added to the mass. The resulting mass is incubated then washed and counted, in the such as gamma counter. If the count is higher than the count obtained for the normal serum control, the specimen contains antibodies to the PreSl-PreS2-S coding regions of FiBV.
ThE~ above procedure for detection of antibodies to the proteins coded for by the PreSl-PreS2--S protein coding region of HBV is believed to be applicable as a diagnostic tool in detecting hepatitis B virus infection.
X340934 ,~, A 3iagnostic test kit for detecting antigens coded for the PreSl-PreS2-S protein coding regions of the HBV genome in a test sample would include the following:
05 1. A solid substrate coated with antibodies to a particles having amino acid sequences corresponding to the PreSl-PreS2-S regions of HBV
surface antigen particles of the present invention;
2. A protein-containing solution to saturate protein binding sites on the solid substrate;
and 3. A given amount of radio-labeled antibody, such as antibody to the HBV surface antigen polr~pept ide;~ .
Another diagnostic test kit for detecting antibodies to the proteins coded for by the PreSl-I'reS2~-S protein coding region of the hepatitis B virus genome in the test sample includes the following:
1. A solid substrate having adsorbed thereon particles containing amino acid sequences corresponding to the PreSl-PreS2-S coding regions of the HBV surface antigen proteins of the present invention, the substrate being exposed to a protein-containing solution to saturate non-specific protein binding sites on a solid substrate; and 2. A given amount of radio-labeled anti-bodies to human IgG or IgM.
Radio-labeled antibodies used in the above described test kits can be packaged in solution form or in lyop~zilized form suitable for reconstitution.
In addition, enzyme-lin};ed or fluorescent-labeled 1340934 ' antibodies can be substituted for the described radio-labeled antibodies.
The above-described test kit and process for detecting antibodies to the PreSl-PreS2-S coding 05 region pol:~peptides of hepatitis B virus can be utilized in ap~~lications such as detecting HEV
infection :Ln a patient by taking serum from the patient and applying the above-described test or using the above-described test; and predicting recovery from HBV infection by taking serum from an infected patient and applying the described antibody detection procedures.
ThE~ test procedures and test kits for antibody detection can be used for making qualitative comparisons between different HBV vaccines by taking serum from 'vaccinated patients and then utilizing the above-described test procedures or kits for antibody detection. In general, all known immunoassays using this antigen as reagent can be performed using the PreSl, Pre;S2, or S-containing particles of this invention. Generally, all known immunoassays using antibody-containing serum or reagents can be performed using antibody serum produced through the use of a peptide produced by recombinant DNA
techniques of the present invention. These immunoassays include all those disclosed by Langone and Van Vunakis in Methods of Enzymology, Academic Press, Vols. 70, 73 and 74 and those assays disclosed in disclosures of the following U.S. Patents:
4,459,359; 4,.'343,896; 4,331,761; 4,292,403;
4,228,240; ~I,157,280; 4,152,411; 4,169,012; 4,016,043;
3,839,153; :3,654 ,090; and Re. 31,006 and Vols. 70, 73 and 74 of Methods of Enzymology.
This invention also relates to a co-trans-fected host: eukaryotic cell co-transfected with the 05 recombinant vector of this invention and a second recombinant DNA vector comprising the PreS2-S
protein coding region, or only the S protein coding region, anc an MII~1T-promoter. Preferably, the second recombinant vector additionally comprises a transcription termination site and, optionally, a selection marker. Optionally, the co-transfected host is co-transfected with the recombinant DNA
vector of this invention, the second recombinant DNA
vector and a third recombinant DNA vector comprising an MMT-promoter and a selection marker. Preferably, the third recombinant vector additionally comprises a transcription termination site. Preferably, the second and optional third recombinant DNA vector also contain a replicon for growth and amplification in a procaryotic host cell, such as a bacterial host cell, when such host is transformed with said second or optional third recombinant DNA vector.
Additionally, this invention relates to a method of preparing such co-transfected host cell which comprises co-transfecting a eukaryotic cell with the recombinant DNA vector of this invention, the second recombinant DNA vector described above, and, optio;~ally, the third recombinant DNA vector described above.. The second and optional third recombinant DNA vectors described above can be constructed by conventional techniques. If the optional third vector is not employed, the second recombinant DNA. vector preferably additionally comprises a selection marker, such as a drug resistance marker. Preferably, if the second and/or optional third recombinant DNA vector comprise a 05 transcriptiot: termination site, such site is the mg-PAS-t termination site. Preferably, in the second recombinant DNF, vector described above, the MMT-promoter is located in such vector immediately upstream of the PreS2-S protein coding region.
Preferably, in the optional third recombinant DNA
vector described above, the PMMT-promoter is located in such vector immediately upstream of the selection marker. Preferably, neither the second recombinant vector nor the optional third recombinant vector described above contain any non-HBV viral DLdA
segments and are non-oncogenic. Thus, upon transfection into a host, such preferred vectors integrate into the host chromosome and are replicated passively with the host genome.
the vector of this invention, the second recombinant DNA vector described above, and, optionally, the third recombinant DNA vector described above .are co-transfected into a eukaryotic host in pairwise combinations, or all three vectors together, using conventional methods. Alternatively, the vector of this invention, the second recombinant DNA vector and, optionally, the third recombinant DNA
vector are transfected into a eukaryotic host in a series of steps. Initially, the vector of this invention is transfected into a eukaryotic host according to the method of this invention to produce the transfected host of this invention.
1340g34.'~
Subsequently, ouch transfected host is then transfected with the second recombinant DNA vector described above, and, optionally, the third recombinant DNA vector described above according to 05 conventional r.~ethods to produce the co-transfected host of this invention.
Prefera~:>ly, the vector of this invention, the second recombinant DNA vector and the optional third recc>mbinant DNA vector comprise selection markers different from each other to enable identification of the newly-transfected host at each subsequent transfection step leading to the final transfected host of the invention. Such secondary transfection steps are carried out in order to increase t:he gene copy number of the PreSl-PreS2-S protein coding region comprised by the multiply transfected host cell. Preferably, neither the second recombinant vector nor the optional third recombinant vector comprise a replicon capable of functioning in a eukaryotic cell.
This invention also relates to a method for preparing a. particle, produced by the co-transfected host of this invention, comprising at least one protein coded for by the entire PreSl-PreS2-S
protein coding region which comprises: a) cultivating the co-transfected host eukaryotic cell of this invention under culture medium conditions which enable such host to express such proteins; and b) isolating ouch particle. Preferably, such co-transfected host is able to secrete such proteins assembled into such particles into the medium.
Preferably, such method additionally comprises the ~ 340934 ' addition of heav~~ metal ions or steroid hormones to such culture medi~.um to enhance the expression of the PreSl-PreS2-S protein coding region contained by the co-tr~~nsformed vectors. Heavy metal ions, 05 particularly zinc or cadmium, are most preferred.
The optima7_ concentration of heavy metal ions or steroid hormones contained in the medium can be determined by conventional techniques. This invention also relates to the particles prepared by such method. If the co-transfected host of this invention secretes such particles directly into the culture medium, such particles can then be isolated from the culture medium of the co-transformed host of this invention by conventional protein isolation techniques. If the co-transfected host does not secrete such particles into the culture medium, then they are obtained from a culture lysate of such co-transfected host by conventional culture lysate techniques. Such particles are isolated in glycosylated form and are composed of proteins coded for by the Pre S1-PreS2-S protein coding region, the PreS2-S protein coding region, and the S
protein coding region.
This invention also relates to a vaccine comprising an :immunoprotective amount (Preferably 1-20 ug) of the particles produced by the co-transfected host of this invention.
Th.= particle produced by the co-transfected host of the subject invention contains no viral DNA
components and therefore is free from undesirable side effects commonly found in naturally-derived vaccines, such as unintentional infection with the virus, allergic reactions, fevers, and the like. The vaccine of the present invention comprising an immunoprotective amount of the particles produced by the co-tranafected host of this invention can be used 05 to improve the F-iBV immune response and to overcome non-responsiveness to hepatitis B virus vaccines which do nc~t include at least one protein coded for by the entire PreSl-PreS2-S protein coding sequence.
A 'vaccine of this invention can be prepared by combining an immunoprotective amount of the particles produced by the co-transfected host of this invention in an appropriate buffer, such as phosphate buffered saline.. The vaccine may additionally comprise an adjuvant, such as aluminum hydroxide and the like.
Alternatively, the polypeptides comprised by the particles produced by the co-transfected host of the present invention can be disassociated from said particles and reassociated in a lipid vesicle. A
hepatitis B vaccine can be prepared using said lipid vesicles comprising the polypeptides of said particles of the present invention. The vaccine comprising the lipid vesicles may additionally comprise an adjuv~ant, such as aluminum hydroxide.
The particles produced by the co-transfected host of the invention comprising the vaccine of this invention can b~e employed with a physiologically acceptable diluant (medium), such as phosphate buffered sa~_ine.
ThE~ vaccine of this invention comprising an immunoprotective .amount of the particles of this invention can be prepared and used in the same general manner as disclosed in U.S. Patent 4,118,479.
Lhc~ vaccine can be administered by subcu-taneous, intradermal or intramuscular injection.
05 While the preferred route would depend on the particular 'vaccine, it is believed that intramuscular injection is generally more suitable. Frequency of administration will vary depending upon the vaccine.
This invention also relates to a method for detecting v:.he presence of antibodies to proteins coded for by the PreSl-PreS2-S protein coding region in a sample of mammalian blood sera which comprises:
a) contacting the sample with a solid substrate coated with non-labeled particles produced by the co-transfected host of this invention;
b) incubating and washing said contacted sample;
c) contacting said contacted sample with labeled particles of this invention thereby producing a labeled contacted sample;
d) incubating and washing said labeled contacted sample; and e) determining the extent of labeled particle in the labeled contacted sample.
This invention also relates to a method for detecting the presence of proteins coded for by the PreSl-PreS2--S protein coding region in a sample of mammalian blood sera which comprises:
a) producing a composition containing an antibody produced by an immunogen comprised by the particles of this invention;
b) contacting the sample with a first portion of the composition and the immunogen which has been labeled, incubating and washing the first portion;
05 c) contacting an antigen-free control with a second portion of the composition and the immunogen vahich has been labeled, incubating and washing the second portion;
d) adding the same amount of staphylococci bearing protein A to tt~~ composition of steps b) and c) above, incubating both compositions and separating liquid from solids; and e) determining the extent of labeled immunogen in each of the resultant compositions from step d) above.
This invention also relates to a diagnostic kit for d.=_tecting the presence of antibodies to proteins coded for by the PreSl-PreS2-S protein coding region in a sample of mammalian blood sera which comprises:
a) unlabeled proteins comprising the particle of this invention attached to a solid support; an~3 b) labeled antibodies to human IgG or IgM, for example.
This invention also relates to a diagnostic kit for detecting the presence of proteins coded for by the Pr.=_Sl-PreS2-S protein coding region in a sample of mammalian blood sera which comprises:
a) antibodies, produced by the particles of this invention, attached to a solid support; and b) labeled antibodies, produced by the particles of this invention.
Th:is invention relates to a recombinant DNA
concatamer comprising: a) a plasmid vector carrying the PreSl-PreS2-S protein coding region and at least one additional plasmid vector comprising one of 05 the following coding regions: PreSl-PreS2-S
protein coding region or PreS2-S protein coding region or S protein coding region; and b) the MMT
promoter. l3uch concatamer preferably additionally comprises a selection marker and a transcription termination site.
This invention also relates to a eukaryotic host cell transfected with the concatamer of this invention. Such host is preferably a mammalian cell. Additionally, the invention relates to a method of preparing such transfected host cell with the concatamer ~of this invention which comprises transfectinc3 a eukaryotic cell with the concatamer of this invention.
This invention also relates to a method for preparing a particle, produced by the transfected host of this invention, comprising at least one protein coded for by the entire PreSl-PreS2-S
protein ceding region, which comprises: a) cultivating the transfected eukaryotic host cell of this invent:ion, which comprises the concatamer of this invention, under culture medium conditions which enable sucl-, host to express such proteins; and b) isolating such particle. Preferably, such co-transfec~ted host is able to secrete such proteins assembled into such particles into the medium.
Preferably, such method additionally comprises the addition of heav~~ metal ions or steroid hormones to such culture medium to enhance the expression of the 134Q934 .
PreSl-PreS2-S protein coding region contained by the transfected vectors. Heavy metal ions, particularly zinc or cadmium, are most preferred.
The optima:L cone entration of heavy metal ions or 05 steroid hormones contained in the medium can be determined by conventional techniques. This invention also relates to the particles prepared by such method. If the host transfected with the concatamer of this invention secretes such particles directly into the culture medium, such particles can then be i~colated from the culture medium of the transfected host of this invention by conventional protein isolation techniques. If the transfected host does not secrete such particles into the culture medium, then they are obtained from a culture lysate of such transfected host by conventional culture lysate tecr~nique:~. Such particles are isolated in glycosylated forms and are composed of proteins coded for by the PreSl-PreS2-S protein coding region, the PreS2-S protein coding region, and the S
protein coding region.
This invention also relates to a vaccine comprising an i.mmunoprotective amount (preferably 1-20 ug) of the particles produced by the host transfected with the concatamer of this invention.
Thc~ particle produced by the host transfected with the concata.mer host of the subject invention contains no viral DNA components and therefore is free from undesirable side effects commonly found in naturally-dE~rived vaccines, such as unintentional infection with the virus, allergic reactions, fevers, and the like. The vaccine of the present invention ~34~934.
comprising an immunoprotective amount of the particles produced by the host transfected with the concatamer of this invention can be used to improve the HBV
immune response and to overcome non-responsiveness to 05 hepatitis F3 virL~ vaccines 4rhich do not include at least one protein coded for by the entire PreSl-PreS2-S protein coding sequence.
A vaccine of this invention can be prepared by combining an immunoprotective amount of the particles ~~roduced by the host transfected with the concatamer of 'this invention in an appropriate buffer, such a;~ phosphate buffered saline. The vaccine may additionally comprise an adjuvant, such as aluminum hydroxide and the like.
Alternatively, the polypeptides comprised by the particles produced by the host transfected with the concatamer of the present invention can be disassociated from said particles and reassociated in a lipid vesicle. A hepatitis B vaccine can be prepared using aaid lipid vesicles comprising the polypeptide;~ of said particles of the present invention. The vaccine comprising the lipid vesicles may additionally comprise an adjuvant, such as aluminum hydroxide.
Th~~ particles produced by the host trans-fected with the concatamer of this invention comprising the vaccine of this invention can be employed with a physiologically acceptable diluant (medium), such as phosphate buffered saline.
Th.=_ vaccine of this invention comprising an immunoprotec~tive amount of the particles of this invention can be prepared and used in the same general manner as disclosed in U.S. Patent 4,118,479.
The vaccine can be administered by subcu-taneous, intradermal or intramuscular injection.
05 ~,Thile the preferred route would depend on the particular vaccine, it is believed that intramuscular injection is generally more suitable. Frequency of administration will vary depending upon the vaccine.
This invention also relates to a method for detecting the presence of antibodies to proteins coded for by the PreSl-PreS2-S protein coding region in a sample of mammalian blood sera which comprises:
a) contacting the sample with a solid substrate coated with non-labeled particles produced by the hosi_ transfected with the concatamer of this invention;
b) incubating and washing said contacted sample;
c) contacting said contacted sample with labeled particle; of this invention thereby producing a labeled contacted sample;
d) incubating and washing said labeled contacted sample; and e) determining the extent of labeled particle in the labeled contacted sample.
This invention also relates to a method for detecting the presence of proteins coded for by the Pre~l-PreS2-S protein coding region in a sample of mammalian blood sera which comprises:
a) producing a composition containing an antibody produced by an immunogen comprised by the particles of this. invention;
~ ~40g34 b) contacting the sample with a first portion of the composition and the immunogen which has been labeled, incubating and washing the first portion;
05 c) contacting an antigen-free control with a second portion of the composition and the immunogen which has been labeled, incubating and washing the second portion;
d) adding the same amount of staphylococci bearing protein A to the composition of steps b) and c) above, incubal=ing both compositions and separating liquid from solids; and e) determining the extent of labeled immunogen in each of the resultant compositions from step d) above.
This invention also relates to a diagnostic kit for detecting the presence of antibodies to proteins coded for by the PreSl-PreS2-S protein coding reg_Lon in a sample of mammalian blood sera 2C which comprises:
a) unlabeled proteins comprising the particle of this invention attached to a solid support; and b) labeled antibodies to human IgG or IgM, for example.
This invention also relates to a diagnostic kit for detecting the presence of proteins coded for by the PreSl-PreS2-S protein coding region in a sample of mammalian blood sera which comprises:
a) antibodies, induced by the particles of this invention, attached to a solid support; and 1 34Q g~4 b) labeled antibodies, produced by the particles o~- this invention.
First Preferred Embodiment 05 In a first preferred embodiment, one vector of this invention includes a vector construction incorporating gene sequences from recombinant plasmids pBPV342-12 (Law et al, PMolecular and Cellular Biol, 'Vol, 3, p. 2110, 1983) and pAOI
(Cummings, :..yV. et al, Proc. Natl. Aca. Sci., U.S.A., Vol. 77, p. 1842, 1980). Some of the genetic elements from these plasmi.ds are combined to create a vector of this invention, designated pDMl (see Figure 3).
Flasmid pDMl., which contains the gene segment carrying the PreSl-FreS2-S protein coding region under the control of the natural promoter system for said protein cod~:ng region, the neomycine-resistance gene, the 1~IMT promoter and an SV40 PAS-t function, is then introduced by tra,nsfection into any one of a number of eukaryotic cells, such as mammalian cells, to produce high-level expression and, preferably, secretion of the particles of this invention.
Pla.smid pBPV342-12 (see Figure 1) contains gene sequences including the MI,4T-promoter, the neomycine-resistance gene, an SV40 PAS-t function and the bovine ;?apilloma virus (BPV) genome. During the construction. of plasmid pDLMl, plasmid pBPV342-12 is subjected to BamHI digestion, with the result that the plasmi<i is split into two DNA fragments.
Following ~cepara~tion of the fragments, the 7.95 kilobase (kb) fragment, which comprises the entire BPV genome, is discarded. The elimination of BPV DNA
1340934 ~
is highly <3dvantageous since it precludes inclusion of any BPV DNA or proteins in the particle of this invention which is to be used for vaccine formulation,. The remaining BamHI fragment (6.65 kb) 05 from pBPV342-12, which contains the gene sequences for the MME:'-promoter, the neomycine resistance gene and the SV40 P.AS-t region, is then ligated, by conventiona7_ techniques, to a DNA sequence derived from plasmid pAOI, but which contains an intact PreSl-PreS2--S protein coding region (discussed below; see also Figure 2).
Plasmid pAOI (see Figure 2), which contains the entire hepatitis B viral genome, is subjected to EcoRI digestion to produce a 3.2 kb fragment. The 3.2 ?;b fragment is ligated to itself to produce tandem repeats comprising a long concatemeric DLdA
structure containing gene sequences encoding both the core arid surface antigens of hepatitis B virus. The method for t=his manipulation of the hepatitis B viral gene sequen~~es, which is necessary to overcome the permutation caused by molecular cloning in plasmid pAOI and to restore a functional organization with respect to the PreSl-PreS2-S protein coding region, is that of Cummings et al in Proc. Natl.
Acad. Sci. USA, Vol. 77, p. 1842, 1980. Through subsequent E3c~lII digestion, the HBV core antigen gene segment is eliminated, leaving a DPJA fragment (2.78 kb) carrying the PreSl-PreS2-S protein coding region of t)ze hef>atitis B virus genome together with the natural promot=er system (PHB) of such region.
The linear pBPV342-12 BamHI fragment (see Figure 1) and the pAOI-derived III fragment (see Figure 2) are then ligated to produce the recombinant plasmid, pl~Ml (see Figure 3), which retains the natural promoter (PHB) for the PreSl-PreS2-S
protein coding region.
Second Preferred Embodiment In a second preferred embodiment of this invention, 'the natural promoter in pDMl is excised by a BglII and BamHI digestion such that the P~ulT-promoter 10 is positioned directly ahead of the PreS2-S protein coding region to control transcription of said coding region by the P4P~'.C-promoter. Following BamFiI + Bc~.lII
digestion three discrete DNA fragments are obtained (see Figures 4A a:nd 4B):
15 1) a _'~.1 kb fragment containing a polya-denylation site a:nd the MMT-promoter;
2) a 3.0 kb fragment containing the neomycine resistance gene and the natural promoter;
and 20 3) a 1.4 kb fragment containing the PreS2-S protein coding region.
Fo7_lowing purification, the 5.1 and the 1.4 kb fragments are ligated using conventional techniques to produce recombinant plasmids (6.46 kb) 25 containing the F>reS2-S protein coding region, now under the control of the P~IMT-promoter. Because the relative orientations of the two ligated fragments are random and two different plasmid types are produced, only one of which exhibits the correct 30 transcriptional reading orientation from the MMT-promoter into the surface antigen gene sequences, the correct functional plasmid, pDM2, is ascertained 1340934' by EcoRI + XbaI digestion of purified plasmid candidates. The plasmid with the correct orientation yields a 2.1 and a 4.4 kb fragment following this digestion.
05 A bacterial strain, HE101, containing pDM2, was deposited at Deutsche Sammlung fur Mikro-organismen, Gotti.ngen, under the depository number DSf9 3285 en April 4, ).985.
Third Preferred Embodiment In a third preferred embodiment, the rstsT-promoter is spliced to a HBV gene segment containing the PreSl-PreS2-S protein coding region in :such a way as to eliminate the natural promoter system and place said coding sequence under the direct contro:L of the MMT-promoter (see Figure 9).
Fourth Preferred Embodiment In a fourth preferred embodiment, the mouse globin gene mg-PF,S-t segment, recovered from plasmid pBR322. G2, is substituted as a polyadenylation termination (PAS-t) signal for the SV40 PAS-t region. The substitution of mg-PAS-t provides DNA
transfer vectors without any non-HPV viral genomic portions (sere Figures 9, 10 and 11).
Fifth Preferred Embodiment A ~_ifth preferred embodiment is a method of preparing pl.asmid DNA mixed concatamers, amplifying 1344934 ' the gene copy number' by ligation of high ratios of plasmids containing gene constructs encoding the PreSl-PreS2-S, PreS2-S or S protein coding regions to plasmids containing the drug-resistance marker, for subsequent transfection into cell lines.
Plasmids pBR.322. G2, POLINK 23456, pBlg/2.8 and pMMT-neo plasmid were deposited at the American Type Culture Collection, Rockville, Maryland, under the accession numbers 67073, 67072, 67075 and 67074, :LO respectively on 11 April 1986.
In i:he following Examples, the preparation of the recombinant DNA vectors, hosts and particles of this invention, as well as the incorporation into mammalian ce:Ll lines is described in more detail.
The following examples are for illustrative purposes only and are not :intended to limit the invention in any way.
EXAMPLES
MATERIALS AND PROCEDURES
A. ORIGIN ArfD DESCRIPTION OF MAMtZALIAN CELL
LINES.
1. L-Cells.
NCTC clonee 929 was derived in March 1948 by K.K. Sanford,, W.R. Earle and G.D. Likely (J. Nat.
Cancer Inst., Vo7_. 9, pp. 229, 1948) from the parental strain L established in 1940 by W.R. Earle (J. Nat. Cans=er Inst., Vol. 4, pp. 165, 1943). The parent strain L was derived from normal subcutaneous areolar and adipose tissue of a 100 day-old male C3H/An mouse, and clone 929 was established (by the capil7_ary technique for single cell isolation) from the 95th suhcul_ture generation of the parent strain.
LM(TK ), a sub-line of mouse L cells, was 05 isolated by D. R. Dubbs and S. Kit (S. Kit et al, Exp.
Cell Res. , Vol. 31, pp. 297-312, 1963; and D.R. Dubbs & S. Kit, E;xp. Cell P,es., Vol. 33, p. 19, 1964), and is deficier,.t in thymidine kinase. It is unable to grow in medium containing HAT. No spontaneous reversion of HAT-resistance has been observed in this cell line, and it seems most likely that a deletion involving t::~is gene has occurred.
2Tumber of ;serial Subcultures from Tissue of Origin:
64~; clone, 553.
Freeze Medium: Culture medium, 90s; DtZSO 100; anti-biotic-free.
Viability: 81-960 (dye exclusion).
Culture Me3ium: DL~4ELM + loo FBS (Dulbecco's and Vogt's Modified E;agle's Medium + 10°s FBS).
2O Growth Characteristics of Thawed Cells: An innoculum of 6-8 x 10 Lls in 5 ml of above culture medium per T-25 f:Lask, yields a 500-fold increase within 7 days at 37°C, provided the medium is renewed two times week7_y and. the pH is adjusted to 7.3 with a humidified mixture of 5°s or 10°s carbon dioxide in air. Subcultures are prepared by scraping or shaking. Cells also grow well on other media (Waymouth's, Eac)le's, NCTC135, etc.) supplemented with l00-30o hor:~e serum.
Plating Efficiency: 700 Morphology: Fibz~obl_ast-like.
1340934'.
Karyology: Chromosome Frequency Distribution 100 Cells : 2n ~- 40 .
Cells: 2 2 1 4 2 9 4 12 16 17 4 13 2 Chromosomes: 5G-58 59 60 61 62 63 64 65 66 67 68 69 Cells: 2 2 1 1 6 Chromosomes: 70 71-76-82-125-241 Long metacentric chromosome with secondary constriction noted in 77/100 cells.
Sterility: Tests. for mycoplasma, bacteria and fungi were negative.
Species: Confirmed as mouse by mixed agglutination and hemagglutination tests.
Virus SL1SCE'_l~tibility: Susceptible to pseudorabies virus and vesicular stomatitis (Indian Strain) virus. t~d'hen the cells were cultured in the above culture medium, Herpes simplex B virus, and vaccinia produced c~~topat7;~ic effects in the first passage only. The :susceptibility to certain viruses may vary v~ith the cu:Lture medium employed. Not susceptible to poliovirus type 1, Coxsackie virus type B-5 and polyoma virus.
Tumorigenici.ty: An innoculum of 1 x lOG cells per mouse was injected subcuntaneously into nude mice.
In non-irradiated mice, 0/25 tumors were produced.
In mice x-irradiated (425 r, whole body) 11/18 tumors (sarcomas) were produced at the site of injection.
Reverse Trar~scripi_ase: Positive.
Submitted, Prepared and Characterized By: American Type Culture CollE~ction, Rockville, tZaryland.
2. Vero Cells.
The Vero cell line was initiated from the kidney of a normal, adult, African green monkey on "March 27, 1'62, by Y. Yasumuar and Y. Kawakita at the Chiba University in Chiba, Japan (Nippon Rinsho, Vol.
21, p. 1209, 1963).
Ldumber of Serial Subcultures from Tissue of Origin:
Freeze IMedium: f9:inimum essential medium (Eagle) with non-essenti~il amino acids and Earle's BSS, 85%; fetal bovine serum, 5'a dimethyl sulfoxide (DPMSO), 10%;
antibiotic-free.
Viability: Appro:Kimately 97% (dye exclusion).
Culture Medium: DPiEM, 5% FBS
Growth Characteristics of Thawed Cells: An innoculum of 3 x 10'~ via.ble cells in 3 ml of the above culture me~Lium per T-25 flask, yields a 30-fold increase wii~hin i' days at 37°C, provided the medium is renewed i:hree times weekly and the pH is adjusted to 7.4 with a humidified mixture of 50 or loo carbon dioxide in air. Subcultures are prepared by trypsinization.
Plating Efficiency: Approximately 24% in above cul-ture medium.
Morphology: Epithelial Fibroblast-like.
Karyology: Chromosome Frequency Distribution 50 Cells: 2n = 60.
Cells: 2 1 2 2 3 4 5 7 2 1 17 2 2 Chromosomes: 4'7-49 59 51 52 53 54 55 56 57 58 59 60 Sterility: Tests for mycoplasma, bacteria and fungi were negative.
Species: C~~nfirmed as monkey by immunofluorescence test.
Virus SusceF>tibil:ity: Susceptible to poliovirus type 3, Getah, NPTdumu, Pixuna, Ross River, Semliki, 1 3 40 93 4 ~, Paramaribo, Kokobera, Modoc, Murutucu, Germiston, Guaroa, Pongola and Tacaribe Arboviruses. Not susceptible to Stratford, Apeu, Caraparu, 2~adrid, Nepuyo and 0ssa Arboviruses.
05 Reverse Transcriptase: I7ot detected.
Submitted, Prepaz-ed and Characterized By: American Type Culture Collection, Rockville, Maryland.
3. CHO~-KI Cells.
The CHO-KI cells were derived as a subclone from the parental CHO cell line initiated from a bior~sy of an ovary of an adult Chinese hamster by T.T. Puck in 1957 (J. Exp. Med., Vol. 108, p. 945, 1958). The CHO~-KI cells have a requirement for proline and a modal chromosome number oz 20 (Proc.
T7at. Acad. Sci., Vol. 60, p. 1275, 1968). The cells apparently lack the active gene form needed for proline synthesis and the block in the biosynthetic chain lies in the step converting glutamic acid to glutamic gamma-se:mialdehyde. The reversion frequency ~.o proline independence is approximately 10 0 (Genetics, Vol. 55, p. 513, 1967).
r7umber of Serial Subcultures from Tissue of Origin:
Approximately 400;~ 7 at ATCC.
Freeze Medium: Culture medium, 92$; glycerol, 8%;
antibiotic-free.
Viability: Approximately 90°s (dye exclusion).
Culture Medium: F-12 Medium (Ham) 90%; fetal bovine serum, 100; antibiotic-free.
Growth Characteri:~tics of Thataed Cells: An innoculum of_ 105 viakle cells/ml in the above culture medium at 37°C increases 15-20 fold within 7 days.
Plating Efficiency: Approximately 90~; in above cul-ture medium.
iiorphology: Epithelial-like.
Karvology: Chrc>mosome Frequency Distribution 50 Cells : 2n =- 22 .
Cells: 2 2 35 3 4 1 1 1 1 05 Chromosomes: 13 19 20 21 22-24-32-39-42 Sterility: Tests for mycoplasma, bacteria and fungi were negative.
Species: Confirmed as Chinese hamster by cytotoxic-antibody dye-exclusion test and isoenzyme analysis.
Virus Susceptibility Susceptible to vesicular stomatitis (Indiana Strain) and Getah arboviruses.
lJot susceptible to poliovirus type 2, Ilodoc and Button Vlilli.am arboviruses.
F'.everse Transcriptase: Not detected.
Special Characteristics: The reference cells require proline for growth.
Submitted B~~: T.T. Puck, Eleanor Roosevelt Institute for Cancer Research, University of Colorado Medical Center, Denver, Colorado.
4. rdIH/3T3 Cells.
They TTIH,/3T3, a continuous cell line of highly contact-inhibited cells was established from NIH Swiss mouse embryo cultures in the same manner as the origina:L random bred 3T3 (ATCC CCL 92) and the inbred BALB/c3T3 (ATCC CCL 163). The established NIH/3T3 line was subjected to more than 5 serial cycles of subcloning in order to develop a subclone with morphologic characteristics best suited for transformation assays. The earliest available passages of this subclone are around 120 beyond the primary embryo culture. The NIH/3T3 is highly sensitive to sarcoma virus focus formation and leukemia virus propagation and has proven to be very useful in DNA transfection studies. J. Virol., Vol.
4, pp. 549-553, 1969; Cell, Vol. 16, pp. 63-75 and 05 347-356, 19 79.
B . MEI>IA, B1:JFFEP,S AND SOLUTIONS .
1. For Mammalian Cell Culture.
a. Dulbecco's t~Zodified Eagle Mledium, High Glucose (DMEM).
GIBCO dry powdered medium is prepared ancL supp:Lemented with:
26 mM NaHC03 2 mM L-glutamine (Gibco) 1 mM Na-pyruvate (Gibco) 60 ~g/ml gentamycine or 100 units/ml penicillin +
100 ~ug/ml 1 streptomycin b. Ham's F12.
'0 GIBCO dry powdered medium is prepared and supp7~~emented v~ith:
14 mM NaHC03 60 lag/ml gentamycin c. Cell Freezing Medium.
90% (v/v) FBS-DMEM
10% (v/v) Dimethylsulfoxide Freeze medium is prepared just before use. All media are filter sterilized through 0.45 and 0.2 micron filters (Gilman). Sterility of media is assessed by incubating an aliquot of antibiotic-free medium at 37°C for 1 week. Media used in static culture is supplemented with 10% FBS. Fetal bovine 1 3 40 93 4 ' serum (FBS) is heat-inactivated at 56°C for 30 minutes. Sterile medium is stored at 40°C prior to use.
d. Trypsin Solution 05 0.5 g/1 trypsin 0.2 g/1 EDTA (tetra sodium) in I:ank's Balanced Salt Solution Since EDTA was found to be toxic for Vero cells, EDTA--free trypsin solution is used with these cells.
e. PBS (phosphate 183 mM rdaCl buf:Eered saline, 8.6 mM Na2HP04 GIBCO) 2.2 mM ICH2P04 f. TNE. 160 mM NaCl 10 mM Tris, pH 7.5 1 mM EDTA
2. For Molecular Biology.
a. Solutions for SDS-PAGE Procedures.
Acrylamide (Stock) 300 (w/w) Acrylamide 2 r.
( BioRad) 0. 8 0 ( w/w ) N' N-Pieth-ylene-bisacrylamide ( BioRad ) 4 x buffer for 1.5 M Tris-C1, pH 8.8 separation gel 0.40 (w/v) sodium-dodecylsulfate (SDS) (Serva) 4 x buffer for 0.5 M Tris-C1, pH 6.8 spacer gel 0 . 4~ ( w/v ) SDS
3 x sample buffer 22.3% (v/v) glycerol 0.03% (w/v) Brom-phenolblue 6.7% (w/v) SDS
05 ZO ;~ electrode 0.25% M Tris, pH 8 . 2 buff=er 1.90 M glycine 1% (w/v) SDS
b. Solutions for Silver Staining Proce-duress.
Solution I 50% (v/v) methanol 10% (v/v) acetic acid Solution II 10% (v/v) methanol 5% (v/v) acetic acid Solution III 10% glutaraldehyde Solution IV 20% AgN03 in H20 (Stock) Solution V 3% (w/v/) Na2C03 0.1% (v/v) formalde-hyde c. Solutions for Nick Translation.
10 x: Reaction 500 mM Tris-C1, buffer: pH 7.2 100 mM MgSO~
1 mM DTT
Nucleotide Mix: 100 mM dGTP
100 mM dATP
100 mM dTTP
in 10 mM Tris, pH 7 . 5 d. Solutions for Hybridization.
20 x SSC 3 ~i NaCl 0.3 M Na3-citrate 50 x Denhardt's 1% (w/w) Ficoll 400*
05 solution (PL-Pharmacia) 1~ (w/w) Polyvinyl-pyrrolidone (Sigma) to (w/w) BSA
Sterile filtrated a:nd stored at -20C
Pre'.hybridization 50~ (v/v) Formamide mix 5 x Denhardt's solu-tion 5 x SSC
50 mPi Na-phosphate pH 7.0 250 ~g/ml denatured salmon sperm DNA
Hybridization 50$ Formamide 2~ m:ix 5 x SSC
20 mI~1 Na-phosphate pH 7.0 1 x Denhardt's solu-tion 100 ~g/ml denatured salmon sperm DNA
e. Buf:Eers for Restriction Enzymes. Planu-facturers sf~ecifications, or:
Low Salt Buffer Medium Salt Buffer High Salt Buffer SmaI BglII BamHI
H~~~aI PstI PvuI
XbaI EcoRI SalI
S~eI BstEII (60C) *Trade mark ~ 340934 ~
Restrict=ion Buffers Low rledium High Tr i s , pH 7 . 4 1 Omr1 6 . 6mri 6 . 6mrM
rzgCl2 10 6 . 6 6 . 6 05 salt 20 ( KC1 ) 60 ( NaC1 ) 150 ( NaCl ) -mercaptoethanol 10 6.6 6.6 f. media.
1. L-Broth: 10 g Bactotryptone 5 g yeast extract 10 g NaCl 1 liter H20 2. L-Broth agar plates: L-Broth containing 15 g/1 Difo agar.
3. L-Broth-amp agar plates:
L-Broth agar containing 20-50 ~g/ml ampicillin.
C . ELTZYriES .
The following enzymes are used:
1. Dec>xyribonuclease I (~dorthington) 2. D~1~~-Polymerase I ("Klenow-Fragment") (Boehrin<3er r-sannheim) 3. T4-DNA L:igase (Boehringer Mannheim) 4. Re~;trict:ion enzymes EcoRI, :~baI, BamHI, Bc~II, BstEII, ~I, Sal_I, ~~aI, SmaI, PvuI, PstI (Boehringer Mannheim, BRL, New England BioLabs) 5. Ly~;ozyme ( SIGr~A) 6. Pranase D. AP~1ALYTIC'.AL APdD QUANTITATIVE PROCEDURES.
1. I~~ Bind:ing Assay.
The concentration of total protein in preparation; of purified particles of this invention 1340934 :.
is determined using the Bio-Rad protein assay kit.
This method utilizes the Bradford assay, a spectrophotcmetric: means of measuring Coomassie Blue binding to protein. Ovalbumin is used as a standard.
05 2. Radioimmune Assay (RIA).
In the LdML RIA 125-I "sandwich" radioimmunoassay, beads coated with mouse antibody to hepatitis B
surface antigen I;anti-HBs) are incubated with serum or plasma and appropriate controls. Particles containing po7Lypeptides encoded by the PreSl-PreS2-S protein coding region are bound to the solid F>hase antibody. After aspiration of the unbound material and washing of the bead, human 7_25I-Anti-HEs is~ allowed to react with the antibody-antigen ~~omplex on the bead. The beads are then washed to remove unbound 1251-Anti-HBs.
The radioactivity remaining on the beads is counted in a gamma scintillation counter. Specimens giving counts per minute (cpm) greater than or equal to the cut-off value determined by multiplying the negative co;ztrol mean count rate (NCx) by a factor are considered reactive.
3. ImmunoprEacipitation.
Cells are grown to 100% confluency in a T75 flaslc. The culture medium is then replaced with 5 ml of methionine-free medium containing 400 uCi of L-[35S] mE:thionine and 400 uCi of L-[35S]
cysteine (Nl~id) for overnight incubation. The medium is concentrated 10-fold by fractional precipitation with polyethylene glycol. Concentrated protein (about 106 cpm) is incubated with 10 ~.zl of pre-immune guinea pig serum or with 1 and 10 ~1 of -69- 1 3 4 0 g 3 4 anti-HBsAg guinea pig serum in 50 ,ul of TEN (lOmM Tris, pH
7.4, 1mM EDTA, 1..0 mM NaCl) - 0.5o Tween 20* for 1 hour at 37°C. The immunoglobulin is bound to 20 ~.sl of protein A-Sepharose* Cl-9:B (Pharmacia) for 1 hour at 37°C, washed once with TEN-Twe~en 20 and 500 mM LiCl and again with TEN-Tween 20*. Samples are electrophoresed in the SDS-PAGE
system described below.. Gels are fixed for 60 minutes in a solution of 10% tricholoro-acetic acid, 10% glacial acetic acid, and 30o methanol and incubated in Enlightning (NEN) for 30 minutes. Ciels are dried and autoradiographed with KODAK XAR-5* film.
4. SDS-Polyacrylamide Gel Electrophoresis (PAGE).
Sample and Gel Preparation. For analysis by PAGE, aliquots of purified particles of this invention are adjusted to 10 ,ug/ml. From each sample, 10 ,ul are taken and mixed with 10 ,ul 1M DTT in 10% SDS and 10 ,ul sample buffer. This mixture is boiled 5, 10 or 15 minutes just before loading in order to break the 20 nm particles and to generate monomeric molecules. After adding 10,u1 of loading buffer, the mixture is electrophoresed in a 0.75 mm thick slab gel (12 x 18 x 0.1 cm) of 1.2.5% polyacrylamide - 0.4%
bisacrylamide, using the Laemmli (1970) buffer system at l5mA and 100 V for 6 hours. Protein bands are visualized by silver staining (see Silver Staining section below).
5. Silver Stainina~.
The PAGE gel is stained. with silver according to Merril et al. (1981). The protein is fixed in the gel with a solution of 50o methanol and 10% acetic * Trademark x - ~o -acid for 30 minutes. The gel is washed with a solution of 10~ methanol and 5o acetic acid for 30 minutes. After incubation in loo glutaraldehyde for 30 minues, the gel is washed in deionized H20 05 overnight. Following incubation in 0.1% AgN03 for 30 minutes, the gel is subjected to a short wash in H20. The atained gel is developed in 100 ml of 30 2da2C03 and 30 ~1 formaldehyde and fixed in to acetic acid. They gel is sealed in a plastic bag and photographed.
E. Genetic Engineering Procedures.
1 . Rec=ombinant DNA Procedures .
a. Restriction Endonuclease Digestion of Purified DNA.
Per manufacturer's specifications of the particular endonuclease.
b. Plasmid DLdA Isolation.
Large Scale CsCl Plasmid Preparation:
1. Grow 1 liter of plasmid-bearing ce7_ls to 0.5 OD60C in L-Broth, amplify 12--20 hours with 200 ~zg/ml chloramphenicol.
2. Centrifuge in Sorval RCSb, 8,000 rpm for 20 minutes.
3. Resuspend in 18 ml cold 250 suc=rose, 50mM Tris, pH 8Ø
4. Transfer to 250 ml Erlenmeyer flask. lKeep on ice.
5. Add 6 ml 5mg/ml lysozyme in 250mP~
Tris, pH 8.0, let stand 10-15 minutes.
6. Add 6 ml 250mM EDTA, pH 8.0 and min: gent=ly; incubate 15 minutes on ice.
7. Add 30 ml detergent mix:
0.01% Triton X-100*
60mPt EDTA, pH 8.0 5 Omit Tr i s , pH 8 . 0 05 8. Incubate 30 minutes on ice.
Most preferably, the vector of this invention does not comprise an autonomous replicating sequence (~_eplicon) capable of functioning in a eulcaryotic host cell transfected therewith. A
primary reason i:or using a non-replicating vector system in eaukaryotic host cells is that all vector systems capable of autonomous and extrachromosomal replication in a mammalian host eukaryotic cell transfected therewith comprise replicons which are derived from oncogenic viruses. It is desirable to employ vectors comprising DNA not derived from oncogenic viruses for expressing DNA sequences encoding polypept:ides of pharmaceutical importance, such as the px-oteins coded for by the PreSl-PreS2-S protein coding region.
They vector of this invention comprises the MMT-promoter. The MMT-promoter is a non-viral, strong transcriptional promoter. The coding sequence of the MMT-promoter is described by Pavlakis and Hamer, Proc. Nat.l. Acad. Sci., Vol. 11, p. 397, 1983. The MMT-promoter is regulable by heavy metal ions, such a.s zinc. and cadmium, and by steroid ~ 34O 9,3,~ v hormones, such as dexamethasone. Such regulation is well known (see, e.g.. Yagle and Palmiter, Molecular and Cellular Biol" Vol. 5, p. 291, 1985).
This invention also relates to a transfected C5 host eukary«tic cell transformed with a recombinant DNA vector «f this invention. Preferably, such host is a mamm~ilian cell, most preferably a Chinese hamster ovary (CHO) cell line, a vero cell line, an L-cell line, or a mouse or rat fibroblastic cell line. Such host may be prepared by transfecting a eukaryotic cell with a recombinant DNA vector of this invention b~~ conventional techniques, such as by the method of Graham and van der Eb, Virology. Vol. 52, p. 456, 1973, or ~~~igler, h:. , Cell, Vol. 14, p. 725, 1978.
This invention also relates to a method for preparing a particle comprising proteins coded for by the PreSl-PreS2-S protein coding region wherein at least one of such proteins corresponds to a polypeptide coded for by the entire PreSl-PreS2-S protein coding region which comprises: a) cultivating the transfected host of this invention under culture medium conditions which enable such host to ex~~ress such proteins, and b) isolating such particle. Preferably, such co-transfected host is able to secrete such proteins assembled into such particles into the medium. Preferably, heavy metal ions or steroid hormones, such as dexamethasone, are added to such culture medium to induce the MMT-promoter and thereby enhance expression of such coding region. Heavy metal ions such as cadmium or zinc are most preferred. The optimal concentration 134934 ' of heavy metal ions or steroid hormone contained in the medium can be determined by conventional techniques.
This invention also relates to the particles 05 prepared b~~ such method. If the transfected host cell of this invention secretes such particles directly into the' culture medium, such particles can then be isolated from the culture medium of the transfected host of this invention by conventional protein isolation techniques. If the transfected host cell of this invention does not secrete such particles, 1=hey are obtained from a culture lysate of such host by conventional culture lysate techniques.
Such particles are obtained in glycosylated form and are compo=>ed of proteins coded for by the PreSl-PreS2--S protein coding region, including at least one protein coded for by the entire PreSl-PreS2--S protein coding region.
Th:~s invention also relates to a vaccine comprising an immunoprotective amount of the particle of this invention. By "immunoprotective amount" is meant that quantity (preferably 1-20 ug) of the particles of this invention required to induce and mantain a lc=vel of antibody in the host sufficient to neutralize the infectious agent and prevent proliferation and subsequent disease by said infectious agent.
The particle of the subject invention contains no viral DNA components and therefore is free from undesirable side effects commonly found in naturally-derived vaccines, such as unintentional infection with the virus, allergic reactions, fevers, and the like. The vaccine of the present invention 1340934 .
comprising an immunoprotective amount of the particles c~f th:is invention can be used to improve the HBV immune response and to overcome non-respon-siveness to hepatitis B virus vaccines which do not 05 include proteins coded for by the entire PreSl-PreS2-S protein coding sequence.
A vaccine of this invention can be prepared by combining a.n immunoprotective amount of the particles of this invention in an appropriate buffer, such as phosphate buffered saline. The vaccine may additionall comprise an adjuvant, such as aluminum hydroxide a.nd the' like.
Alternat=ively, the polypeptides comprised by the particles of the present invention can be disassociated from said particles and reassociated in a lipid ve~~icle. A vaccine against hepatitis B virus can be pre;~ared using said lipid vesicles comprising the polypeptides of the present invention. The lipid vesicles in the appropriate concentration can be used as a vaccine with or without adjuvant, such as aluminum hydroxide.
Th.e particles of the vaccine of this invention can be employed with a physiologically acceptable diluant (medium), such as phosphate buffered saline. ' The vaccine of this invention, comprising an immunoprotective amount of the particles of this invention, can be prepared and used in the same general mariner as disclosed in U.S. Patent 4,118,479.
1340934 ?
Thc~ vaccine can be administered by subcu-taneous, intradermal or intramuscular injection.
V~Thile the preferred route would depend on the particular 'vaccine, it is believed that intramuscular 05 injection is generally more suitable. Frequency of administrat_~.on will vary depending upon the vaccine.
This invention also relates to a method for detecting t=he presence of antibodies to proteins coded for by the PreSl-PreS2-S protein coding region in a sample of mammalian blood sera which comprises:
a) contacting the sample with a solid substrate coated with non-labeled particles of this invention;
b) incubating and washing said contacted sample;
c) convtacting said contacted sample with labeled part=icles of this invention thereby producing a labeled cc>ntacted sample;
d) incubating and washing said labeled contacted sample; and e) determining the extent of labeled particle in the labeled contacted sample.
This invention also relates to a method for detecting the presence of proteins coded for by the PreSl-PreS2-~S protein coding region in a sample of mammalian: blood sera which comprises:
a) producing a composition containing an antibody produced by an immunogen comprised by the particles of this invention;
b) cons=acting the sample with a first portion of the composition and the immunogen which has been l~3beled, incubating and washing the first portion;
c) contacting an antigen-free control with a second portion of the composition and the immunogen 05 which has been :Labeled, incubating and washing the second portion;
d) adding the same amount of staphylococci bearing protein P, to the composition of steps b) and c) above, incubating both compositions and separating liquid from solids; and e) determining the extent of labeled immunogen in each of the resultant compositions from step d) above.
Th_Ls invention also relates to a diagnostic kit for dE~tecting the presence of antibodies to proteins coded for by the PreSl-PreS2-S protein coding region in a sample of mammalian blood sera which compr:~ses:
a) unlabeled proteins comprising the ?0 particle o:~ this invention attached to a solid support ; an~i b) labeled antibodies to human IgG or IgM, for example..
This invention also relates to a diagnostic kit for detecting the presence of proteins coded for by the PreSl-Pr~~S2-S protein coding region in a sample of mammalian blood sera which comprises:
a) antibodies, produced by the particles of this invention, attached to a solid support; and b) labeled antibodies, produced by the particles of. this invention.
1340934 "~
Thc~ present invention also includes diagnostic tests for direct detection of hepatitis B
surface antigens and antibodies, raised to such antigens. Radioimmunoassay (RIA) or enzyme-linked 05 immunosorbant a w ay (ELISA) are employed to detect HBV antigens containing proteins coded for the PreSl-PreS2--S coding regions in sera of HBV
infected animals, such as humans. One diagnostic test includes the following:
1. A solid substrate containing binding sites thereon, such as polystyrene beads, is coated with antibad.ies to particles containing amino acid sequences corresponding to those of the PreSl-PreS2--S containing polypeptides;
2. The coated beads are then washed with, for example, Tris buffered saline to remove excess antibodies;
3. The beads are then contacted with a protein-cone=aining solution, such as bovine serum albuMin (FSA) or gelatin, to saturate protein binding sites on the beads (to reduce non-specific binding).
A convenient concentration of such protein-containing solution can be employed, such as 1 mg/ml to 50 mg/ml;
4. The beads are then washed to remove excess BSA or gelatin;
5. The beads are then incubated with serum samples suspected to contain HBV or HBV surface antigens (normal serum is utilized as a control);
6. The beads are then washed with a solution, such as Tris buffered saline solution, and mixed with a radio-labeled antibody, such as 125I
labeled antibody to HBV surface antigens;
1344934 _.
7. The beads are then incubated; and 8. The beads are then washed and counted in a gamma counter.
Particles of the present invention coded for 05 by the Pre~51-PreS2-S protein coding region can be employed as a diagnostic tool to detect antibodies to the PreSl-F'reS2-S regions of HBV surface antigen proteins in a given sample. The PreSl-PreS2-S
containing particles are adsorbed on a solid substrate containing binding sites thereon, for example, polystyrene beads. The substrate is thereafter contacted with a substance, for example, gelatin, BSA or powdered milk, to saturate the non-specific. binding sites thereon. Thereafter, the substrate i.s wa:~hed with a buffered solution and thereafter the buffer is removed. A specimen, such as human serum, diluted with animal serum, is added to the substrate. The resulting mass is then incubated and washed. Thereafter, radio-labeled, such as iodinated (125I), antibodies to human IgG
or IgM is added to the mass. The resulting mass is incubated then washed and counted, in the such as gamma counter. If the count is higher than the count obtained for the normal serum control, the specimen contains antibodies to the PreSl-PreS2-S coding regions of FiBV.
ThE~ above procedure for detection of antibodies to the proteins coded for by the PreSl-PreS2--S protein coding region of HBV is believed to be applicable as a diagnostic tool in detecting hepatitis B virus infection.
X340934 ,~, A 3iagnostic test kit for detecting antigens coded for the PreSl-PreS2-S protein coding regions of the HBV genome in a test sample would include the following:
05 1. A solid substrate coated with antibodies to a particles having amino acid sequences corresponding to the PreSl-PreS2-S regions of HBV
surface antigen particles of the present invention;
2. A protein-containing solution to saturate protein binding sites on the solid substrate;
and 3. A given amount of radio-labeled antibody, such as antibody to the HBV surface antigen polr~pept ide;~ .
Another diagnostic test kit for detecting antibodies to the proteins coded for by the PreSl-I'reS2~-S protein coding region of the hepatitis B virus genome in the test sample includes the following:
1. A solid substrate having adsorbed thereon particles containing amino acid sequences corresponding to the PreSl-PreS2-S coding regions of the HBV surface antigen proteins of the present invention, the substrate being exposed to a protein-containing solution to saturate non-specific protein binding sites on a solid substrate; and 2. A given amount of radio-labeled anti-bodies to human IgG or IgM.
Radio-labeled antibodies used in the above described test kits can be packaged in solution form or in lyop~zilized form suitable for reconstitution.
In addition, enzyme-lin};ed or fluorescent-labeled 1340934 ' antibodies can be substituted for the described radio-labeled antibodies.
The above-described test kit and process for detecting antibodies to the PreSl-PreS2-S coding 05 region pol:~peptides of hepatitis B virus can be utilized in ap~~lications such as detecting HEV
infection :Ln a patient by taking serum from the patient and applying the above-described test or using the above-described test; and predicting recovery from HBV infection by taking serum from an infected patient and applying the described antibody detection procedures.
ThE~ test procedures and test kits for antibody detection can be used for making qualitative comparisons between different HBV vaccines by taking serum from 'vaccinated patients and then utilizing the above-described test procedures or kits for antibody detection. In general, all known immunoassays using this antigen as reagent can be performed using the PreSl, Pre;S2, or S-containing particles of this invention. Generally, all known immunoassays using antibody-containing serum or reagents can be performed using antibody serum produced through the use of a peptide produced by recombinant DNA
techniques of the present invention. These immunoassays include all those disclosed by Langone and Van Vunakis in Methods of Enzymology, Academic Press, Vols. 70, 73 and 74 and those assays disclosed in disclosures of the following U.S. Patents:
4,459,359; 4,.'343,896; 4,331,761; 4,292,403;
4,228,240; ~I,157,280; 4,152,411; 4,169,012; 4,016,043;
3,839,153; :3,654 ,090; and Re. 31,006 and Vols. 70, 73 and 74 of Methods of Enzymology.
This invention also relates to a co-trans-fected host: eukaryotic cell co-transfected with the 05 recombinant vector of this invention and a second recombinant DNA vector comprising the PreS2-S
protein coding region, or only the S protein coding region, anc an MII~1T-promoter. Preferably, the second recombinant vector additionally comprises a transcription termination site and, optionally, a selection marker. Optionally, the co-transfected host is co-transfected with the recombinant DNA
vector of this invention, the second recombinant DNA
vector and a third recombinant DNA vector comprising an MMT-promoter and a selection marker. Preferably, the third recombinant vector additionally comprises a transcription termination site. Preferably, the second and optional third recombinant DNA vector also contain a replicon for growth and amplification in a procaryotic host cell, such as a bacterial host cell, when such host is transformed with said second or optional third recombinant DNA vector.
Additionally, this invention relates to a method of preparing such co-transfected host cell which comprises co-transfecting a eukaryotic cell with the recombinant DNA vector of this invention, the second recombinant DNA vector described above, and, optio;~ally, the third recombinant DNA vector described above.. The second and optional third recombinant DNA vectors described above can be constructed by conventional techniques. If the optional third vector is not employed, the second recombinant DNA. vector preferably additionally comprises a selection marker, such as a drug resistance marker. Preferably, if the second and/or optional third recombinant DNA vector comprise a 05 transcriptiot: termination site, such site is the mg-PAS-t termination site. Preferably, in the second recombinant DNF, vector described above, the MMT-promoter is located in such vector immediately upstream of the PreS2-S protein coding region.
Preferably, in the optional third recombinant DNA
vector described above, the PMMT-promoter is located in such vector immediately upstream of the selection marker. Preferably, neither the second recombinant vector nor the optional third recombinant vector described above contain any non-HBV viral DLdA
segments and are non-oncogenic. Thus, upon transfection into a host, such preferred vectors integrate into the host chromosome and are replicated passively with the host genome.
the vector of this invention, the second recombinant DNA vector described above, and, optionally, the third recombinant DNA vector described above .are co-transfected into a eukaryotic host in pairwise combinations, or all three vectors together, using conventional methods. Alternatively, the vector of this invention, the second recombinant DNA vector and, optionally, the third recombinant DNA
vector are transfected into a eukaryotic host in a series of steps. Initially, the vector of this invention is transfected into a eukaryotic host according to the method of this invention to produce the transfected host of this invention.
1340g34.'~
Subsequently, ouch transfected host is then transfected with the second recombinant DNA vector described above, and, optionally, the third recombinant DNA vector described above according to 05 conventional r.~ethods to produce the co-transfected host of this invention.
Prefera~:>ly, the vector of this invention, the second recombinant DNA vector and the optional third recc>mbinant DNA vector comprise selection markers different from each other to enable identification of the newly-transfected host at each subsequent transfection step leading to the final transfected host of the invention. Such secondary transfection steps are carried out in order to increase t:he gene copy number of the PreSl-PreS2-S protein coding region comprised by the multiply transfected host cell. Preferably, neither the second recombinant vector nor the optional third recombinant vector comprise a replicon capable of functioning in a eukaryotic cell.
This invention also relates to a method for preparing a. particle, produced by the co-transfected host of this invention, comprising at least one protein coded for by the entire PreSl-PreS2-S
protein coding region which comprises: a) cultivating the co-transfected host eukaryotic cell of this invention under culture medium conditions which enable such host to express such proteins; and b) isolating ouch particle. Preferably, such co-transfected host is able to secrete such proteins assembled into such particles into the medium.
Preferably, such method additionally comprises the ~ 340934 ' addition of heav~~ metal ions or steroid hormones to such culture medi~.um to enhance the expression of the PreSl-PreS2-S protein coding region contained by the co-tr~~nsformed vectors. Heavy metal ions, 05 particularly zinc or cadmium, are most preferred.
The optima7_ concentration of heavy metal ions or steroid hormones contained in the medium can be determined by conventional techniques. This invention also relates to the particles prepared by such method. If the co-transfected host of this invention secretes such particles directly into the culture medium, such particles can then be isolated from the culture medium of the co-transformed host of this invention by conventional protein isolation techniques. If the co-transfected host does not secrete such particles into the culture medium, then they are obtained from a culture lysate of such co-transfected host by conventional culture lysate techniques. Such particles are isolated in glycosylated form and are composed of proteins coded for by the Pre S1-PreS2-S protein coding region, the PreS2-S protein coding region, and the S
protein coding region.
This invention also relates to a vaccine comprising an :immunoprotective amount (Preferably 1-20 ug) of the particles produced by the co-transfected host of this invention.
Th.= particle produced by the co-transfected host of the subject invention contains no viral DNA
components and therefore is free from undesirable side effects commonly found in naturally-derived vaccines, such as unintentional infection with the virus, allergic reactions, fevers, and the like. The vaccine of the present invention comprising an immunoprotective amount of the particles produced by the co-tranafected host of this invention can be used 05 to improve the F-iBV immune response and to overcome non-responsiveness to hepatitis B virus vaccines which do nc~t include at least one protein coded for by the entire PreSl-PreS2-S protein coding sequence.
A 'vaccine of this invention can be prepared by combining an immunoprotective amount of the particles produced by the co-transfected host of this invention in an appropriate buffer, such as phosphate buffered saline.. The vaccine may additionally comprise an adjuvant, such as aluminum hydroxide and the like.
Alternatively, the polypeptides comprised by the particles produced by the co-transfected host of the present invention can be disassociated from said particles and reassociated in a lipid vesicle. A
hepatitis B vaccine can be prepared using said lipid vesicles comprising the polypeptides of said particles of the present invention. The vaccine comprising the lipid vesicles may additionally comprise an adjuv~ant, such as aluminum hydroxide.
The particles produced by the co-transfected host of the invention comprising the vaccine of this invention can b~e employed with a physiologically acceptable diluant (medium), such as phosphate buffered sa~_ine.
ThE~ vaccine of this invention comprising an immunoprotective .amount of the particles of this invention can be prepared and used in the same general manner as disclosed in U.S. Patent 4,118,479.
Lhc~ vaccine can be administered by subcu-taneous, intradermal or intramuscular injection.
05 While the preferred route would depend on the particular 'vaccine, it is believed that intramuscular injection is generally more suitable. Frequency of administration will vary depending upon the vaccine.
This invention also relates to a method for detecting v:.he presence of antibodies to proteins coded for by the PreSl-PreS2-S protein coding region in a sample of mammalian blood sera which comprises:
a) contacting the sample with a solid substrate coated with non-labeled particles produced by the co-transfected host of this invention;
b) incubating and washing said contacted sample;
c) contacting said contacted sample with labeled particles of this invention thereby producing a labeled contacted sample;
d) incubating and washing said labeled contacted sample; and e) determining the extent of labeled particle in the labeled contacted sample.
This invention also relates to a method for detecting the presence of proteins coded for by the PreSl-PreS2--S protein coding region in a sample of mammalian blood sera which comprises:
a) producing a composition containing an antibody produced by an immunogen comprised by the particles of this invention;
b) contacting the sample with a first portion of the composition and the immunogen which has been labeled, incubating and washing the first portion;
05 c) contacting an antigen-free control with a second portion of the composition and the immunogen vahich has been labeled, incubating and washing the second portion;
d) adding the same amount of staphylococci bearing protein A to tt~~ composition of steps b) and c) above, incubating both compositions and separating liquid from solids; and e) determining the extent of labeled immunogen in each of the resultant compositions from step d) above.
This invention also relates to a diagnostic kit for d.=_tecting the presence of antibodies to proteins coded for by the PreSl-PreS2-S protein coding region in a sample of mammalian blood sera which comprises:
a) unlabeled proteins comprising the particle of this invention attached to a solid support; an~3 b) labeled antibodies to human IgG or IgM, for example.
This invention also relates to a diagnostic kit for detecting the presence of proteins coded for by the Pr.=_Sl-PreS2-S protein coding region in a sample of mammalian blood sera which comprises:
a) antibodies, produced by the particles of this invention, attached to a solid support; and b) labeled antibodies, produced by the particles of this invention.
Th:is invention relates to a recombinant DNA
concatamer comprising: a) a plasmid vector carrying the PreSl-PreS2-S protein coding region and at least one additional plasmid vector comprising one of 05 the following coding regions: PreSl-PreS2-S
protein coding region or PreS2-S protein coding region or S protein coding region; and b) the MMT
promoter. l3uch concatamer preferably additionally comprises a selection marker and a transcription termination site.
This invention also relates to a eukaryotic host cell transfected with the concatamer of this invention. Such host is preferably a mammalian cell. Additionally, the invention relates to a method of preparing such transfected host cell with the concatamer ~of this invention which comprises transfectinc3 a eukaryotic cell with the concatamer of this invention.
This invention also relates to a method for preparing a particle, produced by the transfected host of this invention, comprising at least one protein coded for by the entire PreSl-PreS2-S
protein ceding region, which comprises: a) cultivating the transfected eukaryotic host cell of this invent:ion, which comprises the concatamer of this invention, under culture medium conditions which enable sucl-, host to express such proteins; and b) isolating such particle. Preferably, such co-transfec~ted host is able to secrete such proteins assembled into such particles into the medium.
Preferably, such method additionally comprises the addition of heav~~ metal ions or steroid hormones to such culture medium to enhance the expression of the 134Q934 .
PreSl-PreS2-S protein coding region contained by the transfected vectors. Heavy metal ions, particularly zinc or cadmium, are most preferred.
The optima:L cone entration of heavy metal ions or 05 steroid hormones contained in the medium can be determined by conventional techniques. This invention also relates to the particles prepared by such method. If the host transfected with the concatamer of this invention secretes such particles directly into the culture medium, such particles can then be i~colated from the culture medium of the transfected host of this invention by conventional protein isolation techniques. If the transfected host does not secrete such particles into the culture medium, then they are obtained from a culture lysate of such transfected host by conventional culture lysate tecr~nique:~. Such particles are isolated in glycosylated forms and are composed of proteins coded for by the PreSl-PreS2-S protein coding region, the PreS2-S protein coding region, and the S
protein coding region.
This invention also relates to a vaccine comprising an i.mmunoprotective amount (preferably 1-20 ug) of the particles produced by the host transfected with the concatamer of this invention.
Thc~ particle produced by the host transfected with the concata.mer host of the subject invention contains no viral DNA components and therefore is free from undesirable side effects commonly found in naturally-dE~rived vaccines, such as unintentional infection with the virus, allergic reactions, fevers, and the like. The vaccine of the present invention ~34~934.
comprising an immunoprotective amount of the particles produced by the host transfected with the concatamer of this invention can be used to improve the HBV
immune response and to overcome non-responsiveness to 05 hepatitis F3 virL~ vaccines 4rhich do not include at least one protein coded for by the entire PreSl-PreS2-S protein coding sequence.
A vaccine of this invention can be prepared by combining an immunoprotective amount of the particles ~~roduced by the host transfected with the concatamer of 'this invention in an appropriate buffer, such a;~ phosphate buffered saline. The vaccine may additionally comprise an adjuvant, such as aluminum hydroxide and the like.
Alternatively, the polypeptides comprised by the particles produced by the host transfected with the concatamer of the present invention can be disassociated from said particles and reassociated in a lipid vesicle. A hepatitis B vaccine can be prepared using aaid lipid vesicles comprising the polypeptide;~ of said particles of the present invention. The vaccine comprising the lipid vesicles may additionally comprise an adjuvant, such as aluminum hydroxide.
Th~~ particles produced by the host trans-fected with the concatamer of this invention comprising the vaccine of this invention can be employed with a physiologically acceptable diluant (medium), such as phosphate buffered saline.
Th.=_ vaccine of this invention comprising an immunoprotec~tive amount of the particles of this invention can be prepared and used in the same general manner as disclosed in U.S. Patent 4,118,479.
The vaccine can be administered by subcu-taneous, intradermal or intramuscular injection.
05 ~,Thile the preferred route would depend on the particular vaccine, it is believed that intramuscular injection is generally more suitable. Frequency of administration will vary depending upon the vaccine.
This invention also relates to a method for detecting the presence of antibodies to proteins coded for by the PreSl-PreS2-S protein coding region in a sample of mammalian blood sera which comprises:
a) contacting the sample with a solid substrate coated with non-labeled particles produced by the hosi_ transfected with the concatamer of this invention;
b) incubating and washing said contacted sample;
c) contacting said contacted sample with labeled particle; of this invention thereby producing a labeled contacted sample;
d) incubating and washing said labeled contacted sample; and e) determining the extent of labeled particle in the labeled contacted sample.
This invention also relates to a method for detecting the presence of proteins coded for by the Pre~l-PreS2-S protein coding region in a sample of mammalian blood sera which comprises:
a) producing a composition containing an antibody produced by an immunogen comprised by the particles of this. invention;
~ ~40g34 b) contacting the sample with a first portion of the composition and the immunogen which has been labeled, incubating and washing the first portion;
05 c) contacting an antigen-free control with a second portion of the composition and the immunogen which has been labeled, incubating and washing the second portion;
d) adding the same amount of staphylococci bearing protein A to the composition of steps b) and c) above, incubal=ing both compositions and separating liquid from solids; and e) determining the extent of labeled immunogen in each of the resultant compositions from step d) above.
This invention also relates to a diagnostic kit for detecting the presence of antibodies to proteins coded for by the PreSl-PreS2-S protein coding reg_Lon in a sample of mammalian blood sera 2C which comprises:
a) unlabeled proteins comprising the particle of this invention attached to a solid support; and b) labeled antibodies to human IgG or IgM, for example.
This invention also relates to a diagnostic kit for detecting the presence of proteins coded for by the PreSl-PreS2-S protein coding region in a sample of mammalian blood sera which comprises:
a) antibodies, induced by the particles of this invention, attached to a solid support; and 1 34Q g~4 b) labeled antibodies, produced by the particles o~- this invention.
First Preferred Embodiment 05 In a first preferred embodiment, one vector of this invention includes a vector construction incorporating gene sequences from recombinant plasmids pBPV342-12 (Law et al, PMolecular and Cellular Biol, 'Vol, 3, p. 2110, 1983) and pAOI
(Cummings, :..yV. et al, Proc. Natl. Aca. Sci., U.S.A., Vol. 77, p. 1842, 1980). Some of the genetic elements from these plasmi.ds are combined to create a vector of this invention, designated pDMl (see Figure 3).
Flasmid pDMl., which contains the gene segment carrying the PreSl-FreS2-S protein coding region under the control of the natural promoter system for said protein cod~:ng region, the neomycine-resistance gene, the 1~IMT promoter and an SV40 PAS-t function, is then introduced by tra,nsfection into any one of a number of eukaryotic cells, such as mammalian cells, to produce high-level expression and, preferably, secretion of the particles of this invention.
Pla.smid pBPV342-12 (see Figure 1) contains gene sequences including the MI,4T-promoter, the neomycine-resistance gene, an SV40 PAS-t function and the bovine ;?apilloma virus (BPV) genome. During the construction. of plasmid pDLMl, plasmid pBPV342-12 is subjected to BamHI digestion, with the result that the plasmi<i is split into two DNA fragments.
Following ~cepara~tion of the fragments, the 7.95 kilobase (kb) fragment, which comprises the entire BPV genome, is discarded. The elimination of BPV DNA
1340934 ~
is highly <3dvantageous since it precludes inclusion of any BPV DNA or proteins in the particle of this invention which is to be used for vaccine formulation,. The remaining BamHI fragment (6.65 kb) 05 from pBPV342-12, which contains the gene sequences for the MME:'-promoter, the neomycine resistance gene and the SV40 P.AS-t region, is then ligated, by conventiona7_ techniques, to a DNA sequence derived from plasmid pAOI, but which contains an intact PreSl-PreS2--S protein coding region (discussed below; see also Figure 2).
Plasmid pAOI (see Figure 2), which contains the entire hepatitis B viral genome, is subjected to EcoRI digestion to produce a 3.2 kb fragment. The 3.2 ?;b fragment is ligated to itself to produce tandem repeats comprising a long concatemeric DLdA
structure containing gene sequences encoding both the core arid surface antigens of hepatitis B virus. The method for t=his manipulation of the hepatitis B viral gene sequen~~es, which is necessary to overcome the permutation caused by molecular cloning in plasmid pAOI and to restore a functional organization with respect to the PreSl-PreS2-S protein coding region, is that of Cummings et al in Proc. Natl.
Acad. Sci. USA, Vol. 77, p. 1842, 1980. Through subsequent E3c~lII digestion, the HBV core antigen gene segment is eliminated, leaving a DPJA fragment (2.78 kb) carrying the PreSl-PreS2-S protein coding region of t)ze hef>atitis B virus genome together with the natural promot=er system (PHB) of such region.
The linear pBPV342-12 BamHI fragment (see Figure 1) and the pAOI-derived III fragment (see Figure 2) are then ligated to produce the recombinant plasmid, pl~Ml (see Figure 3), which retains the natural promoter (PHB) for the PreSl-PreS2-S
protein coding region.
Second Preferred Embodiment In a second preferred embodiment of this invention, 'the natural promoter in pDMl is excised by a BglII and BamHI digestion such that the P~ulT-promoter 10 is positioned directly ahead of the PreS2-S protein coding region to control transcription of said coding region by the P4P~'.C-promoter. Following BamFiI + Bc~.lII
digestion three discrete DNA fragments are obtained (see Figures 4A a:nd 4B):
15 1) a _'~.1 kb fragment containing a polya-denylation site a:nd the MMT-promoter;
2) a 3.0 kb fragment containing the neomycine resistance gene and the natural promoter;
and 20 3) a 1.4 kb fragment containing the PreS2-S protein coding region.
Fo7_lowing purification, the 5.1 and the 1.4 kb fragments are ligated using conventional techniques to produce recombinant plasmids (6.46 kb) 25 containing the F>reS2-S protein coding region, now under the control of the P~IMT-promoter. Because the relative orientations of the two ligated fragments are random and two different plasmid types are produced, only one of which exhibits the correct 30 transcriptional reading orientation from the MMT-promoter into the surface antigen gene sequences, the correct functional plasmid, pDM2, is ascertained 1340934' by EcoRI + XbaI digestion of purified plasmid candidates. The plasmid with the correct orientation yields a 2.1 and a 4.4 kb fragment following this digestion.
05 A bacterial strain, HE101, containing pDM2, was deposited at Deutsche Sammlung fur Mikro-organismen, Gotti.ngen, under the depository number DSf9 3285 en April 4, ).985.
Third Preferred Embodiment In a third preferred embodiment, the rstsT-promoter is spliced to a HBV gene segment containing the PreSl-PreS2-S protein coding region in :such a way as to eliminate the natural promoter system and place said coding sequence under the direct contro:L of the MMT-promoter (see Figure 9).
Fourth Preferred Embodiment In a fourth preferred embodiment, the mouse globin gene mg-PF,S-t segment, recovered from plasmid pBR322. G2, is substituted as a polyadenylation termination (PAS-t) signal for the SV40 PAS-t region. The substitution of mg-PAS-t provides DNA
transfer vectors without any non-HPV viral genomic portions (sere Figures 9, 10 and 11).
Fifth Preferred Embodiment A ~_ifth preferred embodiment is a method of preparing pl.asmid DNA mixed concatamers, amplifying 1344934 ' the gene copy number' by ligation of high ratios of plasmids containing gene constructs encoding the PreSl-PreS2-S, PreS2-S or S protein coding regions to plasmids containing the drug-resistance marker, for subsequent transfection into cell lines.
Plasmids pBR.322. G2, POLINK 23456, pBlg/2.8 and pMMT-neo plasmid were deposited at the American Type Culture Collection, Rockville, Maryland, under the accession numbers 67073, 67072, 67075 and 67074, :LO respectively on 11 April 1986.
In i:he following Examples, the preparation of the recombinant DNA vectors, hosts and particles of this invention, as well as the incorporation into mammalian ce:Ll lines is described in more detail.
The following examples are for illustrative purposes only and are not :intended to limit the invention in any way.
EXAMPLES
MATERIALS AND PROCEDURES
A. ORIGIN ArfD DESCRIPTION OF MAMtZALIAN CELL
LINES.
1. L-Cells.
NCTC clonee 929 was derived in March 1948 by K.K. Sanford,, W.R. Earle and G.D. Likely (J. Nat.
Cancer Inst., Vo7_. 9, pp. 229, 1948) from the parental strain L established in 1940 by W.R. Earle (J. Nat. Cans=er Inst., Vol. 4, pp. 165, 1943). The parent strain L was derived from normal subcutaneous areolar and adipose tissue of a 100 day-old male C3H/An mouse, and clone 929 was established (by the capil7_ary technique for single cell isolation) from the 95th suhcul_ture generation of the parent strain.
LM(TK ), a sub-line of mouse L cells, was 05 isolated by D. R. Dubbs and S. Kit (S. Kit et al, Exp.
Cell Res. , Vol. 31, pp. 297-312, 1963; and D.R. Dubbs & S. Kit, E;xp. Cell P,es., Vol. 33, p. 19, 1964), and is deficier,.t in thymidine kinase. It is unable to grow in medium containing HAT. No spontaneous reversion of HAT-resistance has been observed in this cell line, and it seems most likely that a deletion involving t::~is gene has occurred.
2Tumber of ;serial Subcultures from Tissue of Origin:
64~; clone, 553.
Freeze Medium: Culture medium, 90s; DtZSO 100; anti-biotic-free.
Viability: 81-960 (dye exclusion).
Culture Me3ium: DL~4ELM + loo FBS (Dulbecco's and Vogt's Modified E;agle's Medium + 10°s FBS).
2O Growth Characteristics of Thawed Cells: An innoculum of 6-8 x 10 Lls in 5 ml of above culture medium per T-25 f:Lask, yields a 500-fold increase within 7 days at 37°C, provided the medium is renewed two times week7_y and. the pH is adjusted to 7.3 with a humidified mixture of 5°s or 10°s carbon dioxide in air. Subcultures are prepared by scraping or shaking. Cells also grow well on other media (Waymouth's, Eac)le's, NCTC135, etc.) supplemented with l00-30o hor:~e serum.
Plating Efficiency: 700 Morphology: Fibz~obl_ast-like.
1340934'.
Karyology: Chromosome Frequency Distribution 100 Cells : 2n ~- 40 .
Cells: 2 2 1 4 2 9 4 12 16 17 4 13 2 Chromosomes: 5G-58 59 60 61 62 63 64 65 66 67 68 69 Cells: 2 2 1 1 6 Chromosomes: 70 71-76-82-125-241 Long metacentric chromosome with secondary constriction noted in 77/100 cells.
Sterility: Tests. for mycoplasma, bacteria and fungi were negative.
Species: Confirmed as mouse by mixed agglutination and hemagglutination tests.
Virus SL1SCE'_l~tibility: Susceptible to pseudorabies virus and vesicular stomatitis (Indian Strain) virus. t~d'hen the cells were cultured in the above culture medium, Herpes simplex B virus, and vaccinia produced c~~topat7;~ic effects in the first passage only. The :susceptibility to certain viruses may vary v~ith the cu:Lture medium employed. Not susceptible to poliovirus type 1, Coxsackie virus type B-5 and polyoma virus.
Tumorigenici.ty: An innoculum of 1 x lOG cells per mouse was injected subcuntaneously into nude mice.
In non-irradiated mice, 0/25 tumors were produced.
In mice x-irradiated (425 r, whole body) 11/18 tumors (sarcomas) were produced at the site of injection.
Reverse Trar~scripi_ase: Positive.
Submitted, Prepared and Characterized By: American Type Culture CollE~ction, Rockville, tZaryland.
2. Vero Cells.
The Vero cell line was initiated from the kidney of a normal, adult, African green monkey on "March 27, 1'62, by Y. Yasumuar and Y. Kawakita at the Chiba University in Chiba, Japan (Nippon Rinsho, Vol.
21, p. 1209, 1963).
Ldumber of Serial Subcultures from Tissue of Origin:
Freeze IMedium: f9:inimum essential medium (Eagle) with non-essenti~il amino acids and Earle's BSS, 85%; fetal bovine serum, 5'a dimethyl sulfoxide (DPMSO), 10%;
antibiotic-free.
Viability: Appro:Kimately 97% (dye exclusion).
Culture Medium: DPiEM, 5% FBS
Growth Characteristics of Thawed Cells: An innoculum of 3 x 10'~ via.ble cells in 3 ml of the above culture me~Lium per T-25 flask, yields a 30-fold increase wii~hin i' days at 37°C, provided the medium is renewed i:hree times weekly and the pH is adjusted to 7.4 with a humidified mixture of 50 or loo carbon dioxide in air. Subcultures are prepared by trypsinization.
Plating Efficiency: Approximately 24% in above cul-ture medium.
Morphology: Epithelial Fibroblast-like.
Karyology: Chromosome Frequency Distribution 50 Cells: 2n = 60.
Cells: 2 1 2 2 3 4 5 7 2 1 17 2 2 Chromosomes: 4'7-49 59 51 52 53 54 55 56 57 58 59 60 Sterility: Tests for mycoplasma, bacteria and fungi were negative.
Species: C~~nfirmed as monkey by immunofluorescence test.
Virus SusceF>tibil:ity: Susceptible to poliovirus type 3, Getah, NPTdumu, Pixuna, Ross River, Semliki, 1 3 40 93 4 ~, Paramaribo, Kokobera, Modoc, Murutucu, Germiston, Guaroa, Pongola and Tacaribe Arboviruses. Not susceptible to Stratford, Apeu, Caraparu, 2~adrid, Nepuyo and 0ssa Arboviruses.
05 Reverse Transcriptase: I7ot detected.
Submitted, Prepaz-ed and Characterized By: American Type Culture Collection, Rockville, Maryland.
3. CHO~-KI Cells.
The CHO-KI cells were derived as a subclone from the parental CHO cell line initiated from a bior~sy of an ovary of an adult Chinese hamster by T.T. Puck in 1957 (J. Exp. Med., Vol. 108, p. 945, 1958). The CHO~-KI cells have a requirement for proline and a modal chromosome number oz 20 (Proc.
T7at. Acad. Sci., Vol. 60, p. 1275, 1968). The cells apparently lack the active gene form needed for proline synthesis and the block in the biosynthetic chain lies in the step converting glutamic acid to glutamic gamma-se:mialdehyde. The reversion frequency ~.o proline independence is approximately 10 0 (Genetics, Vol. 55, p. 513, 1967).
r7umber of Serial Subcultures from Tissue of Origin:
Approximately 400;~ 7 at ATCC.
Freeze Medium: Culture medium, 92$; glycerol, 8%;
antibiotic-free.
Viability: Approximately 90°s (dye exclusion).
Culture Medium: F-12 Medium (Ham) 90%; fetal bovine serum, 100; antibiotic-free.
Growth Characteri:~tics of Thataed Cells: An innoculum of_ 105 viakle cells/ml in the above culture medium at 37°C increases 15-20 fold within 7 days.
Plating Efficiency: Approximately 90~; in above cul-ture medium.
iiorphology: Epithelial-like.
Karvology: Chrc>mosome Frequency Distribution 50 Cells : 2n =- 22 .
Cells: 2 2 35 3 4 1 1 1 1 05 Chromosomes: 13 19 20 21 22-24-32-39-42 Sterility: Tests for mycoplasma, bacteria and fungi were negative.
Species: Confirmed as Chinese hamster by cytotoxic-antibody dye-exclusion test and isoenzyme analysis.
Virus Susceptibility Susceptible to vesicular stomatitis (Indiana Strain) and Getah arboviruses.
lJot susceptible to poliovirus type 2, Ilodoc and Button Vlilli.am arboviruses.
F'.everse Transcriptase: Not detected.
Special Characteristics: The reference cells require proline for growth.
Submitted B~~: T.T. Puck, Eleanor Roosevelt Institute for Cancer Research, University of Colorado Medical Center, Denver, Colorado.
4. rdIH/3T3 Cells.
They TTIH,/3T3, a continuous cell line of highly contact-inhibited cells was established from NIH Swiss mouse embryo cultures in the same manner as the origina:L random bred 3T3 (ATCC CCL 92) and the inbred BALB/c3T3 (ATCC CCL 163). The established NIH/3T3 line was subjected to more than 5 serial cycles of subcloning in order to develop a subclone with morphologic characteristics best suited for transformation assays. The earliest available passages of this subclone are around 120 beyond the primary embryo culture. The NIH/3T3 is highly sensitive to sarcoma virus focus formation and leukemia virus propagation and has proven to be very useful in DNA transfection studies. J. Virol., Vol.
4, pp. 549-553, 1969; Cell, Vol. 16, pp. 63-75 and 05 347-356, 19 79.
B . MEI>IA, B1:JFFEP,S AND SOLUTIONS .
1. For Mammalian Cell Culture.
a. Dulbecco's t~Zodified Eagle Mledium, High Glucose (DMEM).
GIBCO dry powdered medium is prepared ancL supp:Lemented with:
26 mM NaHC03 2 mM L-glutamine (Gibco) 1 mM Na-pyruvate (Gibco) 60 ~g/ml gentamycine or 100 units/ml penicillin +
100 ~ug/ml 1 streptomycin b. Ham's F12.
'0 GIBCO dry powdered medium is prepared and supp7~~emented v~ith:
14 mM NaHC03 60 lag/ml gentamycin c. Cell Freezing Medium.
90% (v/v) FBS-DMEM
10% (v/v) Dimethylsulfoxide Freeze medium is prepared just before use. All media are filter sterilized through 0.45 and 0.2 micron filters (Gilman). Sterility of media is assessed by incubating an aliquot of antibiotic-free medium at 37°C for 1 week. Media used in static culture is supplemented with 10% FBS. Fetal bovine 1 3 40 93 4 ' serum (FBS) is heat-inactivated at 56°C for 30 minutes. Sterile medium is stored at 40°C prior to use.
d. Trypsin Solution 05 0.5 g/1 trypsin 0.2 g/1 EDTA (tetra sodium) in I:ank's Balanced Salt Solution Since EDTA was found to be toxic for Vero cells, EDTA--free trypsin solution is used with these cells.
e. PBS (phosphate 183 mM rdaCl buf:Eered saline, 8.6 mM Na2HP04 GIBCO) 2.2 mM ICH2P04 f. TNE. 160 mM NaCl 10 mM Tris, pH 7.5 1 mM EDTA
2. For Molecular Biology.
a. Solutions for SDS-PAGE Procedures.
Acrylamide (Stock) 300 (w/w) Acrylamide 2 r.
( BioRad) 0. 8 0 ( w/w ) N' N-Pieth-ylene-bisacrylamide ( BioRad ) 4 x buffer for 1.5 M Tris-C1, pH 8.8 separation gel 0.40 (w/v) sodium-dodecylsulfate (SDS) (Serva) 4 x buffer for 0.5 M Tris-C1, pH 6.8 spacer gel 0 . 4~ ( w/v ) SDS
3 x sample buffer 22.3% (v/v) glycerol 0.03% (w/v) Brom-phenolblue 6.7% (w/v) SDS
05 ZO ;~ electrode 0.25% M Tris, pH 8 . 2 buff=er 1.90 M glycine 1% (w/v) SDS
b. Solutions for Silver Staining Proce-duress.
Solution I 50% (v/v) methanol 10% (v/v) acetic acid Solution II 10% (v/v) methanol 5% (v/v) acetic acid Solution III 10% glutaraldehyde Solution IV 20% AgN03 in H20 (Stock) Solution V 3% (w/v/) Na2C03 0.1% (v/v) formalde-hyde c. Solutions for Nick Translation.
10 x: Reaction 500 mM Tris-C1, buffer: pH 7.2 100 mM MgSO~
1 mM DTT
Nucleotide Mix: 100 mM dGTP
100 mM dATP
100 mM dTTP
in 10 mM Tris, pH 7 . 5 d. Solutions for Hybridization.
20 x SSC 3 ~i NaCl 0.3 M Na3-citrate 50 x Denhardt's 1% (w/w) Ficoll 400*
05 solution (PL-Pharmacia) 1~ (w/w) Polyvinyl-pyrrolidone (Sigma) to (w/w) BSA
Sterile filtrated a:nd stored at -20C
Pre'.hybridization 50~ (v/v) Formamide mix 5 x Denhardt's solu-tion 5 x SSC
50 mPi Na-phosphate pH 7.0 250 ~g/ml denatured salmon sperm DNA
Hybridization 50$ Formamide 2~ m:ix 5 x SSC
20 mI~1 Na-phosphate pH 7.0 1 x Denhardt's solu-tion 100 ~g/ml denatured salmon sperm DNA
e. Buf:Eers for Restriction Enzymes. Planu-facturers sf~ecifications, or:
Low Salt Buffer Medium Salt Buffer High Salt Buffer SmaI BglII BamHI
H~~~aI PstI PvuI
XbaI EcoRI SalI
S~eI BstEII (60C) *Trade mark ~ 340934 ~
Restrict=ion Buffers Low rledium High Tr i s , pH 7 . 4 1 Omr1 6 . 6mri 6 . 6mrM
rzgCl2 10 6 . 6 6 . 6 05 salt 20 ( KC1 ) 60 ( NaC1 ) 150 ( NaCl ) -mercaptoethanol 10 6.6 6.6 f. media.
1. L-Broth: 10 g Bactotryptone 5 g yeast extract 10 g NaCl 1 liter H20 2. L-Broth agar plates: L-Broth containing 15 g/1 Difo agar.
3. L-Broth-amp agar plates:
L-Broth agar containing 20-50 ~g/ml ampicillin.
C . ELTZYriES .
The following enzymes are used:
1. Dec>xyribonuclease I (~dorthington) 2. D~1~~-Polymerase I ("Klenow-Fragment") (Boehrin<3er r-sannheim) 3. T4-DNA L:igase (Boehringer Mannheim) 4. Re~;trict:ion enzymes EcoRI, :~baI, BamHI, Bc~II, BstEII, ~I, Sal_I, ~~aI, SmaI, PvuI, PstI (Boehringer Mannheim, BRL, New England BioLabs) 5. Ly~;ozyme ( SIGr~A) 6. Pranase D. AP~1ALYTIC'.AL APdD QUANTITATIVE PROCEDURES.
1. I~~ Bind:ing Assay.
The concentration of total protein in preparation; of purified particles of this invention 1340934 :.
is determined using the Bio-Rad protein assay kit.
This method utilizes the Bradford assay, a spectrophotcmetric: means of measuring Coomassie Blue binding to protein. Ovalbumin is used as a standard.
05 2. Radioimmune Assay (RIA).
In the LdML RIA 125-I "sandwich" radioimmunoassay, beads coated with mouse antibody to hepatitis B
surface antigen I;anti-HBs) are incubated with serum or plasma and appropriate controls. Particles containing po7Lypeptides encoded by the PreSl-PreS2-S protein coding region are bound to the solid F>hase antibody. After aspiration of the unbound material and washing of the bead, human 7_25I-Anti-HEs is~ allowed to react with the antibody-antigen ~~omplex on the bead. The beads are then washed to remove unbound 1251-Anti-HBs.
The radioactivity remaining on the beads is counted in a gamma scintillation counter. Specimens giving counts per minute (cpm) greater than or equal to the cut-off value determined by multiplying the negative co;ztrol mean count rate (NCx) by a factor are considered reactive.
3. ImmunoprEacipitation.
Cells are grown to 100% confluency in a T75 flaslc. The culture medium is then replaced with 5 ml of methionine-free medium containing 400 uCi of L-[35S] mE:thionine and 400 uCi of L-[35S]
cysteine (Nl~id) for overnight incubation. The medium is concentrated 10-fold by fractional precipitation with polyethylene glycol. Concentrated protein (about 106 cpm) is incubated with 10 ~.zl of pre-immune guinea pig serum or with 1 and 10 ~1 of -69- 1 3 4 0 g 3 4 anti-HBsAg guinea pig serum in 50 ,ul of TEN (lOmM Tris, pH
7.4, 1mM EDTA, 1..0 mM NaCl) - 0.5o Tween 20* for 1 hour at 37°C. The immunoglobulin is bound to 20 ~.sl of protein A-Sepharose* Cl-9:B (Pharmacia) for 1 hour at 37°C, washed once with TEN-Twe~en 20 and 500 mM LiCl and again with TEN-Tween 20*. Samples are electrophoresed in the SDS-PAGE
system described below.. Gels are fixed for 60 minutes in a solution of 10% tricholoro-acetic acid, 10% glacial acetic acid, and 30o methanol and incubated in Enlightning (NEN) for 30 minutes. Ciels are dried and autoradiographed with KODAK XAR-5* film.
4. SDS-Polyacrylamide Gel Electrophoresis (PAGE).
Sample and Gel Preparation. For analysis by PAGE, aliquots of purified particles of this invention are adjusted to 10 ,ug/ml. From each sample, 10 ,ul are taken and mixed with 10 ,ul 1M DTT in 10% SDS and 10 ,ul sample buffer. This mixture is boiled 5, 10 or 15 minutes just before loading in order to break the 20 nm particles and to generate monomeric molecules. After adding 10,u1 of loading buffer, the mixture is electrophoresed in a 0.75 mm thick slab gel (12 x 18 x 0.1 cm) of 1.2.5% polyacrylamide - 0.4%
bisacrylamide, using the Laemmli (1970) buffer system at l5mA and 100 V for 6 hours. Protein bands are visualized by silver staining (see Silver Staining section below).
5. Silver Stainina~.
The PAGE gel is stained. with silver according to Merril et al. (1981). The protein is fixed in the gel with a solution of 50o methanol and 10% acetic * Trademark x - ~o -acid for 30 minutes. The gel is washed with a solution of 10~ methanol and 5o acetic acid for 30 minutes. After incubation in loo glutaraldehyde for 30 minues, the gel is washed in deionized H20 05 overnight. Following incubation in 0.1% AgN03 for 30 minutes, the gel is subjected to a short wash in H20. The atained gel is developed in 100 ml of 30 2da2C03 and 30 ~1 formaldehyde and fixed in to acetic acid. They gel is sealed in a plastic bag and photographed.
E. Genetic Engineering Procedures.
1 . Rec=ombinant DNA Procedures .
a. Restriction Endonuclease Digestion of Purified DNA.
Per manufacturer's specifications of the particular endonuclease.
b. Plasmid DLdA Isolation.
Large Scale CsCl Plasmid Preparation:
1. Grow 1 liter of plasmid-bearing ce7_ls to 0.5 OD60C in L-Broth, amplify 12--20 hours with 200 ~zg/ml chloramphenicol.
2. Centrifuge in Sorval RCSb, 8,000 rpm for 20 minutes.
3. Resuspend in 18 ml cold 250 suc=rose, 50mM Tris, pH 8Ø
4. Transfer to 250 ml Erlenmeyer flask. lKeep on ice.
5. Add 6 ml 5mg/ml lysozyme in 250mP~
Tris, pH 8.0, let stand 10-15 minutes.
6. Add 6 ml 250mM EDTA, pH 8.0 and min: gent=ly; incubate 15 minutes on ice.
7. Add 30 ml detergent mix:
0.01% Triton X-100*
60mPt EDTA, pH 8.0 5 Omit Tr i s , pH 8 . 0 05 8. Incubate 30 minutes on ice.
9. Centrifuge 25,000 rpm. 90 minutes in 5;728 :rotor, 4C.
10. To supernatant fluid, add pronase to 250~uc3/ml and incubate 30 minutes, 37C.
11. Phenol extract once with 1/2 volume phenol equilibrated with TE (lOmM
Tris, pH 8.0, 1mM EDTA).
Tris, pH 8.0, 1mM EDTA).
12. Remove aqueous layer; add podium acetate to 300mM; add 2 volumes cold 100s etr~anol; mix thoroughly. Hold at -20C
overnight:.
overnight:.
13. Centrifuge; resuspend in 6 ml TE-10 ( 10mr~ Tr i s , 1 OmtM EDTA, pH 8 . 0 ) .
14. Add 9.4g CsCl, 0.65 ml of 6 mg/ml 2~:? et:;.i3iur~~ bromide; make up to 10 ml volume with sterile double-distilled water.
15. Fill Beckman heat-sealable gradient tubes; centrifuge 48,000 rpm, 40 hr. in Ti70.1 Beckman rotor.
16. Visualize plasmid bands with UV
and remove plasmid DNA with syringe and 18 gauge needle by piercing the side of the tube.
and remove plasmid DNA with syringe and 18 gauge needle by piercing the side of the tube.
17. Remove ethidium bromide from the plasmid :Fraction by 3 successive extractions with equal volumes of isobutanol.
*Trade mark 1340934 ~~
*Trade mark 1340934 ~~
18. Dialyze against one 2-liter lot of 1 O:nM Tr :i s , pH 7 . 4 , lm?M EDTA, pH 7 . 5 , 5mLvi LJaCl for 2 hours or more at 4°C.
19. Phenol extract once with 1/3 vol.
05 ph~anol equilibrated with TE as above.
05 ph~anol equilibrated with TE as above.
20 . Add NaAc to 300mM, add 2 vol . 100 0 ethanol; precipitate at -20°C overnight, or at -70°C for 30 minutes.
c. Nick Translation. Nick-translation is performed according to Rigby et al (J. Mol. Biol., Vol. 113, pp. 237-251, 1977). The reaction mixture for 32P-labeling of DNA usually contains 0.5 dug of, for example, the EcoRI-XbaI fragment of pD~l2, in a total volume of :30 ~.Z1 with 50 mM Tris, pH 7.8, 5 mM
Mc~Cl2, 10 mTM mercaptoethanol, 0.1 mIYI dATP, 0.1 mM
dGTP, 0.1 mT1 dT'TP, 50 uCi 32P-dCTP, 10 units DNA
polymerase I, 3 ~zl of a 2 x 10 5 fold dilution of 1 mg/ml DNase I a:nd is incubated for 90 minutes at 15°C, yielding .'3 x 106 to 12 x 106 total cpm, i . a . 1 x 10 '~ to 5 x 107 cpm/~g DLdA.
d. Transformation of Competent Bacterial Cells. Fro:n a dense overnight culture, 1 ml of the bacterial cell suspension is taken for innoculation of 100 ml growth medium (L-broth). The cells are grown at 37°C to a density of OD550 - 0.7 which is reached within 2 hours with vigorous shaking in a 500 ml Erlenmeyer flask. Growth is stopped by chilling the culture on ice for 10 minutes. From this culture, 3 rnl is taken for harvesting the exponential bacterial cells at 3,000 rpm for 5 minutes. The cells are resuspended in 1.5 ml of 50 mM CaCl2 in 10 mP~ Tris, pH 8.0, and incubated on ice for another -73- ~ ~ 40 93 4 :, 15 minutes. The cells are harvested once more by centrifugation of: 3,000 rpm for 5 minutes and resuspended in 200 ,ul of 50 mM CaCl2 in 10 mM Tris, pH 8.0, and kept S at 4°C overnight.
Thereafter, the l.igation mixture was filled up with 10 mM
Tris, pH 7.5, 1 ~~M EDTA, to a total volume of 70 ,ul and added to the 200 ~.sl bacterial cell suspension for DNA
take-up.
The mixture was incubated on ice for 30 minutes, then 1 ml L-broth was added. and t:he mixture was incubated at 42°C
for 2 minutes and. at 37°C for 40 minutes. After the incubation, the cells were spread on agar plates containing 50 ,ug ampic:illin/ml agar at volumes of 50 ,ul up to 300 ,ul of the cell suspension per plate. The agar plates were incubated at 37°C overnight. After this incubation period, single isolated bacterial colonies were formed .
e. Southern Blot F~nalysis. To characterize the organization within the: host cell genome of the vectors of this invention, chromosomal DNA from cell lines producing particles of this invention is isolated and digested with the appropriate restriction enzymes) and analyzed by the method of Southern (J. Mol. Biol., Vol.
98, pp. 503-517, 1975) using a 3'P-labeled DNA probe.
Following digestion of the chromosomal DNA (20 ,ug) with restriction enzymes EcoRI and XbaI, the resulting fragments are separated. by 0.7% agarose gel electrophoresis. Thereafter, the DNA is denatured by exposing to 366 nm W light for 10 minutes and by incubation in a solution of 0.5N NaOH and 1 M NaCl for 45 minutes. The gels are neutralized by X
~340~34 _ 74 -incubation in 0. _'>tz Tris, 1.5 ri NaCl, pH 7. 5 for 60 minutes. Tze DNA is transferred to a nitrocellulose filter by soaking in 3 IM NaCl, 0.3 t1 NaCitrate (20XSSC) for 20 hours through the gel by covering the 05 top of the nitrocellulose filter with a staple of dry paper towel:. Th;e nitrocellulose filter is kept for 2 hours in a vacuum oven at 80°C.
The rad.ioact:ive DNA probe from the EcoRI-~~baI
fragment of the pDtil plasmid ( 4. 3 kb ) i s prepared by nick-translation.
For hybridization with the DNA probe, the nitrocellulose falter is sealed in a plastic bag containing 10 rn.l of prehybridization mixture . 500 formamide, _'s x SSC, 50 mM sodium-phosphate, pH 7.0, 5 x Denhardt's solution, 250 ~g/ml denatured salmon sperm DNA. The :Filter is incubated in this mixture for 4 hours at 45°C, after which the pre-hybridi2;ation mixture is replaced by the hybridizatic>n mixture: 50% formamide, 5 x SSC, 20 mM
?G Na phosphate, pH 7.0, 1 x Denhardt's solui.ion, 100 ~ug/ml denatured salmon sperm DNA, 5 x 105 cpm/ml 32P-probe. The filter, after incubating in the hybridization mix for 18 hours at 45°C, is washed three times, 5 minutes each, in 0.1 x SSC, 0.1% SDS
at 50°C. The filter is dried at 60°C for 10 minutes and exposed to two X-ray films (Y,AR-5, KODAK) between two intensifying screens and kept at -80°C. The first X-ray film is developed after 3 days' exposure;
the second film after 7 days' exposure.
HlTbridization of the labeled DNA probe to one of cellular DN~~ restriction fragments from transfectants which produce part=icles of this invention - 75 _ demonstrates that the cellular DNA does in fact contain th~= integrated vector of this invention.
Since the cellular DNA restriction pattern, as elucidated by the Southern blot procedure, is 05 identical to that. of the original plasmid construct, no major rearrangement of plasmid DNA occurred upon intec3ration into the host cell DNA.
2. Transfection of Mammalian Cell Lines and IdE:ntification of Clones Producing Particles of this Invention.
Cells a:=a transfected using the calcium phosphate precipitation method of vaigler et al (Cell, Vol. 14, p. 725, 1978). Cells are then split 1:6 into new culture dishes, and cells which have received the drug-resistance marker gene (neo) are selected by groc~ath in 6418-containing medium. After 10 days of grovath in :elective medium, drug-resistant colonies are cloned into micro-titer plates. Levels of expression of the particles of the present invention ara assessed by the P,IA technique after the cells have reached. confluency.
Tissue culture dishes (100 mm diameter) are innoculated with 5 x 105 cells and incubated overnight at. 37°C. Four hours prior to transfection, the culture medium is replaced with fresh medium.
DNA to be transfected is suspended in 1 ml of solution containing 250 mP? CaCl2, 140 mPM NaCl, 25 ml~I Hepes, f>H7.1 and 0.75 mP-~ NaHP04. This calcium phosphate - DNA precipitate is added to the cells in culture. After an overnight incubation, fresh medium is added to the cell culture and the cells are incubated for an addition two days.
_ 76 _ F. Procedures for Static Cell Culture of fiammalian Cells.
An ampule of cells held at 196°C in liquid nitrogen is thawed quickly by placing the ampule in a 05 37°C water bath for 5 minutes. One ml of the freshly-thawed cell suspension is diluted by slowly adding 10 ml of the appropriate culture medium.
Thereafter, the cells are harvested by centrifugation, resuspended in 10 ml of culture medium. T~lereafter, the cells are harvested by centrifugation, resuspended in 10 ml of culture medium, transferred to a T25 culture flask, and grown in an incubator at 37°C and 5o C02. V~Tith these conditions, the doubling time is in the range of 15 to 20 hours for :L-cells and CHO cells, and 30 to 40 hours for Vero cells. L-cells and CHO cells can be '.kept alive and growing beyond 1008 confluency, whereas Vero cells are passaged at 100 confluency.
G. Procedures for L~lammalian Cell Growth in ACUSYST-P
2G and ~~arvest o:f Particles of this Invention.
1. Growth and Preparation of Innoculum.
Cells are maintained in T75 or T150 flasks containing D.rsEM-1.0°s FBS. Roller bottles ( 850 cm2 ) are innoculated with 10-15 x 106 cells harvested from T-flasks. After 3 to 5 days of growth, approximately 10~~ total cells are harvested from six roller 'bottles and used to innoculate six hollow fiber cartridges (HFC's) in an ACUSYST-P* made by Endotronics, Inc. of Minneapolis, Minnesota U.S.A.
2. ACt~SYST-1? Culture and Harvest of Particles of this :Invention.
After i:nnoculation, the HFC's are inserted into the ACUSYST-~P culi=ure system, and the medium flo4a *Trade mark 1 340 93 t~ ~.
rate through the cartridges is adjusted to and mintained at 50 ml/hr. Medium is assayed daily for pH, pO2, glucose and lactate. After 2 to 3 weeks of growth, a 600 ml fluid volume is harvested two 05 times weekly from the extracapillary space of each ACUSYST-P hollow fiber cartridge. Production of particles of they subject invention is assayed by the radioimmunoassay (RIA). Since freezing destroys RIA
assayable activity, harvests are stored at 4°C prior to purification.. No protease activity can be detected under these conditions as assayed by Azocoll substrate proteo:Lysis (SIG??A) .
- 3. ~ualitv Control Procedures.
'= a. _Te:~ts for the Presence of Bacteria and Fungi. A pool of cells containing about 5 x 105 cells per ml, suspended in the cell culture medium from which they were harvested, is tested for the presence of bacteria and fungi. One ml of cells is streaked onto trypticase soy agar (BBL) plates, and another 1 nil is innoculated into thioglycolate medium (DIFCO) to test for the presence of aerobic as well as anaerobic bacteria. Fungal contamination is assayed by streaking 1 ml of cells onto Sabouraud _ (DIFCO) agar plat=es. These tests were performed on a regular basis to ensure sterility of growth medium and absences of bacterial and fungal contamination in cell cultures.
b. Te=>t for the Presence of Myconlasma:
Fluorescence Staining The presence of mycoplasmal DNA in the cell cytoplasm is determined by a staining method using they fluorochrome, Hoechst 33258* This DNA-staining dye fluoresces under ultraviolet light *Trad'.e Marks 1340934 ~
_ 78 _ and provider the basis for a very rapid and sensitive test for mycoplasmas. The procedure involves coverslip cultures of the cells to be tested which are used when 5(J-70s confluent. After fixing and 05 staining, the coverslips are examined with a fluorescent microscope. Cytoplasmic fluorescence indicates the pre:~ence of mycoplasma.
The stack of the Hoechst 33258 bisbenzamide fluorochrome solution is made by dissolving 5 mg in 100 ml PBS using a magnetic stirrer. It is sterilized by filtration through a 0.22 um membrane stored in the dark at 4°C, and diluted a thousand-fold with PBS before use. The medium from coverslip cultures, which are 50-70% confluent, is aspirated from th.e cells and fixed with two changes of ethanol:acetic acid (3:1) at 4°C. Following one wash with deionized water and incubation for 30 minutes at 3'7°C with diluted bisbenzamide fluorochrome (Hoechst-33258), the coverslips are rinsed tail deionized water. The coverslips are mounted, ce~.l-side down, on a slide using a glycerol mountant (22.2 ml. 2.1~ citric acid; 1 ml H20; 27.8 ml 2.8o Na2HPA4; 50 ml glycerol pH 5.5).
c. Testis for the Presence of Viruses.
1) Tests in Cell Cultures. Tester cells are examinE~d for normal morphology during an incubation period of at least 14 days after innoculation with a suspension of the cell line being tested. In addition, on days 3-5, and again after the 12th da:y, at least 4% of the innoculated tester cultures is washed and tested for hemadsorption using red cells from sheep or humans. Tests are read after _ 1340934.
_ 79 -the cultures have' incubated for 30 minutes at 3°-4°C
and again after 30 minutes at 34°-37°C. Results of tests for virus-~~nfected cells which absorb erythro-cytes indicate no viral contamination.
05 2) Tests in Animals. Cells to be tested are suspended in 140 mM NaCl, 2.7 mM KC1, 8.1 mL~i Na~HP04, pH 7.2 at a concentration of 106 per ml and are innoculated into different groups of animals. I::~ one group, at least 10 animals from at least two litters of suckling mice are innoculated.
Each animal is innoculated with 0.1 ml cells intraperitoneally and 0.01 ml intracerebrally. The mice are observed daily for at least 14 days. Each mouse that dies after the first 24 hours of the test, or is sacrificed because of illness, is necropsied and examined for evidence of viral infection. Such examination involves additional testing by intracerebral and. intraperitoneal subinnoculation of appropriate tissue suspensions into an additional group of at. least five suckling mice following by daily observations for 14 days. In addition, a blind passage is made from a single pool of the emulsified tissue (minus skin and viscera) of all mice surviving the original. 24-day test.
In another group, 10 adult mice are also innoculated. Each animal is innoculated with 0.5 ml cells intrape:ritoneally and 0.03 cells intracerebrally. The animals are observed for four weelcs and any that become sick or show abnormality is examined to establish the cause of illness. Results of the tests :in animals for the presence of contarlinating viruses indicate no viral contamination.
'3~+~934 _80-d. Testing for Tumorigenicity. A suitable test for tumorigenicity using nude mice (nu/nu) involves innocula.ting 20 animals within 24 hours of birth with ~.1 ml. of potent serum. The injection is 05 given either by the intramuscular or subcutaneous route and is repeated on days 2, 7 and 14 of life.
One million reference tumor cells routinely produces progressive7_y growing tumors and metastases. One million viable cEalls of the candidate cell line are innoculated by tree subcutaneous route at any site at which developing tumors can be palpitated (the limbs are suitable). The animals are observed for 21 days for evidence of nodule formation at the site of injection, and measurements are made periodically to determine whether there has been progressive growth.
At the end of the 21-day observation period, all animals are sacrificed and examined for gross evidence of tumor formation at the site of injection and in other organs such as the lymph nodes, lungs, vidneys and liver. All tumor-like lesions ana all innoculation site's are examined histopathologically.
In addition, since some cell lines may form metastases without evidence of local tumor growth, the lungs and regional lymph nodes of all animals are examined hi:~tologically.
For the purpose of this test, a progressively growing tumor is defined as a palpable nodule that increases in size' over the 21-day observation period and that shows. viable and mitotically active innoculated cell; when examined histologically. The presence ~~f microscopically viable cells in association with a stationary or regressing nodule is not consideoed a progressively growing tumor.
1340934 ' H. Purification of the Particles of this Invention.
1. Fractional Precipitation with Polyethylene ~'col ( F?EG ) .
Piedium f:rom t'he extracapillary space is harvested 05 from the AC:USYST-P* culture system and pooled into volumes of 3000 ml. To each volume, 180 g of PEG
8000 (SIGP~A) are added, dissolved by stirring at room temperature for 20 minutes and stirred for another 3 hours at ~,°C. The precipitate is collected by centrifugation in 500 ml bottles in a GS 3 rotor at 4500 rpm (:3000 x g) for 30 minutes at 10°C. The supernatant is collected and 180 g of PEG 8000* are again added and dissolved at room temperature as described above. The solution is stirred at 4°C for an additional 3 hours. The precipitate from this solution is collected as described above except that centrifugation is performed at 9000 rpm (15,000 x g) for 60 minutes. The pellet is resuspended in 20 ml of phosphate buffe=red saline (PBS).
~0 2. Gel Filtration Chromatography.
The material obtained after PEG precipitation, and redissolved in PBS, is submitted to gel filtration c=hromatography using BioRad A-5m*resin at 4°C. Column dimensions are 25 x 1000 mm, and the bed volume is 480 ml. In a typical fractionation run, 1000 ~g of PEG-precipitated particles of this invention in a volume of 10 to 15 ml is loaded and eluted with PBS or THE at a speed of 6 drops/minute (18 ~l/hr) in fractions of 3 ml. Figure 6 shows the profile of RIA-as;sayable material eluted from an A-5m column. The solid line indicates the 280 nm *Trade mark 134~~34 absorbance of each fraction, and the dots indicate amount of n.aterial calculated from the RIA results.
3. Isopycnic Centrifugation in CsCl.
About 30 fractions covering the first peak 05 resulting :From gel filtration column chromatography and containing partially purified particles of this invention are pooled (approximately 100 ml). This solution is adjusted to a density of 1.30 g/cc with CsCl and subsequently transferred to nitrocellulose tubes fitting into a SVJ 27/28 rotor (Beckman).
Gradients are seat by underlaying 4 ml of a CsCl solution of 1.35 g/cc and by overlaying 4 ml of 1.25 g/cc and 4 ml o:E 1.20 g/cc density. Gradients are run at 28, 030 rpm for 50 hours at 10°C, fractionated, and the purified particles in the 1.20 g/cc density layer at the tc>p of the gradient (Figure 7) is collected. Purified particles are well separated from the small amount of contaminating protein banding in the mp.ddle of the gradient. The solution was desalte~~ by dialysis, first against water, then against 3 changes of saline, over a 24-hour period.
As a further quality control measure, a portion of this gradient-purified material is submitted to linear CsCl-gradient centrifugation. A single peak of purified particles appears as expected at 1.2 g/cc.
I. Preparation o:E the Adjuvant of Purified Particles of this Invention and Stability Testing.
In order to prepare an adjuvant of the vaccine of this invention, 1/10,000 volume Thimerosol, and 1/10 volume of filter-sterilized 0.2 M A1 K(S04)2:12 H20 are added to the desired concentration of antigen in sterile saline. The pH is adjusted to 5.0 1340934 ' with steri7_e 1N NaOH and the suspension is stirred at room temper=ature for 3 hours. The alum-precipitated antigen is recov~'red by centrifugation for 10 minutes at 2000 rpm, resuspended in sterile normal saline 05 containing 1:10,000 Thimerosol, and aliquoted under sterile conditions.
To determine the stability of the particles of this invention absorbed to alum, the antigen was recovered ;~y re~solubilizing the alum in 3~ sodium citrate fo.Llowed by successive dialyses against 30 sodium citrate and PBS. The quantity of particles of this inven~~ion :is then determined by parallel-line radioimmune assay against dilutions of a purified FiBsAg standard ir.~ PBS-50o newborn calf serum.
Table 1 Stability Testing Parameters Samples: two each of bulk and alum absorbed 30 Sample Concentration: 5 ~c;/ml Storage Container: glass vials Storage Temperature: 2°-8°C
Tests: (a) Qualitative: SDS-PAGE
(b) Quantitative: Radioimmunoassay Example 1 E:K ression of Particles Comprising PreSl-Pr~sS2-S Protein Coding Region Poly~peptides A. Preparation of Recombinant Plasmid pMMT-neo.
The plasmid pBPV342-12 was digested with the restriction-endonuclease BamHI (see Figure 1 and Materials and Procedures). Two DNA molecules were generated: one molecule (7.95 kb) comprises the entire gent>me of bovine papilloma virus (BPV); the other molecule (6.65 kb) contains part of the bacterial plasmi.d pML2 (pBR322 deleted for the "poison sequence"), the mouse metallothionein promoter (pLMI~iT), the neomycine resistance gene (neo) and the SV9~0 PAS-t (see Figure 1). When ligated to itself (circularized), this fragment gives rise to plasmid pP~M3T-neo (see also Figure 5).
The rea~~tion was performed in a total volume of 400 y~l of reaction buffer (see P~iaterials and nrocec~ures) at a final concentration of 0.2 ~g/~l pBPV342-12 I»1A; and 80 units of BamHI at 37°C for 4 hours. The completion of the digest was checked by agarose gel electrophoresis in a 0.7o agarose gel (see Materials a.nd Procedures). The reaction was stopped by adding 40~u1 of 8 t-1 LiCl and the DNA was X340934 ~, precipitated with 1 ml of ethanol at -80°C for 30 minutes. The precipitated DNA was resuspended in 100 ~1 of 10 mM Tris, pH 7.8.
B. Isolation of a Fragment Containing the Entire 05 PreSl-PreS2-S Protein Coding Region Along with the Natural Promoter of Transcription.
The prE~paration and cloning of the HBV genome, subtype adw, is described by Cummings, I.W. et al in Proc. LJatl. Aca. Sci., U.S.A., Vol. 77, p. 1842, 1980. The circular HBV genome was linearized at the unique EcoRI site for cloning into bacteriophage lambda gtWES and for subcloning in a bacterial plasmid to yield plasmid pA01 (see Figure 2). Since the EcoRI restriction site lies within the PreSl-PreS2-S protein coding region, the latter is interrupted when in the EcoRI linear form. To regenerate an intact PreSl-PreS2-S protein coding region, the 3.2 kb EcoRI insert of pA01 was isolated and separated from the plasmid DNA by digesting 100 ~zg of pA01 in a total volume of 400 j~l of reaction buffer (seep Materials and Procedures) containing 200 units EcoRI for 4 hours at 37°C. The 3.2 kb insert DNA was separated from the plasmid DNA by preparative 0.7~ agarose gel. electrophoresis (see Materials and Procedures). They DNA was electro-eluted from the agarose gel on DE 81*Whatman ion exchange filter from which the DNA is removed in a high salt solution.
The DNA was purified by one phenol/chloroform extraction and two ethanol precipitations. The purified 1:W ear 3.2 kb EcoRI fragment encoding the complete HBV genome was then subjected to self-ligation at a high DNA concentration in order to favor formation c>f concatamers: 30 ~ g of EcoRI
*Trade mark:
fragment D:VA ways ligated in a total volume of 50 ~ul of reaction buffer containing 1.8 units T4 DNA ligase and 2 mM A'rP at 15°C for 1 hour and at 4°C overnight;
thereafter, th.e DNA was purified by two 05 phenol/chloroform extractions, one chloroform extraction, followed by two ethanol precipitations.
The pellet--_d DNA was resuspended in 20 ~ul of 10 mP4 Tris, pH 7.8, a,nd digested with restriction enzyme BglII in a total_ volume of 50 ~ul of reaction buffer ( see Materials and Procedures ) containing 50 units of BglII at 37°C for 4 hours.
The DNl?, fragments generated by this reaction were separated by 1.5% agarose gel electrophoresis. The 2.7e kb suze Bc~lII fragment containing the intact PreSl-PreS2-S protein coding region, together with its natural promoter system necessary for expression of the surface antigen polypeptides, was isolated from the agarose gel and purified as described above (see Figure 2).
C. Insertion of the Fragment Containing the PreSl-PreS2-~~ Protein Coding Region into the pPM4T-neo Plas;mid: Construction of pDMl.
From a DNA solution prepared as described under part A. above, 1 ~ul was ta);en and mixed with 5 Jal of the 2 . 78 kb ~,I I fragment ( 0. 25 yg/~1 ) described under part :B, above.
The mixture was submitted to a ligation reaction at a tota.L volume or 10 ~ul of reaction buffer containing 0.9 units of T4 DNA ligase at 15°C for 1 hour and at 4°C overnight.
Bacterial cells, preferably HB101, were made competent for taking up DNA in a transformation l3t,D934 -, reaction a~ccord:ing to the method described in Materials a.nd Procedures. The ligation mixture was made 10 mM Tris, pH 7.5, 1 mM EDTA, in a total volume of 70 ~Z1 and added to 200,/ul competent bacterial cell 05 suspension for DhfA take-up.
The mixture was incubated on ice for 30 minutes, then 1 ml L-broth was added and the mixture was incubated at 42°C for 2 minutes and at 37°C for 40 minutes. After t=he incubation, the cells were spread on LB agar plate's containing 50 ~tzg ampicillin/ml at volumes of 50 ~ul up to 300 ~ul of the cell suspension per plate. The agar plates were incubated at 37°C
overnight. After this incubation period, single isolated h~acterial colonies were screened for the presence o:. the pMMT-neo bacterial plasmid containing the desired 2.713 kb BglII DNA fragment insert (see Figure 3).
Screen=~~ng was preferentially performed by the colony hybridization procedure. For this procedure, single colonies were picked with a toothpick and transferred to a.n LB-ampicillin-containing agar plate in a grief fornnat to allow identification of the clones. Tile plate was incubated overnight at 37°C.
The colonies were transferred to a nitrocellulose filter by layering the filter on the agar surface until the filter was wet. The filter was quickly peeled off and sequentially transferred to three layers of Whatman 3M paper, each of them either soalced in 0.5 1'~I LdaOfi, or 1 M Tris, pH 7.0; 1.5 M
NaCl/0.5 r9 Trig;, pH 7.4, or 2 X SSC. The filters were placed on the soaked paper, colony-side up, during the lysis of the cells and the following neutralization and fixing procedures.
_88_ After the filters were dried in air, they were ba~;ed at 80°C .in vacuum. Thereafter, the nitro-cellulose filter; were submitted to DNA hybridization with a radioactively labeled DNA probe made from the 05 2.78 kb BglII fragment containing the PreSl-PreS2-S protein coding region, prepared as described in part B above, by nick-translation (see Materials a:nd Procedures).
The nitrocellulose filter was incubated in a pre-hybridization mix containing 50% formamide, 20 mr-1 sodium phosphate buffer, pEi 6.6; 1 X Denhardts solution; 1.00 ~g~/ml denatured salmon sperm DNA and 1 x 107 cpm 32P-labeled DLdA which was r.:elted in U.2 N LdaOH ~st 68°C for 10 minutes.
The filter was incubated in this mixture for 16 hours at 45°C. Thereafter, the filter was washed twice in 2 X SSC, 0.1'6 UDJ, for 5 minutes each cycle at room temperature and twice in O.1XSSC, 0.1% SDS, for 15 minutes each cycle at 50°C. The filters were 2U exposed to an ?;-ray film (preferably 3M) between two screens at -80°C for two days.
Colonies containing the recombinant plasmid carrying th.e cloned 2.78 kb Bc~lII fragment appeared as black spots. Four out of 50 colonies were identified and t7ze plasmid DNA of these colonies was isolated in min:ipreparations according to Birnboim and Doly, l~Tucl. Acids Res., Vol. 7, p. 1513, 1979, and analyzed by restriction endonuclease digestion by a BglII rind XbaI double digest. The analyses confirmed t:he intended construction explained above (see Figure 3).
D. Substitution of the Natural Promoter for the PreSl-P:reS2-S Protein Coding Regions by the Metallothionein Promoter: Construction of pDf-I2.
The plaamid pDMl described above was submitted to 05 complete digestion with the restriction enzymes BglII
and BamHI in a total. volume of 100 ~.Z1 containing 20 ,fig of plasmid DLJA and first with 50 units B~lII in reaction buffer containing 6.7 mM Tris, pH 7.8; 6.7 mM MgCl2; 6.7 ml'-1 ~ -mercaptoethanol, at 37°C for 4 hours; then the salt concentration was raised to 150 mM NaCl and the digestion was completed by addition of 40 units BamHI at 37°C overnight. Three fragments were generated, t:he large fragment being 5.1 kb, the medium fragment being 2.97 kb and the small fragment being 1.4 kb. These fragments were separated by preparative 0.7~ agarose gel electrophoresis from v.~hich the (5.1 kb and 1.4 kb) fragments were isolated electrophor~etically using DE 81* Whatman ion exchange filter.
2U The DD1A wa.s removed from this filter by incubation in high salt for 4 hours at 4°C and purified by phenol/chloroform extraction and two ethanol precipitations. The DNA was resuspended in ~1 of 10 mM Tris, pH 7.8. One ~1 was checked for purity and estimation of quantity by agarose gel electrophoresis.
The large (5.1 kb) fragment containing the metallothionein F~romoter at the BglII end was ligated to the small (1.4 kb) fragment containing the PreS2-S protein coding region, but not the natural promoter o:r the PreSl portion of that gene. The large fragment also contained the natural *Trade mark 1340934 ;
termination-polyadenylation signal (hb-PAS-t) necessary for e~;pression of the hepatitis B PreS2-S
protein coding region (see also Figure 4).
BecausE~ the large fragment also contains the C5 bacterial ;~lasmid, it is possible that self-ligation of this fragment will propagate itself without incorporating 'the small size BamHI fragment.
Therefore, 10 ,~a7L of the total 20 ~.zl preparation of the large fragment were subjected to treatment with alkaline phosphatase by adding 28 units of this enzyme and incubating at 37°C for 20 minutes. The reaction was stopped by adding 1 ~ul 50 mtd EGTA and incubating at E>8°C for 10 minutes. The DNA was purified by two ethanol precipitations in 0.8 M LiCl.
The pelleted DNA was resuspended in 10 jzl 10 mM
Tris, pH 7.8, of which 5 ~1 was taken as a control and checked for self-ligation and another 5 ~1 was mixed with 5 ~1 of the electroeluted small (1.4 kb) BamHI fragment.. Ligation of each sample was performed in a total volume of 20 ~1 as described above. Transformation on HB101 bacterial cells was carried out. as above.
~'welve single colonies were picked and grown in 2 ml cultures and the plasmid DNA was isolated according to Birnboim and Doly, supra. The plasmid DNA was checked for the insertion and orientation of the small aize BamHI fragment by a double digest with the restriction enzymes EcoRI + XbaI.
Two of the twelve plasmid DNAs have the small (1.4 kb) BamI-i:I fragment inserted in the same orientation as the metallothionein promoter.
These plasmid. DNAs are grown in mass culture and prepared for introduction by co-transfection into eukaryotic c=ells.
Alterna~=ively, the EcoRI/BglII (1.9 kb) fragment 05 from plasmi.d pMMT-neo carrying the metallothionein promoter is used for substituting the short (32 b.p.) EcoRI-BamiiI fragment in front of the PreS2-S
protein coding rE~gion in pDMl to give plasmid pDM3, as illustrai=ed in Figure 5.
E. Introduction of the Recombinant DNA Vectors Obtained Under Parts C. and D. above into Mammalian Cells and the Establishment of Cell Lines Producing Particles of this Invention.
The recombinant plasmids, pDMl and pDM2, were co-transfeci=ed into mouse L cells, vero (African green monkey) cells, CHO cells, and 3T3 mouse fibrobl_asts by standard transfection procedures (Graham and van der Eb, Virology, Vol. 52, p. 456, 1973, supra) (See Materials and Procedures). Cells w~~ich take up and maintain the plasmid DL~tA are resistant 1=o the drug 6418 and survive in the selective medium containing this drug. Cells which do not ta~;e up the DNA do not survive in the selective medium. These cells are detected as single clones on the surface of a culture plate. The cells were picked with a cloning cylinder and propagated to prepare for mass culture in a conventional manner in a usual nutrient medium, e.g., Dulbecco's modified Eagle medium supplemented with loo calf serum and 500 ~.zg G418 per ml (see Materials and Procedures) . Five of these clones were established as cell lines, and designated ENDO-I, -II, -III, -IV, and -V, and frozen stocks were prepared (see Materials and Procedures).
The production rate of these cell lines in static culture was determined by radioimmune assay (RIA; see Materials and Procedures) and compared to the 05 production rate c>f known cell lines. On the average, the cell lines ~sccording to the invention produced 500 ng to 1.,000 :ng of the particle of this invention per ml of culture medium per day.
F. Acusyst-P Culture of Cell Lines Producing the Particles of the Invention.
Approximately 109 total cells grown in DMEM+10%
FBS in s:ix roller bottles (see Materials and Procedures) were used to innoculate six hollo~. fiber cartridges in t:he Acusyst-P* culture system (see Materials and Procedures). Approximately 600 ml fluid volume was harvested two times weekly from the extracapillary space of each Acusyst-P hollow fiber cartridge and stared at 4°C prior to purification.
The concentration of the particles of this invention was assayed by RIA (see f~Iaterials and Procedures).
G. Purification Particles of the Invention from Acusyst-P Culture tdedium.
Particles containing proteins coded for by the PreSl-PreS2-S protein coding region produced by the novel cell lines were purified by polyethylene glycol (PF~G) precipitation, gel filtration and isopycnic ultracentrifugation in CsCl gradients (see below).
1. Fractional Precipitation with Polyethylene G1 cy of (PEG).
Medium from the extracapillary space was harvested from the ACUSYST-P culture system and *Trade mark ~ ~ 4~ 93 4 _ 93 pooled into volumes of 3000 ml. To each volume, 180 g of PEG 8000 (SIGMA) were added, dissolved by stirring at room temperature for 20 minutes and stirred for another 3 hours at 4°C. The precipitate 05 was collected by centrifugation in 500 ml bottles in a GS 3 rotor at 4500 rpm (3000 x g) for 30 minutes at 10°C. The supernatant was collected and 180 g of PEG
8000* are again added and dissolved at room temperature as described above. The solution was stirred at 4°C for an additional 3 hours. The precipitate from this solution was collected as described above except that centrifugation was performed a.t 9000 rpm (15,000 x g) for 60 minutes.
The pellet was resuspended in 20 ml of phosphate buffered saline (PBS).
2. Gel Filtration Chromatography.
The mai_erial obtained after PEG precipitation, and redissolved in PBS, was submitted to gel filtration chromatography using BioRad* A-5m resin at 4°C. Column dimensions were 25 x 1000 mm, and the bed volume was X180 ml. In a typical fractionation run, 1000 dug of PEG-precipitated particles of this invention in a volume of 10 to 15 ml is loaded and eluted with PBS or THE at a speed of 6 drops/minute (18 ml/hr) in fractions of 3 ml. Figure 6 shows the profile of the particles of this invention eluted from an A-5m co:Lumn. The solid line indicates the 280 nm ab~;orbance of each fraction, and the dots indicate the quantity of the particles of this invention in each fracton as calculated by RIA.
*Trade lark 3. Is~cpycnic Centrifugation in CsCl.
About 30 fractions covering the first peak resulting from gel filtration column chromatography (see Figure 6) and containing partially purified 05 particles of. this invention were pooled (approximate=ly 100 ml). This solution was adjusted to a density of 1.30 g/cc with CsCl and subsequently transferred to nitrocellulose tubes fitting into a S4J 27 or S4J 28 rotor (Beckman) . Gradients were set by underlaying 4 ml of a CsCl solution of 1.35 g/cc and by overlaying 4 ml of 1.25 g/cc and 4 ml of 1.20 g/cc density. Gradients were run at 28,000 rpm for 50 hours a.t 10"C, fractionated, and the purified antigen in the 1..20 g/cc density layer at the top of the gradient was collected. The particles of this invention were well separated from the small amount of contaminating protein banding in the middle of the gradient (see Figure 7). The solution was desalted by dialysis against three changes of saline over a 24-hour peruod.
As a quality control measure, a portion of this gradient-purified material was submitted to linear CsCl-gradient centrifugation. A single peak of the particles of this invention appeared as expected at 1.2 g/cc.
H. Characterization of the Particles of this Invention Produced by Cell Lines.
The purity anal physical identity of the polypep tides of the particles of this invention in prepara tions are established by conventional laboratory techniques, such as radioimmune assay, immunoprecipi-tation and ~>DS-PAGE (see P4aterials and Procedures).
1. Purity I>eterminations.
a. Determination of Levels of Contamina-ting Plasma Proteins.
The levels of various serum proteins in prepara 05 tions of purified prticles of this invention are assessed by the :standard RIA technique using specific antibodies against bovine serum albumin, IgA, IgG, IgM, etc. The results shown in Table 2 show no detectable immunoglobulin contamination and only minor albumin contamination.
Table 2 Determination of Contaminatin P:Lasma Proteins by RIA
Protein % of Total Proteins Albumin 0.05 IgG 0.2 IgA 0.2 Ic~M 0.2 b. Determination of Levels of Contaminating Host Cell or PreSl-PreS2-S Nucleic Acid Seguences in Purified Preparations of the Particles of this Invention by the Dot Blot Procedure.
A dot-blot hybridization is performed on purified particles of this invention in order to check for contaminating PreSl-PreS2-S DNA sequences or host chromosomal DNA ;sequences. For each spot, a 100 ng quantity of puri:Eied particles of this invention in Jal 0.25 LJ LJaOF3 is heated at 6S°C for 10 minutes.
As a positive control, 20 pg of recombinant plasmid pDMl DNA of this invention in 40 ~1 0.25 N NaOH is treated the same way. Immediately after denaturation in NaOH, the solution is spotted onto a 05 nitrocellulose filter. The following treatment of the filter and the hybridizing procedures are analogous to those described for the Southern Blot procedure, except that the 32P-labeled probe is a 50:50 mixture of chromosomal DNA and plasmid DNA of this invention containing the PreSl-PreS2-S
protein coding rE:gion. As expected, the hybridizing probe gives a strong signal with the recombinant plasmid DN~~ of this invention, but only a weak background signal with the purified particle preparation. This hybridization pattern indicates no PreSl-PreS2-S D1~1A or chromosomal DNA in the sample.
2. Immunoprecipitation and SDS-Polyacrylamide _Ge1 Analysis.
To det:ermin~e the pattern of PreSl-PreS2-S
polypeptide;~ produced by transfectants of this invention, proteins produced by such transfectants are biosynthetically labeled, immunoprecipitated and analyzed on SDS-PAGE gels (see Materials and Procedures). ~C'he electrophoretic pattern of proteins, isolated from culture medium by immuno-precipitation with antibodies to naturally derived HBV, was visuali2,ed by the silver staining procedure.
The two major proteins correspond in size to nonglycosylated and glycosylated polypeptides encoded by the S pr~~tein coding region (24 kd and 27 kd, _ 97 _ respectively). Two minor proteins of 33 kd and 36 kd correspond in si~;e to the two polypeptides encoded by the PreS2-:~ prot.ein coding region. This method is not sensitive enough to detect the small quantities 05 of polypeptidea containing the PreSl-PreS2-S
polypeptides, but the presence of the proteins encoded by the entire PreSl-PreS2-S protein coding region wa:a detected by the western immunoblot procedure (see, i.nfra). The weak bands at the top of the gel result from aggregated protein and are also visible with naturally-derived HBV particles. Thus, cells transfected with the PreSl-PreS2-S protein coding region secrete proteins which not only react with antibodies against the natural product, but also are identical in size. The presence of proteins which are i~3entical in size to the naturally-occurring glycosylated PreSl-PreS2-S, PreS2-S and S poly peptides from the hepatitis E virus suggests that glycosylati~~n pathways function normally in these cells.
3. Immunoblot (4Jestern) .
To establish that each of the polypeptides species appearing in the silver stained SDS-PAGE gel system reacts with specific anti-HBV antibody, the 4destern immunoblot procedure was employed. Proteins separated in a SDS-PAGE were transferred to a nitrocellulose filter (Schleicher & Schull) by electroblotting (BioRad) at 4°C, 100 V, 3 hours.
After transfer, t:he filter is saturated with proteins in order to avoid nonspecific binding of peroxidase-labeled antibody. To do this, the filter is incubated for 1 hour at room temperature in 20~
- 1340934 ' newborn calf serum in 1?BS, and the polypeptides of the particle of this inveni:ion were made visible by HBV-specific antibodies conjugated with peroxidase. The S filter is placed on parafilm and covered with a solution of the conjugated antibodies for 3 hours at room temperature in a wet chamber. The filter is washed six times with 0.5% Tween* in lOmM Tris and 5 mM CaCl and subsequently covered with the chromogen, POD
(o-phenylenediaminedihydrochloride) in hydrogen peroxide (0.3g/1) in citrate-phosphate-buffer. Stopping solution is 0.5 N sulfuric acid. The protein bands corresponding to all six viral pclypept:ides appear, i.e., all the proteins coded for by the PreSl-PreSz-S protein coding region.
4. Amino Acid Composition of Purified Proteins Coded for by the PreSl-PreS,~-S Protein Coding Region Comprising the Particles of this Invention.
Following acid hydroly~~is, the amino acid composition of the purified proteins coded for by the PreSl-PreS2-S
protein coding region i.s determined and compared with the polypeptide composition. calculated from the nucleotide sequence of the S protean coding region, the PreS2-S
protein coding region or the PreSl-PreS2-S protein coding region. The results presented in Table 3 correlate well with values predicted from the DNA sequences as well as with published results (Shiraishi et al, J. Gen. Virol., Vol. 48, p. 31, 1980). In particular, the relatively high percentage of serine, proline and leucine is characteristic of these polypeptides.
*Trade mark Table 3 Amino A-id Composition of Purified Particles of this Invention Residue Percent EXPECTED
Ar~tILJO DETER-PreS -S PreS -PreS -S
ACIDS* MINED S 2 1 2 ASIJ + 5.5 4.2 5.2 7.3 ASP
THR 7.5 8.0 7.9 7.8 SER 12.5 11.3 12.0 10.8 GLN + GLU 7.5 4.2 4.5 5.1 1'RO 12..5 10.8 10.5 12.6 GLY 8.6 6.6 7.1 8.3 ALF~ 5 . 0 ? . 8 4 . 5 5 . 4 VAL 4.2 5.2 5.2 4.8 CYS 3.2 6.6 5.2 3.8 r2ET 1.9 2.3 2.2 1.9 ILE 4.9 7.5 7.5 6.7 LEU 12.8 15.5 13.9 12.1 'iYR 2.0 2.8 2.6 1.9 PHE 5.6 7.5 6.0 5.4 LYS 2.1 1.9 1.5 1.3 HIS 0.9 0.5 1.1 1.9 ARG 2.1 2.4 3.0 3.0 !a8.8o 100.1s 99.90 100.10 *Tryptophan is lost in the acid hydrolysis procedure.
- 1C>0 -I. Electro:a .~i::roscony of Purified Hepatitis B
Particl~=s of this Invention_ In the= electron microscope, the purified particles of t7zis invention were visualized as 05 spherical 22 nm particles which are similar to those typically Formed by the assembly of hepatitis B
S protein polypeptides.
J. Preparation of the Adjuvant of Purified Particles of this Invention and Stability Testing.
In order to prepare an adjuvant of the vaccine of this invention, :1/10,000 volume Thimerosol, and 1/10 volume of filter-sterilized 0.2 M Al K(S04)2:12 Fi20 are added to the desired concentration of antigen in sterile saline. The pH is adjusted to 5.0 wlth Sterile 1L~1 P~;faOH and the suspension is stirred at room temperature for 3 hours. The alum-precipitated antigen is recovered by centrifugation for 10 minutes at 2000 rpm, resuspended in sterile normal saline containing 1:10,()00 Thimerosol, and aliquoted under sterile con~3itions.
To determine the stability of the particles of this i.nvent:ion absorbed to alum, the antigen was recovered by re:~olubilizing the alum in 3% sodium citrate followed by successive dialyses against 3%
sodium citrate and PBS. The quantity of particles of this invention was then determined by parallel-line radioimmune assay against dilutions of a purified standard in PBS-~50% newborn calf serum. Stability testing results (Table 4) show the purified particles (either bulk or alum-absorbed) to be stable at 2°-8°C
for at least 7 months.
Table 4 Stability ~l~'eSting SDS-PAGE:
BandingPattern Results Bulk Sample Alum Absorbed Sample S torage i~9on 1 2 1 2 th Start normal normal normal normal 1 " " " "
" " " "
" " " "
" ~, " "
5 " " " "
" " "
" " " "
Quantitative Determination by RIA:
Percent (s) Activity Bulk Sample Alum Absorbed Sample Storage Month 1 2 1 2 Start 101 101 E> 100 101 100 100 ___ 1 3 4 0 9 3 4 K. Seroconversion and EDso of the Particles of this Invention.
The seroconversic>n testis are carried out on five groups of ten adult white r~iice (F?igures 8A, 8B and 8C) . Gm female 1CR strain swiss mice are injected subcutaneously with 1 ml of alum adjuva.nt vaccine containing particles of this invention at the following dilutions: 1/1 (5.0 ,ug), 1/4 (1.3 ,ug) , 1/16 (0.31 ,ug) , 1/64 (0. 16 ,ug) , and 1/256 (0.078 ,ug). Mice are bled after 28 days, and anti-body titer is quantitated by the AUSAB test in comparison with the HBIG standard.. The serum samples from each animal were diluted four-fold serially and tested in triplicate.
The percentage of mice exhibiting a positive serotype declined with increasing dilution of particles of this invention. The dose re~~ulting in 50% of the animals exhibiting a seropositi.ve response (EDso) is about 0.05-0.25 ~cg of vaccine containing particles of this invention. In general., the seroconversion results obtained with the vaccine of this invention are indistinguishable from those obtained with the naturally derived vaccine.
Example 2 Expression cf Part:icles Comprising PreSl-PreS2-S
Protein. Codina Recrion Polypeptides A. Preparation of Recombinant Plasmid pENDO-1.
Gene sequences from recombinant plasmids, pBglII2.8, pBR322.(3G2 (a plasmid made by subcloning into the EcoRI
site of pBR322 the 7 kb EcoRI (3G2 fragment from plasmid pMB9.(3G2 described by Tilghman, x - l03 - 1340934 S.M. et al, Proceeding Natl. Acad. Sci., Vol. 75, p. 725, 1978), pDM2, POLI:NK 23456 (see infra), and the pMMT-neo plasmid, are used in the steps to prepare vector constructions to produce recombinant expression vectors having no non-HPV vira7L genomic portions.
Referring to Figures 9A and 9B, the pBglII2.8 plasmid contains a gene segment: (1.34 kb BstEII-HpaI fragment) containing the PreSl-PreS2-S protein coding region and carries the natural strong promoter for transcription of the PreS2-S protein coding region, but lacks the natural (weak) promoter for transcription of the PreSl-PreS2-S
protein coding region, as well as the transcription termination functions and the polyadenylation signal.
Plasmid pBglII2.8 was ~;ubjected to BstEII and HpaI
digestion to produce two DNA fragments. Following separation by 3.5% polyacrylamide gel electrophoresis of the fragments, one of them (1.34 kb), which contains the PreSl-PreSz-S protein coding region, was purified and retained. The H~aI end. is blunt. The BstEII end was made blunt by filling in (ma.de double-stranded) using DNA
polymerase I (Klenow fragment) and the four dNTP's using conventional techniques to give rise to a DNA fragment with two blunt ends.
A polylinker plasmid, POLINK 23456, having a number of restriction sites contained within the polylinker region (below) was employed.
X
__ 1340934 ~
:C~aI Bc'_I foal C:laI _3in3III hen= _3am:~Il S_ohI _Sac. SaII
G=~:.TTCT=..G?.TC'_"GnTC~GTTA_aCA'rC:=:
T~~GCT'='GGiACCGGaTCCCGGGC~TGCG~GCTCGaG'~'~
Scoa_T 3ylII Smal Xhol Xnal waI
?.val The polylinker adapter is carried within the EcoRI-SalI
back-bone fragme:zt (3.1 kb) of pBR328. POLINK 23456 is digested with H~~I to linearize the plasmid. Such HpaI
linearized plasm.id bears two blunt ends. The linear plasmid is then blunt-end ligated with the 1.34 kb DNA
fragment containing the PreSl-PreS2-S protein coding region obtained :From the pBglII2.8 plasmid (above). Since the 1.34 kb fra<~ment can be incorporated into the polylinker plasm:id irm~ither orientation, the correct orientation (cloc=kwise with respect to the natural direction of transcription) is ascertained by digestion with EcoRI, followed by size analysis on to agarose gels.
EcoRI digestion of the plasmid carrying the PreSl-PreS2-S
protein coding region .in the correct orientation gives rise to two fragrnents of 0.4 kb and 4.1 kb; the incorrect orientation giver rise to two fragments of 1.0 kb and 3.5 kb. The new plasrlid containing the PreS1-PreS2-S protein coding region is then digested with HpaI and "partially"
digested with BaryHI. The short (24 b.p.) sequence between the H~aI and BamFiI restriction sites is separated from the HpaI-BamHI "partial" back-bone (5.0 kb) and discarded along with the fz-agmeni~s in the reaction mixture resulting from complete dic~estlOIl.
The pE3R322.~G2 plasmici is digested with Ball (blunt end) and BglII ("sticky" end) to obtain a gene secsuence (1.E. kb) containing the mg-PAS-t polyadenylation-termination sequence from the mouse 05 globin gene. The mg-PAS-t fragment is then ligated into the opened HpaI-BamHI linearized plasmid from above, Ball-to-HpaI and BglII-to-BamHI (BglII and BamF~I "sticky" ends are identicle and therefore are compatible, and suitable for ligation to each other), to form a new intermediate recombinant plasmid (6.0 kb) containing both the PreSl-PreS2-S protein coding region and the mg-PAS-t sequence in the correct orientations and order. The PreSl-PreS2-S protein coding region/mg-PAS-t-containing plasmi~:i is then digested with BglII and SalI, splitting the plasmid into two DNA fragments (2.95 kb and 3.05 kb), as ascertained by 1% agarose gel electrophori=sis. Following digestion of the 3.05 kb fragment with PvuI to produce two smaller fragments (1.08 kb and 1.98 kb), the remaining 2.95 kb BglII-SalI DNA fragment (containing no PvuI site) containing the fused PreSl-PreS2-S protein coding region/mg-P~~S-t segments is purified and retained.
'I'iie pMI~'IT-neo plasmid, the plasmid formed from ligation of the 6.65 fragment obtained in Example 1, Section A (see Figure 9) is then digested with BglII
and SalI resulting in two DNA fragments (4.23 kb and 2.42 kb). The 2.42 kb fragment containing the neomycine :election marker and the SV40 PAS-t is discarded. The remaining 4.23 kb fragment is purified a sing 0.8°s agarose gel electrophoresis and X340934.
is then ligated with the BglII-SalI 2.95 kb PreSl-PreS2-S protein coding region/mg-PAS-t DNA fragment obtained move to form a 7.15 kb recombinant plasmid containing in order: the MMT-promoter, the 05 PreSl-)?reS2-S protein coding region and the mg-PAS-t pol yadenylation and termination sequence, the plasmi3 being referred to as pENDO-1. The pENDO-1 plasmid contains no non-HBV
viral genomic portions ( s~=e Figure 9) .
B. Preparation of Recombinant Plasmid pELTDO-2.
F,eferri:zg to Figure 10, pDM2, which includes the MMT-promoter, thE= PreS2-S protein coding region and the protein X coding region, is digestecz with restriction enzymes SpeI and SalI. The SpeI
restriction the site is in the S protein rection of PreS2-S protein coding region and is unique in pDl~2, as i;s the SalI site downstream of the gene.
Using these two enzymes, the plasmid is split into two DNA fragment;> (1.58 kb and 4.88 kb) and the 1.58 kb fragment, containing the carboxy end of the PreS2-S protein coding region and the protein X
coding region, is discarded, while the 4.88 kb fragment is purified and retained.
The pEN:~O-1 plasmid is also subjected to SpeI and SalI digestion, splitting the plasmid into two DNA
fragments, 1.9 kb and 5.25 kb. The 1.9 kb fragment containing the carboxy end of the S protein region of the PreS2-~S protein coding region, plus the mg-PAS-t rec3ion is purified and retained.
~'lne two retained DLTA fragments (1.° kb and 4.88 kb) are then ligated to produce a new recombinant plasmi:3 (6,.8 kb,) designated pENDO-2 which contains the MMT-promoter, the PreS2-S protein coding region 05 and the mg--PAS-t sequence. Plasmid pENDO-2 contains no non-HBV viral genomic portions (see Figure 10).
C. Preparation of Recombinant Plasmid pENDO-0.
A third new plasmid, referred to as pENDO-0 (see Figure 11), containing a neomycine selection marker, is formed by digesting pLMiMT-neo, the plasmid formed from ligation of the 6.65 kb fragment obtained in Lxample 1,. Section A (see Figure 11), with restriction enzymes SmaI (blunt end) and BamHI. The plasmid is split into two DNA fragments (5.5 kb and 1.15 kb). The 5.5 kb fragment containing the PMMT-promoter and the neomycine selection marker from tile pl,lrlT-neo p7_asmid backbone is retained. The latter fragment is then ligated with the DNA (1.6 kb) fragment ccntaini.ng the Ball-BglII mg-PAS-t sequence as described with reference to the formation of pENDO-1. The resulting 7.2 kb recombinant plasmid, pELlDO-;), now contains the MMT-promoter, the neomycine selection marker and the mg-PAS-t sequence and no viral I)NA sequences (see Figure 11).
D. Introdu~~tion of Plasmid Vectors pENDO-0, pENDO-1 and wENDO-2 into r?ammalian Cells by Co-Transfection.
The recombinant plasmid vectors pENDO-0, pENDO-1 and pENDO-2 are co-transfected into mammalian cells, such as mouse L~-cells, Vero (African green monkey) cells, CHO cell; and 3T3 mouse fibroblasts, using standard co-transfection procedures. 6418-resistant transfectants are isolated and screened for production of the particles of this invention using the RIA method.
E. Preparation of Mixed Concatamers of pENDO-0, 05 pElvlDO-1, pECdL>O-2 Plasmid DNAs for Transfection of Cell Lines.
9Jach of t:he plasmids, pENDO-1, pENDO-2 and pEPTDO-0, contain a unique restriction site, PvuI, which exists within the Amp gene, counterclockwise to the EcorI restriction site (see Figures 9, 10 & 11).
Each o.f the plasmids is digested separately with PvuI
to linearize. Then such plasmids are polymerized to form mixed concatamers with various ratios of the different plasmids. For example, polymerization of the pFNDO-:L and pENDO-0 linearized plasmids using ligase, such as T4 DLdA ligase, can form a mixed concatamer with a ratio of 10:1 pENDO-l:pENDO-0, if the input ratio of the component linearized plasmids is lU:l pENDO-l:pENDO-0. The resulting pEivDO-1/pEL~rDO-o mixed concatamer is transfected into a eukaryotic cel7_, such as a mammalian cell, and the cell subjected to 6418 selection. It is believed, on the average, that every G47_8-resistant (transfected) cell shou7.d cc>ntain about ten copies of the PreSl-PreS2-S protein coding region per one copy of the neo gene.
Similarly, the pENDO-0 fragment is polymerized with the pENDO-2 fragment by ligase, such as T4 DNA
ligase, at a ratio of 1.0:1 pENDO-2:pENDO-0, for example, tc> form a pENDO-2/pENDO-0 mixed concatamer. ' The concatamer i;s then transfected into a eukaryotic cell, such as a mammalian cell. The cells will then be subjected to 6418 selection and on the average each 6418-resistant (transfected) cell will contain about ten copi.e;s of pEIdDO-1 to pENDO-0, in this example.
05 Similarly, pENDO-1, pENDO-2 and pENDO-0 are polymerized by ligase, such as T4 DNA ligase, to form a mixed concatamer at a ratio of 10:10:1, pENDO-l:pENDO-2:pENDO-0, for example. The resulting mixed concatamer is transfected into a eukaryotic cell, such as ma:~,~malian cell, and the cell subjected to 6418 selectic>n. On the average, the resistant (transfected) cells should contain ten copies of pEi;DO-1. to ~~en copies pELdDO-2 to one copy pENDO-0.
Fach of the resulting resistant cells should result in higl-ier yields of the particles containing the proteins encoded by the PreSl-PreS2-S protein coding region.
1340934 ~' - llo -Applications of Non~Re~li.cating_Vectors Comprising the Mouse bletallothionein Promoter to the Expression of Other Proteins in TransfEected Fukaryotic f-lost Cells 0~
The invention also comprises recombinant DNA
transfer vectors comprising the M.MT promoter, any functional DNA sc~quenc~= and additional DNA other than viral DNA.
By the term functional DNA sequence is meant any discrete region of DNA derived directly or indirectly from any source which functions in a eukaryotic host cell transfected with the vector of this invention as a complete gene .expression module, a structural gene, 1~ a promoter or regulatory region.
By complete gene expression unit is meant a structural gene and the promoter and other regulatory regions required for its transcription and translation.
By promoter is meant any region upstream of a structural gene which permits binding of RNA
polymerase and initiation of transcription.
By regulatory region is meant any region which regulates the rate or extent of transcription of the 2~~ structural gene.
By structural gene is meant a coding sequence which serves the template for the synthesis of messenger RNA.
Preferred structural genes include those coding ?0 for polypeptides of pharmaceutical importance such a.s viral antigens, insulin, interferon or other l.ynphokines, rat, human or other growth hormones, Tissue Plasminoge~n Activator, alpha-1-antiprypsin anc).
1340934 , the like. Most preferred is rat or human growth hormone.
This invention relates to a recombinant DNA
vector comprisin~~ the functional DNA sequence and an 0~~ MMT-promoter. The MM'r-promoter may be incorporated into the DNA vec~_or either in addition to the natural promoter for th~~ DNA sequence or in place of the natural promoter. Preferably, the MMT-promoter is located in the D1~1A vector immediately upstream of the 1C~ DNA sequence protein coding regian. Preferably, such vector also comprises a transcription termination sequence and a selection marker. Preferably, such selection marker is a drug-resistance marker, such as the neomycine gene. Preferably, such transcription 1~ termination sequence is an SV40 termination site (sv-PAS-t) or more preferably the DEF region (mg-PAS-t) of th<~ mouse globin gene. Such vector may be prepared by conventional recombinant DNA and other molecular biological techniques. In the most 20 preferable case, using the mg-PAS-t region, the vector does not <~ontain any viral DNA segments and is not oncogenic, i.e., it will not transform (make cancerous) any host. cell into which it is introduced. Such vector, upon transfection into a 25 host, if it does not contain an autonomous replication sequenc<a (replicon) capable of functioning in a host transfected therewith, integrates into the host chromosome and replicates passively with the hash genome.
3C~ Preferably, the recombinant DNA vector of this invention should comprise the following characteristics:
134pg34 .
1) The DNA sequence of interest.
2) An MMT-promoter located immediately upstream of the DNA sequence of interest.
3) The vector should be able to replicate in 0.'i bacteria, or other procaryotic host into which it is transformed, For growth, amplification and preparation of large quantities of the recombinant vector. Thus, such vector should include a bacterial or other procaryotic replicon, i.e., a DNA segment bearing all the functions required for autonomous replication arid maintenance of the vector extrachromosomally in a procaryotic host cell, such as a bacterial host cell, transformed therewith.
Such replicons are well known in the art.
1.'i 4) The vector replicon should be small (i.e., probably smaller than 6-8 kilobase pairs) to enable easy genetic arid molecular biological manipulation thereof.
5) The vector should carry a selection marker, preferably a drug-resistance marker such as ampicillin, fog usc~ in bacterial host cells transformed therewith.
6) The vector should carry a second selection marker, preferably a drug-resistance marker such as 2!5 neomycine, for use as such in eukaryotic host cells transfected therewith.
7) The vector should contain convenient endonuclease restriction sites for cloning.
8) The vector ;should contain a transcription termination and polyadenylation sequence.
9) The gene expression module does not contain any viral segments and is not oncongenic.
Most preferably the vector of this invention does not comprise an autonomous replicating sequence (replicon) capable oi= functioning in a eukaryotic host cell transfected therewith. A primary reason 05 forusing a non-replicating vector system in eukaryotic host cells is that. all known vector systems capable of autonomous and extrachromosomal replication in a mammalion eukaryotic host cell transfected therewith comprise replicons which are derived from onc:ogenic viruses. It is desirable to employ vectors comprising DNA not derived from oncogenic viruses for expressing DNA sequences encoding po7.ypept:ides of pharmaceutical importance.
This invention also relates to a transfected host 1'. eukaryotic cell transformed with a recombinant DNA
vector containin<~ the functional DNA sequence of this invention. Prei=erably, such host is a mammalian cell, most preferably a Chinese Hamster ovary (CHO) cell line, a vero cell line, an L-cell line, or a mouse or rat fiY~robla:~tic cell line. Such host may be prepared by transfe~cting a eukaryotic cell with a recombinant DNP, vector of this invention by conventional techniques.
This invention also relates to a method for preparing the proteins coded for by the DNA sequence of interest. which comprises: a) cultivating the transfected host: of this invention under culture medium condition; which enable such host to express such proteins, and b) isolating such proteins.
Preferably, such transfected host is able to secrete such proteins into the medium. Preferably, heavy metal ions or steroid hormones, such as dexamethasone, are added to such culture medium to induce the MHT-promoter and thereby enhance expression of such coding region. Heavy metal ions such as cadmium or ::inc are most preferred. The OF~ optimal concentration of heavy metal ions or steroid hormone contained in t:he medium can be determined by conventional_ techniques.
This invention also relates to the proteins prepared by suc:z method. If the transfected host cell of this invention secretes such proteins directly into tl~e culture medium, such proteins can then be isolated from the culture medium of the transfected host of this invention by conventional protein isalation techniques. If the transfected 1~ host cell of tr.is invention does not secrete such proteins, they are obtained from a culture lysate of such host by con~~entional culture lysate techniques.
Example 3 2C~ Expression of Human Growth Hormone Preparation of a Recombinant DNA Vector Encoding Human Growth Hormone, Said Vector Comprising the Metallothionein Promoter 2c A. Isolation of a DNP, Fragment Containing the Human Growth Hormone Gene Without the Natural Promoter For Transcription.
The recombinant plasmid pBR322 containing a 2 kb 30 EcoRI DNA fragment insert encoding the human growth hormone gene (in a counterclockwise orientation) is digested with the restriction endonuclease BamHI in a total volume of 500 ;ul containing 100 ~g of plasmid DNA, 240 units BamHI, in BamHI (high salt) buffer.
Two fragments are generated, one being about 5.6 kb and _another fragment. 0.8 kb. The fragments are 05 separated by e:~ectrophoresis in a 0.8$ preparative agarose ge.l. 'fhe larger fragment, containing the structural gene for human growth hormone without the natural promoter for gene, is electro-eluted from the gel and purified by two cycles of phenol/chloroform extraction, two cycles of chloroform extraction and two cycles of ethanol precipitation. The pelleted DNA is resuspended in 20 ~1 10 mM Tris, pH 8Ø
B. Isolation of a DNA,Fr_agment Containing the Metal-1-'~ lothionein Promoter and the pML2 Vector Backbone.
Plasmid pMMT-neo (see Figure 5) is digested with restriction endonucleases BglII and BamHI to produce two fragments, one being 4.5 kb and containing the MMT-promoter and the pML2 vector backbone, and the other being 2.15 kb and containing the neo gene coupled to the s'r-PAS-t segment. In order to prevent self-ligation, the .Linear DNA is treated with alkaline phosphatase in a total volume of 100 ~ul containing 25 dug of the DNA fragment, and, 28 U of alkaline phosphatase. The mixture is incubated 20 minutes at 37°C, and the reaction is stopped by adding 10 ~ul 50 mM EI)T,A and heating for 10 minutes at 68°C. The fragments are separated by electrophoresis in a 0.8$ agarose gel. The 4.5 kb fragment is electro-eluted from the gel as described and is purified by t:wo cycles of phenol/chloroform extraction, two cycles of chloroform extraction and 134093 .
two cycles of e~thano~L precipitation. The pelleted DNA is resuspende~d in 2.0 ~ul 10 mM Tris, pH 8Ø
C. Lilgation of the DL~fA Fragments Isolated _in A. and B. Above.
05 A 1.5 dug quantity of the 5.6 kb fragment from A.
above is taken a;zd mixed with 0.3 dug of the fragment from B. above in a total volume of 10 ~ul containing 0.9 units of T4 DNA-ligase and 1X ligation buffer, plus 100 mM ATP. The' mixture is incubated for 1 hour at 15°C and overnight at 4°C. The ligation mixture is then introduc~sd into the bacterial strain, HB101, using the procedure described in Materials and Procedures. Cells from the transformation mixture are spread onto LB-ampicillin agar plates and isolated colonies are screened for those containing plasmids of 10.1 kb in size that can be linearized with restriction endonuclease BamHI. The orientation is determined by EcoRI digestion. Plasmids containing the insert: in the correct orientation yield two EcoRI fragments, one of 3.5 kb and the other of 6.6 kb. Such plasmids, designated pMMT-hGH, are subjected to large scale growth and purification as described.
D. Introduction of the Recombinant DNA Vector Obtained in C. Af>ove into Mammalian Cells and Establishment of Cell Lines Producing Human Growth Hormone.
The vector, pMMT-hGH, is then transfected into eukaryotic host cells (mammalian) by standard co-transfection techniques to yield transfectants that synthesize the human growth hormone polypeptide. Such transfectants are identified by the RIA technique using antibodies to human growth hormone. Transfectants showing secretion of human growth hormone are established as cell lines using 05 conventional techniques.
Example 4 Expre=~sion of Rat Growth Hormone 1~ Preparation of a Recombinant DNA Vector Comprising the Rat Growth Hormone, Said Vector Containing the Metallothionein Promoter.
A. Conversion c~f the- BglII- Restriction Endonuclease l.'~ Site in pMMT-neo to XhoI.
A 200 fag quantity of pMMT-neo is digested with 500 U of restriction endonuclease BglII in medium salt buffer in ,~ total reaction volume of 1,000 ~ul.
The ends of the 6.65 hb linear plasmid DNA are first 20 made blunt by filling in the BglII ends using DNA
polymerase I (Kl_enow fragment) and the four dNTP's, as described previously, followed by attachment of the following synthetic DNA linker fragment containing the XhoI restriction endonuclease recognition and 2~~ cleavage site (from PL Biochemicals, Milwaukee, Wisconsin 53205):.
5'-CCTCGAGG-3' 3'-GGAGCTCC-5' 3C The filling in reaction is performed in a total volume of 30 ~ul cont,aining 2.5 dug of the 6.65 kb fragment, 200 mM of the four dNTP's, 3 dal NT-buffer, l~ul Klenow fragment. The mixture is incubated at room temperature for 30 minutes. The reaction is stopped by the addition of 2 N1 0.25 M EDTA and the DNA is washed once with an equal volume of phenol/chloroform and then with an equal volume of 0'.i chloroform alone. The DNA in the aqueous phase is further purified by two ethanol precipitations. The precipitated DNA is resuspended in 10 ~..il 10 mM Tris, pH 8.0, at a con~~entration of 50 ng//ul.
The linker fragment is dissolved in 50 ~ul of 10 mM Tris, pH 8.1, at a concentration of 1 pmole/J.zl.
The blunt-ended 6.65 kb fragmen t is ligated to the linker fragment in a total volume of 20 ~ul containing 2 pmoles of the linker fragment, 0.5 dug of the 6.65 kb fragment., 9 units of T4 DNA-ligase (Boehringer 1_'i Mannheim), 30 ~u:L 2X blunt-end ligation buffer, plus 100 mM ATP. The ligation is performed at 15°C for one hour and at ~~°C overnight.
This ligation mixture is used to transform the bacterial strain, HB101, which is made competent as described in Materials and Procedures. Cells from the transformation mixture were spread on LB-ampicillin agar plates. Bacterial colonies able to grow on ampicillin plates are picked and grown up in 2 ml cultures and subjected to plasmid preparations as descrilbed above.
From 12 bacterial clones, four (OK 3, OK 4, OK 11, OK 12) were identified as having a plasmid showing the expected size after digestion with XhoI
alone or together with BamHI and EcoRI. The plasmid DNA from OK 3 and OK 7L1 were propagated in HB101 for large scale plasrnid preparations.
B. Isolation of a DNA Fragment Containing the Metal-lothionein Promoter and the pML2 Vector Backbone.
A 100 dug quantity of each of OK 3 and OK 11 was digested with th<~ restriction endonucleases BamHI and 05 Xhol which yield two fragments, one (5.8 kb) with the MMT-promoter located close to the XhoI end and the pML2-part at the BamHI end, a.nd the other (0.85 kb) containing the sv--PAS-t sequence. The fragments were separated by electrophoresis in 0.8$ agarose.
The 5.8 kb fragment was purified by electro-elution, followed by two cycles of phenol/chl_oroform extraction, two cyclea of chloroform extraction and two cycles of Ethanol precipitation. The pelleted DNA was resuspended i.n 50 ~.zl 10 mM Tris, gH 8Ø
1 '~
C. Isolation of a DNA Fragment Containing the Rat Growth Hormone Ge_n~e_.
Plasmid pBR~s22.rGH, containing a genomic BamHI
insert encoding the rat growth hormone gene (in a counterclockwise orientation), was digested with BamHI and XhoI. The genomic BamHI-XhoI fragment containing the structural gene is isolated from a 0.8$ preparative agarose gel as above.
2~ D. Ligation of the Fragments Isolated as in B. and C_ Ahnve_ The fragments are ligated to each other such that the MMT-promoter will be linked, via the Xhol "sticky" end dii:ectly to the structural gene via its XhoI sticky end, in the correct orientation.
The ligation mixture is used to transform the bacterial strain, HB101, as described above. Trans-1 3 40 9 :~'~
formants forming colonies on LB-ampicillin agar plates are picked and grown up in 2 ml cultures for small scale plasmid preparation. Plasmids yielding the two expected fragments upon digestion with BamHI
0!p and XhoI, designated pMMT-rGH, are then grown up in large scale for transfection into mammalian cells.
E. Introduction of the Re cc>mbinant DNA Vector Obtained in D. Above into Mammalian Cells and Establishment of Cell Lines Producing Rat Growth Hormone.
The vector, pMMT-rGH, is then transfected into eukaryotic host: cells (mammalian) by standard co-transfection techniques to yield transfectants that synthesize the rat growth hormone polypeptide.
1_'. Such transfectamts are identified by the RIA
technique using antibodies to rat growth hormone.
Transfectants sh~~wing secretion of rat growth hormone are established as cell lines using conventional techniques.
2C~ Although the present invention has been described with reference to preferred embodiments, workers skilled in the a.rt will recognize that changes may be made in form and detail without departing from the spirit and scope of thc~ invention.
c. Nick Translation. Nick-translation is performed according to Rigby et al (J. Mol. Biol., Vol. 113, pp. 237-251, 1977). The reaction mixture for 32P-labeling of DNA usually contains 0.5 dug of, for example, the EcoRI-XbaI fragment of pD~l2, in a total volume of :30 ~.Z1 with 50 mM Tris, pH 7.8, 5 mM
Mc~Cl2, 10 mTM mercaptoethanol, 0.1 mIYI dATP, 0.1 mM
dGTP, 0.1 mT1 dT'TP, 50 uCi 32P-dCTP, 10 units DNA
polymerase I, 3 ~zl of a 2 x 10 5 fold dilution of 1 mg/ml DNase I a:nd is incubated for 90 minutes at 15°C, yielding .'3 x 106 to 12 x 106 total cpm, i . a . 1 x 10 '~ to 5 x 107 cpm/~g DLdA.
d. Transformation of Competent Bacterial Cells. Fro:n a dense overnight culture, 1 ml of the bacterial cell suspension is taken for innoculation of 100 ml growth medium (L-broth). The cells are grown at 37°C to a density of OD550 - 0.7 which is reached within 2 hours with vigorous shaking in a 500 ml Erlenmeyer flask. Growth is stopped by chilling the culture on ice for 10 minutes. From this culture, 3 rnl is taken for harvesting the exponential bacterial cells at 3,000 rpm for 5 minutes. The cells are resuspended in 1.5 ml of 50 mM CaCl2 in 10 mP~ Tris, pH 8.0, and incubated on ice for another -73- ~ ~ 40 93 4 :, 15 minutes. The cells are harvested once more by centrifugation of: 3,000 rpm for 5 minutes and resuspended in 200 ,ul of 50 mM CaCl2 in 10 mM Tris, pH 8.0, and kept S at 4°C overnight.
Thereafter, the l.igation mixture was filled up with 10 mM
Tris, pH 7.5, 1 ~~M EDTA, to a total volume of 70 ,ul and added to the 200 ~.sl bacterial cell suspension for DNA
take-up.
The mixture was incubated on ice for 30 minutes, then 1 ml L-broth was added. and t:he mixture was incubated at 42°C
for 2 minutes and. at 37°C for 40 minutes. After the incubation, the cells were spread on agar plates containing 50 ,ug ampic:illin/ml agar at volumes of 50 ,ul up to 300 ,ul of the cell suspension per plate. The agar plates were incubated at 37°C overnight. After this incubation period, single isolated bacterial colonies were formed .
e. Southern Blot F~nalysis. To characterize the organization within the: host cell genome of the vectors of this invention, chromosomal DNA from cell lines producing particles of this invention is isolated and digested with the appropriate restriction enzymes) and analyzed by the method of Southern (J. Mol. Biol., Vol.
98, pp. 503-517, 1975) using a 3'P-labeled DNA probe.
Following digestion of the chromosomal DNA (20 ,ug) with restriction enzymes EcoRI and XbaI, the resulting fragments are separated. by 0.7% agarose gel electrophoresis. Thereafter, the DNA is denatured by exposing to 366 nm W light for 10 minutes and by incubation in a solution of 0.5N NaOH and 1 M NaCl for 45 minutes. The gels are neutralized by X
~340~34 _ 74 -incubation in 0. _'>tz Tris, 1.5 ri NaCl, pH 7. 5 for 60 minutes. Tze DNA is transferred to a nitrocellulose filter by soaking in 3 IM NaCl, 0.3 t1 NaCitrate (20XSSC) for 20 hours through the gel by covering the 05 top of the nitrocellulose filter with a staple of dry paper towel:. Th;e nitrocellulose filter is kept for 2 hours in a vacuum oven at 80°C.
The rad.ioact:ive DNA probe from the EcoRI-~~baI
fragment of the pDtil plasmid ( 4. 3 kb ) i s prepared by nick-translation.
For hybridization with the DNA probe, the nitrocellulose falter is sealed in a plastic bag containing 10 rn.l of prehybridization mixture . 500 formamide, _'s x SSC, 50 mM sodium-phosphate, pH 7.0, 5 x Denhardt's solution, 250 ~g/ml denatured salmon sperm DNA. The :Filter is incubated in this mixture for 4 hours at 45°C, after which the pre-hybridi2;ation mixture is replaced by the hybridizatic>n mixture: 50% formamide, 5 x SSC, 20 mM
?G Na phosphate, pH 7.0, 1 x Denhardt's solui.ion, 100 ~ug/ml denatured salmon sperm DNA, 5 x 105 cpm/ml 32P-probe. The filter, after incubating in the hybridization mix for 18 hours at 45°C, is washed three times, 5 minutes each, in 0.1 x SSC, 0.1% SDS
at 50°C. The filter is dried at 60°C for 10 minutes and exposed to two X-ray films (Y,AR-5, KODAK) between two intensifying screens and kept at -80°C. The first X-ray film is developed after 3 days' exposure;
the second film after 7 days' exposure.
HlTbridization of the labeled DNA probe to one of cellular DN~~ restriction fragments from transfectants which produce part=icles of this invention - 75 _ demonstrates that the cellular DNA does in fact contain th~= integrated vector of this invention.
Since the cellular DNA restriction pattern, as elucidated by the Southern blot procedure, is 05 identical to that. of the original plasmid construct, no major rearrangement of plasmid DNA occurred upon intec3ration into the host cell DNA.
2. Transfection of Mammalian Cell Lines and IdE:ntification of Clones Producing Particles of this Invention.
Cells a:=a transfected using the calcium phosphate precipitation method of vaigler et al (Cell, Vol. 14, p. 725, 1978). Cells are then split 1:6 into new culture dishes, and cells which have received the drug-resistance marker gene (neo) are selected by groc~ath in 6418-containing medium. After 10 days of grovath in :elective medium, drug-resistant colonies are cloned into micro-titer plates. Levels of expression of the particles of the present invention ara assessed by the P,IA technique after the cells have reached. confluency.
Tissue culture dishes (100 mm diameter) are innoculated with 5 x 105 cells and incubated overnight at. 37°C. Four hours prior to transfection, the culture medium is replaced with fresh medium.
DNA to be transfected is suspended in 1 ml of solution containing 250 mP? CaCl2, 140 mPM NaCl, 25 ml~I Hepes, f>H7.1 and 0.75 mP-~ NaHP04. This calcium phosphate - DNA precipitate is added to the cells in culture. After an overnight incubation, fresh medium is added to the cell culture and the cells are incubated for an addition two days.
_ 76 _ F. Procedures for Static Cell Culture of fiammalian Cells.
An ampule of cells held at 196°C in liquid nitrogen is thawed quickly by placing the ampule in a 05 37°C water bath for 5 minutes. One ml of the freshly-thawed cell suspension is diluted by slowly adding 10 ml of the appropriate culture medium.
Thereafter, the cells are harvested by centrifugation, resuspended in 10 ml of culture medium. T~lereafter, the cells are harvested by centrifugation, resuspended in 10 ml of culture medium, transferred to a T25 culture flask, and grown in an incubator at 37°C and 5o C02. V~Tith these conditions, the doubling time is in the range of 15 to 20 hours for :L-cells and CHO cells, and 30 to 40 hours for Vero cells. L-cells and CHO cells can be '.kept alive and growing beyond 1008 confluency, whereas Vero cells are passaged at 100 confluency.
G. Procedures for L~lammalian Cell Growth in ACUSYST-P
2G and ~~arvest o:f Particles of this Invention.
1. Growth and Preparation of Innoculum.
Cells are maintained in T75 or T150 flasks containing D.rsEM-1.0°s FBS. Roller bottles ( 850 cm2 ) are innoculated with 10-15 x 106 cells harvested from T-flasks. After 3 to 5 days of growth, approximately 10~~ total cells are harvested from six roller 'bottles and used to innoculate six hollow fiber cartridges (HFC's) in an ACUSYST-P* made by Endotronics, Inc. of Minneapolis, Minnesota U.S.A.
2. ACt~SYST-1? Culture and Harvest of Particles of this :Invention.
After i:nnoculation, the HFC's are inserted into the ACUSYST-~P culi=ure system, and the medium flo4a *Trade mark 1 340 93 t~ ~.
rate through the cartridges is adjusted to and mintained at 50 ml/hr. Medium is assayed daily for pH, pO2, glucose and lactate. After 2 to 3 weeks of growth, a 600 ml fluid volume is harvested two 05 times weekly from the extracapillary space of each ACUSYST-P hollow fiber cartridge. Production of particles of they subject invention is assayed by the radioimmunoassay (RIA). Since freezing destroys RIA
assayable activity, harvests are stored at 4°C prior to purification.. No protease activity can be detected under these conditions as assayed by Azocoll substrate proteo:Lysis (SIG??A) .
- 3. ~ualitv Control Procedures.
'= a. _Te:~ts for the Presence of Bacteria and Fungi. A pool of cells containing about 5 x 105 cells per ml, suspended in the cell culture medium from which they were harvested, is tested for the presence of bacteria and fungi. One ml of cells is streaked onto trypticase soy agar (BBL) plates, and another 1 nil is innoculated into thioglycolate medium (DIFCO) to test for the presence of aerobic as well as anaerobic bacteria. Fungal contamination is assayed by streaking 1 ml of cells onto Sabouraud _ (DIFCO) agar plat=es. These tests were performed on a regular basis to ensure sterility of growth medium and absences of bacterial and fungal contamination in cell cultures.
b. Te=>t for the Presence of Myconlasma:
Fluorescence Staining The presence of mycoplasmal DNA in the cell cytoplasm is determined by a staining method using they fluorochrome, Hoechst 33258* This DNA-staining dye fluoresces under ultraviolet light *Trad'.e Marks 1340934 ~
_ 78 _ and provider the basis for a very rapid and sensitive test for mycoplasmas. The procedure involves coverslip cultures of the cells to be tested which are used when 5(J-70s confluent. After fixing and 05 staining, the coverslips are examined with a fluorescent microscope. Cytoplasmic fluorescence indicates the pre:~ence of mycoplasma.
The stack of the Hoechst 33258 bisbenzamide fluorochrome solution is made by dissolving 5 mg in 100 ml PBS using a magnetic stirrer. It is sterilized by filtration through a 0.22 um membrane stored in the dark at 4°C, and diluted a thousand-fold with PBS before use. The medium from coverslip cultures, which are 50-70% confluent, is aspirated from th.e cells and fixed with two changes of ethanol:acetic acid (3:1) at 4°C. Following one wash with deionized water and incubation for 30 minutes at 3'7°C with diluted bisbenzamide fluorochrome (Hoechst-33258), the coverslips are rinsed tail deionized water. The coverslips are mounted, ce~.l-side down, on a slide using a glycerol mountant (22.2 ml. 2.1~ citric acid; 1 ml H20; 27.8 ml 2.8o Na2HPA4; 50 ml glycerol pH 5.5).
c. Testis for the Presence of Viruses.
1) Tests in Cell Cultures. Tester cells are examinE~d for normal morphology during an incubation period of at least 14 days after innoculation with a suspension of the cell line being tested. In addition, on days 3-5, and again after the 12th da:y, at least 4% of the innoculated tester cultures is washed and tested for hemadsorption using red cells from sheep or humans. Tests are read after _ 1340934.
_ 79 -the cultures have' incubated for 30 minutes at 3°-4°C
and again after 30 minutes at 34°-37°C. Results of tests for virus-~~nfected cells which absorb erythro-cytes indicate no viral contamination.
05 2) Tests in Animals. Cells to be tested are suspended in 140 mM NaCl, 2.7 mM KC1, 8.1 mL~i Na~HP04, pH 7.2 at a concentration of 106 per ml and are innoculated into different groups of animals. I::~ one group, at least 10 animals from at least two litters of suckling mice are innoculated.
Each animal is innoculated with 0.1 ml cells intraperitoneally and 0.01 ml intracerebrally. The mice are observed daily for at least 14 days. Each mouse that dies after the first 24 hours of the test, or is sacrificed because of illness, is necropsied and examined for evidence of viral infection. Such examination involves additional testing by intracerebral and. intraperitoneal subinnoculation of appropriate tissue suspensions into an additional group of at. least five suckling mice following by daily observations for 14 days. In addition, a blind passage is made from a single pool of the emulsified tissue (minus skin and viscera) of all mice surviving the original. 24-day test.
In another group, 10 adult mice are also innoculated. Each animal is innoculated with 0.5 ml cells intrape:ritoneally and 0.03 cells intracerebrally. The animals are observed for four weelcs and any that become sick or show abnormality is examined to establish the cause of illness. Results of the tests :in animals for the presence of contarlinating viruses indicate no viral contamination.
'3~+~934 _80-d. Testing for Tumorigenicity. A suitable test for tumorigenicity using nude mice (nu/nu) involves innocula.ting 20 animals within 24 hours of birth with ~.1 ml. of potent serum. The injection is 05 given either by the intramuscular or subcutaneous route and is repeated on days 2, 7 and 14 of life.
One million reference tumor cells routinely produces progressive7_y growing tumors and metastases. One million viable cEalls of the candidate cell line are innoculated by tree subcutaneous route at any site at which developing tumors can be palpitated (the limbs are suitable). The animals are observed for 21 days for evidence of nodule formation at the site of injection, and measurements are made periodically to determine whether there has been progressive growth.
At the end of the 21-day observation period, all animals are sacrificed and examined for gross evidence of tumor formation at the site of injection and in other organs such as the lymph nodes, lungs, vidneys and liver. All tumor-like lesions ana all innoculation site's are examined histopathologically.
In addition, since some cell lines may form metastases without evidence of local tumor growth, the lungs and regional lymph nodes of all animals are examined hi:~tologically.
For the purpose of this test, a progressively growing tumor is defined as a palpable nodule that increases in size' over the 21-day observation period and that shows. viable and mitotically active innoculated cell; when examined histologically. The presence ~~f microscopically viable cells in association with a stationary or regressing nodule is not consideoed a progressively growing tumor.
1340934 ' H. Purification of the Particles of this Invention.
1. Fractional Precipitation with Polyethylene ~'col ( F?EG ) .
Piedium f:rom t'he extracapillary space is harvested 05 from the AC:USYST-P* culture system and pooled into volumes of 3000 ml. To each volume, 180 g of PEG
8000 (SIGP~A) are added, dissolved by stirring at room temperature for 20 minutes and stirred for another 3 hours at ~,°C. The precipitate is collected by centrifugation in 500 ml bottles in a GS 3 rotor at 4500 rpm (:3000 x g) for 30 minutes at 10°C. The supernatant is collected and 180 g of PEG 8000* are again added and dissolved at room temperature as described above. The solution is stirred at 4°C for an additional 3 hours. The precipitate from this solution is collected as described above except that centrifugation is performed at 9000 rpm (15,000 x g) for 60 minutes. The pellet is resuspended in 20 ml of phosphate buffe=red saline (PBS).
~0 2. Gel Filtration Chromatography.
The material obtained after PEG precipitation, and redissolved in PBS, is submitted to gel filtration c=hromatography using BioRad A-5m*resin at 4°C. Column dimensions are 25 x 1000 mm, and the bed volume is 480 ml. In a typical fractionation run, 1000 ~g of PEG-precipitated particles of this invention in a volume of 10 to 15 ml is loaded and eluted with PBS or THE at a speed of 6 drops/minute (18 ~l/hr) in fractions of 3 ml. Figure 6 shows the profile of RIA-as;sayable material eluted from an A-5m column. The solid line indicates the 280 nm *Trade mark 134~~34 absorbance of each fraction, and the dots indicate amount of n.aterial calculated from the RIA results.
3. Isopycnic Centrifugation in CsCl.
About 30 fractions covering the first peak 05 resulting :From gel filtration column chromatography and containing partially purified particles of this invention are pooled (approximately 100 ml). This solution is adjusted to a density of 1.30 g/cc with CsCl and subsequently transferred to nitrocellulose tubes fitting into a SVJ 27/28 rotor (Beckman).
Gradients are seat by underlaying 4 ml of a CsCl solution of 1.35 g/cc and by overlaying 4 ml of 1.25 g/cc and 4 ml o:E 1.20 g/cc density. Gradients are run at 28, 030 rpm for 50 hours at 10°C, fractionated, and the purified particles in the 1.20 g/cc density layer at the tc>p of the gradient (Figure 7) is collected. Purified particles are well separated from the small amount of contaminating protein banding in the mp.ddle of the gradient. The solution was desalte~~ by dialysis, first against water, then against 3 changes of saline, over a 24-hour period.
As a further quality control measure, a portion of this gradient-purified material is submitted to linear CsCl-gradient centrifugation. A single peak of purified particles appears as expected at 1.2 g/cc.
I. Preparation o:E the Adjuvant of Purified Particles of this Invention and Stability Testing.
In order to prepare an adjuvant of the vaccine of this invention, 1/10,000 volume Thimerosol, and 1/10 volume of filter-sterilized 0.2 M A1 K(S04)2:12 H20 are added to the desired concentration of antigen in sterile saline. The pH is adjusted to 5.0 1340934 ' with steri7_e 1N NaOH and the suspension is stirred at room temper=ature for 3 hours. The alum-precipitated antigen is recov~'red by centrifugation for 10 minutes at 2000 rpm, resuspended in sterile normal saline 05 containing 1:10,000 Thimerosol, and aliquoted under sterile conditions.
To determine the stability of the particles of this invention absorbed to alum, the antigen was recovered ;~y re~solubilizing the alum in 3~ sodium citrate fo.Llowed by successive dialyses against 30 sodium citrate and PBS. The quantity of particles of this inven~~ion :is then determined by parallel-line radioimmune assay against dilutions of a purified FiBsAg standard ir.~ PBS-50o newborn calf serum.
Table 1 Stability Testing Parameters Samples: two each of bulk and alum absorbed 30 Sample Concentration: 5 ~c;/ml Storage Container: glass vials Storage Temperature: 2°-8°C
Tests: (a) Qualitative: SDS-PAGE
(b) Quantitative: Radioimmunoassay Example 1 E:K ression of Particles Comprising PreSl-Pr~sS2-S Protein Coding Region Poly~peptides A. Preparation of Recombinant Plasmid pMMT-neo.
The plasmid pBPV342-12 was digested with the restriction-endonuclease BamHI (see Figure 1 and Materials and Procedures). Two DNA molecules were generated: one molecule (7.95 kb) comprises the entire gent>me of bovine papilloma virus (BPV); the other molecule (6.65 kb) contains part of the bacterial plasmi.d pML2 (pBR322 deleted for the "poison sequence"), the mouse metallothionein promoter (pLMI~iT), the neomycine resistance gene (neo) and the SV9~0 PAS-t (see Figure 1). When ligated to itself (circularized), this fragment gives rise to plasmid pP~M3T-neo (see also Figure 5).
The rea~~tion was performed in a total volume of 400 y~l of reaction buffer (see P~iaterials and nrocec~ures) at a final concentration of 0.2 ~g/~l pBPV342-12 I»1A; and 80 units of BamHI at 37°C for 4 hours. The completion of the digest was checked by agarose gel electrophoresis in a 0.7o agarose gel (see Materials a.nd Procedures). The reaction was stopped by adding 40~u1 of 8 t-1 LiCl and the DNA was X340934 ~, precipitated with 1 ml of ethanol at -80°C for 30 minutes. The precipitated DNA was resuspended in 100 ~1 of 10 mM Tris, pH 7.8.
B. Isolation of a Fragment Containing the Entire 05 PreSl-PreS2-S Protein Coding Region Along with the Natural Promoter of Transcription.
The prE~paration and cloning of the HBV genome, subtype adw, is described by Cummings, I.W. et al in Proc. LJatl. Aca. Sci., U.S.A., Vol. 77, p. 1842, 1980. The circular HBV genome was linearized at the unique EcoRI site for cloning into bacteriophage lambda gtWES and for subcloning in a bacterial plasmid to yield plasmid pA01 (see Figure 2). Since the EcoRI restriction site lies within the PreSl-PreS2-S protein coding region, the latter is interrupted when in the EcoRI linear form. To regenerate an intact PreSl-PreS2-S protein coding region, the 3.2 kb EcoRI insert of pA01 was isolated and separated from the plasmid DNA by digesting 100 ~zg of pA01 in a total volume of 400 j~l of reaction buffer (seep Materials and Procedures) containing 200 units EcoRI for 4 hours at 37°C. The 3.2 kb insert DNA was separated from the plasmid DNA by preparative 0.7~ agarose gel. electrophoresis (see Materials and Procedures). They DNA was electro-eluted from the agarose gel on DE 81*Whatman ion exchange filter from which the DNA is removed in a high salt solution.
The DNA was purified by one phenol/chloroform extraction and two ethanol precipitations. The purified 1:W ear 3.2 kb EcoRI fragment encoding the complete HBV genome was then subjected to self-ligation at a high DNA concentration in order to favor formation c>f concatamers: 30 ~ g of EcoRI
*Trade mark:
fragment D:VA ways ligated in a total volume of 50 ~ul of reaction buffer containing 1.8 units T4 DNA ligase and 2 mM A'rP at 15°C for 1 hour and at 4°C overnight;
thereafter, th.e DNA was purified by two 05 phenol/chloroform extractions, one chloroform extraction, followed by two ethanol precipitations.
The pellet--_d DNA was resuspended in 20 ~ul of 10 mP4 Tris, pH 7.8, a,nd digested with restriction enzyme BglII in a total_ volume of 50 ~ul of reaction buffer ( see Materials and Procedures ) containing 50 units of BglII at 37°C for 4 hours.
The DNl?, fragments generated by this reaction were separated by 1.5% agarose gel electrophoresis. The 2.7e kb suze Bc~lII fragment containing the intact PreSl-PreS2-S protein coding region, together with its natural promoter system necessary for expression of the surface antigen polypeptides, was isolated from the agarose gel and purified as described above (see Figure 2).
C. Insertion of the Fragment Containing the PreSl-PreS2-~~ Protein Coding Region into the pPM4T-neo Plas;mid: Construction of pDMl.
From a DNA solution prepared as described under part A. above, 1 ~ul was ta);en and mixed with 5 Jal of the 2 . 78 kb ~,I I fragment ( 0. 25 yg/~1 ) described under part :B, above.
The mixture was submitted to a ligation reaction at a tota.L volume or 10 ~ul of reaction buffer containing 0.9 units of T4 DNA ligase at 15°C for 1 hour and at 4°C overnight.
Bacterial cells, preferably HB101, were made competent for taking up DNA in a transformation l3t,D934 -, reaction a~ccord:ing to the method described in Materials a.nd Procedures. The ligation mixture was made 10 mM Tris, pH 7.5, 1 mM EDTA, in a total volume of 70 ~Z1 and added to 200,/ul competent bacterial cell 05 suspension for DhfA take-up.
The mixture was incubated on ice for 30 minutes, then 1 ml L-broth was added and the mixture was incubated at 42°C for 2 minutes and at 37°C for 40 minutes. After t=he incubation, the cells were spread on LB agar plate's containing 50 ~tzg ampicillin/ml at volumes of 50 ~ul up to 300 ~ul of the cell suspension per plate. The agar plates were incubated at 37°C
overnight. After this incubation period, single isolated h~acterial colonies were screened for the presence o:. the pMMT-neo bacterial plasmid containing the desired 2.713 kb BglII DNA fragment insert (see Figure 3).
Screen=~~ng was preferentially performed by the colony hybridization procedure. For this procedure, single colonies were picked with a toothpick and transferred to a.n LB-ampicillin-containing agar plate in a grief fornnat to allow identification of the clones. Tile plate was incubated overnight at 37°C.
The colonies were transferred to a nitrocellulose filter by layering the filter on the agar surface until the filter was wet. The filter was quickly peeled off and sequentially transferred to three layers of Whatman 3M paper, each of them either soalced in 0.5 1'~I LdaOfi, or 1 M Tris, pH 7.0; 1.5 M
NaCl/0.5 r9 Trig;, pH 7.4, or 2 X SSC. The filters were placed on the soaked paper, colony-side up, during the lysis of the cells and the following neutralization and fixing procedures.
_88_ After the filters were dried in air, they were ba~;ed at 80°C .in vacuum. Thereafter, the nitro-cellulose filter; were submitted to DNA hybridization with a radioactively labeled DNA probe made from the 05 2.78 kb BglII fragment containing the PreSl-PreS2-S protein coding region, prepared as described in part B above, by nick-translation (see Materials a:nd Procedures).
The nitrocellulose filter was incubated in a pre-hybridization mix containing 50% formamide, 20 mr-1 sodium phosphate buffer, pEi 6.6; 1 X Denhardts solution; 1.00 ~g~/ml denatured salmon sperm DNA and 1 x 107 cpm 32P-labeled DLdA which was r.:elted in U.2 N LdaOH ~st 68°C for 10 minutes.
The filter was incubated in this mixture for 16 hours at 45°C. Thereafter, the filter was washed twice in 2 X SSC, 0.1'6 UDJ, for 5 minutes each cycle at room temperature and twice in O.1XSSC, 0.1% SDS, for 15 minutes each cycle at 50°C. The filters were 2U exposed to an ?;-ray film (preferably 3M) between two screens at -80°C for two days.
Colonies containing the recombinant plasmid carrying th.e cloned 2.78 kb Bc~lII fragment appeared as black spots. Four out of 50 colonies were identified and t7ze plasmid DNA of these colonies was isolated in min:ipreparations according to Birnboim and Doly, l~Tucl. Acids Res., Vol. 7, p. 1513, 1979, and analyzed by restriction endonuclease digestion by a BglII rind XbaI double digest. The analyses confirmed t:he intended construction explained above (see Figure 3).
D. Substitution of the Natural Promoter for the PreSl-P:reS2-S Protein Coding Regions by the Metallothionein Promoter: Construction of pDf-I2.
The plaamid pDMl described above was submitted to 05 complete digestion with the restriction enzymes BglII
and BamHI in a total. volume of 100 ~.Z1 containing 20 ,fig of plasmid DLJA and first with 50 units B~lII in reaction buffer containing 6.7 mM Tris, pH 7.8; 6.7 mM MgCl2; 6.7 ml'-1 ~ -mercaptoethanol, at 37°C for 4 hours; then the salt concentration was raised to 150 mM NaCl and the digestion was completed by addition of 40 units BamHI at 37°C overnight. Three fragments were generated, t:he large fragment being 5.1 kb, the medium fragment being 2.97 kb and the small fragment being 1.4 kb. These fragments were separated by preparative 0.7~ agarose gel electrophoresis from v.~hich the (5.1 kb and 1.4 kb) fragments were isolated electrophor~etically using DE 81* Whatman ion exchange filter.
2U The DD1A wa.s removed from this filter by incubation in high salt for 4 hours at 4°C and purified by phenol/chloroform extraction and two ethanol precipitations. The DNA was resuspended in ~1 of 10 mM Tris, pH 7.8. One ~1 was checked for purity and estimation of quantity by agarose gel electrophoresis.
The large (5.1 kb) fragment containing the metallothionein F~romoter at the BglII end was ligated to the small (1.4 kb) fragment containing the PreS2-S protein coding region, but not the natural promoter o:r the PreSl portion of that gene. The large fragment also contained the natural *Trade mark 1340934 ;
termination-polyadenylation signal (hb-PAS-t) necessary for e~;pression of the hepatitis B PreS2-S
protein coding region (see also Figure 4).
BecausE~ the large fragment also contains the C5 bacterial ;~lasmid, it is possible that self-ligation of this fragment will propagate itself without incorporating 'the small size BamHI fragment.
Therefore, 10 ,~a7L of the total 20 ~.zl preparation of the large fragment were subjected to treatment with alkaline phosphatase by adding 28 units of this enzyme and incubating at 37°C for 20 minutes. The reaction was stopped by adding 1 ~ul 50 mtd EGTA and incubating at E>8°C for 10 minutes. The DNA was purified by two ethanol precipitations in 0.8 M LiCl.
The pelleted DNA was resuspended in 10 jzl 10 mM
Tris, pH 7.8, of which 5 ~1 was taken as a control and checked for self-ligation and another 5 ~1 was mixed with 5 ~1 of the electroeluted small (1.4 kb) BamHI fragment.. Ligation of each sample was performed in a total volume of 20 ~1 as described above. Transformation on HB101 bacterial cells was carried out. as above.
~'welve single colonies were picked and grown in 2 ml cultures and the plasmid DNA was isolated according to Birnboim and Doly, supra. The plasmid DNA was checked for the insertion and orientation of the small aize BamHI fragment by a double digest with the restriction enzymes EcoRI + XbaI.
Two of the twelve plasmid DNAs have the small (1.4 kb) BamI-i:I fragment inserted in the same orientation as the metallothionein promoter.
These plasmid. DNAs are grown in mass culture and prepared for introduction by co-transfection into eukaryotic c=ells.
Alterna~=ively, the EcoRI/BglII (1.9 kb) fragment 05 from plasmi.d pMMT-neo carrying the metallothionein promoter is used for substituting the short (32 b.p.) EcoRI-BamiiI fragment in front of the PreS2-S
protein coding rE~gion in pDMl to give plasmid pDM3, as illustrai=ed in Figure 5.
E. Introduction of the Recombinant DNA Vectors Obtained Under Parts C. and D. above into Mammalian Cells and the Establishment of Cell Lines Producing Particles of this Invention.
The recombinant plasmids, pDMl and pDM2, were co-transfeci=ed into mouse L cells, vero (African green monkey) cells, CHO cells, and 3T3 mouse fibrobl_asts by standard transfection procedures (Graham and van der Eb, Virology, Vol. 52, p. 456, 1973, supra) (See Materials and Procedures). Cells w~~ich take up and maintain the plasmid DL~tA are resistant 1=o the drug 6418 and survive in the selective medium containing this drug. Cells which do not ta~;e up the DNA do not survive in the selective medium. These cells are detected as single clones on the surface of a culture plate. The cells were picked with a cloning cylinder and propagated to prepare for mass culture in a conventional manner in a usual nutrient medium, e.g., Dulbecco's modified Eagle medium supplemented with loo calf serum and 500 ~.zg G418 per ml (see Materials and Procedures) . Five of these clones were established as cell lines, and designated ENDO-I, -II, -III, -IV, and -V, and frozen stocks were prepared (see Materials and Procedures).
The production rate of these cell lines in static culture was determined by radioimmune assay (RIA; see Materials and Procedures) and compared to the 05 production rate c>f known cell lines. On the average, the cell lines ~sccording to the invention produced 500 ng to 1.,000 :ng of the particle of this invention per ml of culture medium per day.
F. Acusyst-P Culture of Cell Lines Producing the Particles of the Invention.
Approximately 109 total cells grown in DMEM+10%
FBS in s:ix roller bottles (see Materials and Procedures) were used to innoculate six hollo~. fiber cartridges in t:he Acusyst-P* culture system (see Materials and Procedures). Approximately 600 ml fluid volume was harvested two times weekly from the extracapillary space of each Acusyst-P hollow fiber cartridge and stared at 4°C prior to purification.
The concentration of the particles of this invention was assayed by RIA (see f~Iaterials and Procedures).
G. Purification Particles of the Invention from Acusyst-P Culture tdedium.
Particles containing proteins coded for by the PreSl-PreS2-S protein coding region produced by the novel cell lines were purified by polyethylene glycol (PF~G) precipitation, gel filtration and isopycnic ultracentrifugation in CsCl gradients (see below).
1. Fractional Precipitation with Polyethylene G1 cy of (PEG).
Medium from the extracapillary space was harvested from the ACUSYST-P culture system and *Trade mark ~ ~ 4~ 93 4 _ 93 pooled into volumes of 3000 ml. To each volume, 180 g of PEG 8000 (SIGMA) were added, dissolved by stirring at room temperature for 20 minutes and stirred for another 3 hours at 4°C. The precipitate 05 was collected by centrifugation in 500 ml bottles in a GS 3 rotor at 4500 rpm (3000 x g) for 30 minutes at 10°C. The supernatant was collected and 180 g of PEG
8000* are again added and dissolved at room temperature as described above. The solution was stirred at 4°C for an additional 3 hours. The precipitate from this solution was collected as described above except that centrifugation was performed a.t 9000 rpm (15,000 x g) for 60 minutes.
The pellet was resuspended in 20 ml of phosphate buffered saline (PBS).
2. Gel Filtration Chromatography.
The mai_erial obtained after PEG precipitation, and redissolved in PBS, was submitted to gel filtration chromatography using BioRad* A-5m resin at 4°C. Column dimensions were 25 x 1000 mm, and the bed volume was X180 ml. In a typical fractionation run, 1000 dug of PEG-precipitated particles of this invention in a volume of 10 to 15 ml is loaded and eluted with PBS or THE at a speed of 6 drops/minute (18 ml/hr) in fractions of 3 ml. Figure 6 shows the profile of the particles of this invention eluted from an A-5m co:Lumn. The solid line indicates the 280 nm ab~;orbance of each fraction, and the dots indicate the quantity of the particles of this invention in each fracton as calculated by RIA.
*Trade lark 3. Is~cpycnic Centrifugation in CsCl.
About 30 fractions covering the first peak resulting from gel filtration column chromatography (see Figure 6) and containing partially purified 05 particles of. this invention were pooled (approximate=ly 100 ml). This solution was adjusted to a density of 1.30 g/cc with CsCl and subsequently transferred to nitrocellulose tubes fitting into a S4J 27 or S4J 28 rotor (Beckman) . Gradients were set by underlaying 4 ml of a CsCl solution of 1.35 g/cc and by overlaying 4 ml of 1.25 g/cc and 4 ml of 1.20 g/cc density. Gradients were run at 28,000 rpm for 50 hours a.t 10"C, fractionated, and the purified antigen in the 1..20 g/cc density layer at the top of the gradient was collected. The particles of this invention were well separated from the small amount of contaminating protein banding in the middle of the gradient (see Figure 7). The solution was desalted by dialysis against three changes of saline over a 24-hour peruod.
As a quality control measure, a portion of this gradient-purified material was submitted to linear CsCl-gradient centrifugation. A single peak of the particles of this invention appeared as expected at 1.2 g/cc.
H. Characterization of the Particles of this Invention Produced by Cell Lines.
The purity anal physical identity of the polypep tides of the particles of this invention in prepara tions are established by conventional laboratory techniques, such as radioimmune assay, immunoprecipi-tation and ~>DS-PAGE (see P4aterials and Procedures).
1. Purity I>eterminations.
a. Determination of Levels of Contamina-ting Plasma Proteins.
The levels of various serum proteins in prepara 05 tions of purified prticles of this invention are assessed by the :standard RIA technique using specific antibodies against bovine serum albumin, IgA, IgG, IgM, etc. The results shown in Table 2 show no detectable immunoglobulin contamination and only minor albumin contamination.
Table 2 Determination of Contaminatin P:Lasma Proteins by RIA
Protein % of Total Proteins Albumin 0.05 IgG 0.2 IgA 0.2 Ic~M 0.2 b. Determination of Levels of Contaminating Host Cell or PreSl-PreS2-S Nucleic Acid Seguences in Purified Preparations of the Particles of this Invention by the Dot Blot Procedure.
A dot-blot hybridization is performed on purified particles of this invention in order to check for contaminating PreSl-PreS2-S DNA sequences or host chromosomal DNA ;sequences. For each spot, a 100 ng quantity of puri:Eied particles of this invention in Jal 0.25 LJ LJaOF3 is heated at 6S°C for 10 minutes.
As a positive control, 20 pg of recombinant plasmid pDMl DNA of this invention in 40 ~1 0.25 N NaOH is treated the same way. Immediately after denaturation in NaOH, the solution is spotted onto a 05 nitrocellulose filter. The following treatment of the filter and the hybridizing procedures are analogous to those described for the Southern Blot procedure, except that the 32P-labeled probe is a 50:50 mixture of chromosomal DNA and plasmid DNA of this invention containing the PreSl-PreS2-S
protein coding rE:gion. As expected, the hybridizing probe gives a strong signal with the recombinant plasmid DN~~ of this invention, but only a weak background signal with the purified particle preparation. This hybridization pattern indicates no PreSl-PreS2-S D1~1A or chromosomal DNA in the sample.
2. Immunoprecipitation and SDS-Polyacrylamide _Ge1 Analysis.
To det:ermin~e the pattern of PreSl-PreS2-S
polypeptide;~ produced by transfectants of this invention, proteins produced by such transfectants are biosynthetically labeled, immunoprecipitated and analyzed on SDS-PAGE gels (see Materials and Procedures). ~C'he electrophoretic pattern of proteins, isolated from culture medium by immuno-precipitation with antibodies to naturally derived HBV, was visuali2,ed by the silver staining procedure.
The two major proteins correspond in size to nonglycosylated and glycosylated polypeptides encoded by the S pr~~tein coding region (24 kd and 27 kd, _ 97 _ respectively). Two minor proteins of 33 kd and 36 kd correspond in si~;e to the two polypeptides encoded by the PreS2-:~ prot.ein coding region. This method is not sensitive enough to detect the small quantities 05 of polypeptidea containing the PreSl-PreS2-S
polypeptides, but the presence of the proteins encoded by the entire PreSl-PreS2-S protein coding region wa:a detected by the western immunoblot procedure (see, i.nfra). The weak bands at the top of the gel result from aggregated protein and are also visible with naturally-derived HBV particles. Thus, cells transfected with the PreSl-PreS2-S protein coding region secrete proteins which not only react with antibodies against the natural product, but also are identical in size. The presence of proteins which are i~3entical in size to the naturally-occurring glycosylated PreSl-PreS2-S, PreS2-S and S poly peptides from the hepatitis E virus suggests that glycosylati~~n pathways function normally in these cells.
3. Immunoblot (4Jestern) .
To establish that each of the polypeptides species appearing in the silver stained SDS-PAGE gel system reacts with specific anti-HBV antibody, the 4destern immunoblot procedure was employed. Proteins separated in a SDS-PAGE were transferred to a nitrocellulose filter (Schleicher & Schull) by electroblotting (BioRad) at 4°C, 100 V, 3 hours.
After transfer, t:he filter is saturated with proteins in order to avoid nonspecific binding of peroxidase-labeled antibody. To do this, the filter is incubated for 1 hour at room temperature in 20~
- 1340934 ' newborn calf serum in 1?BS, and the polypeptides of the particle of this inveni:ion were made visible by HBV-specific antibodies conjugated with peroxidase. The S filter is placed on parafilm and covered with a solution of the conjugated antibodies for 3 hours at room temperature in a wet chamber. The filter is washed six times with 0.5% Tween* in lOmM Tris and 5 mM CaCl and subsequently covered with the chromogen, POD
(o-phenylenediaminedihydrochloride) in hydrogen peroxide (0.3g/1) in citrate-phosphate-buffer. Stopping solution is 0.5 N sulfuric acid. The protein bands corresponding to all six viral pclypept:ides appear, i.e., all the proteins coded for by the PreSl-PreSz-S protein coding region.
4. Amino Acid Composition of Purified Proteins Coded for by the PreSl-PreS,~-S Protein Coding Region Comprising the Particles of this Invention.
Following acid hydroly~~is, the amino acid composition of the purified proteins coded for by the PreSl-PreS2-S
protein coding region i.s determined and compared with the polypeptide composition. calculated from the nucleotide sequence of the S protean coding region, the PreS2-S
protein coding region or the PreSl-PreS2-S protein coding region. The results presented in Table 3 correlate well with values predicted from the DNA sequences as well as with published results (Shiraishi et al, J. Gen. Virol., Vol. 48, p. 31, 1980). In particular, the relatively high percentage of serine, proline and leucine is characteristic of these polypeptides.
*Trade mark Table 3 Amino A-id Composition of Purified Particles of this Invention Residue Percent EXPECTED
Ar~tILJO DETER-PreS -S PreS -PreS -S
ACIDS* MINED S 2 1 2 ASIJ + 5.5 4.2 5.2 7.3 ASP
THR 7.5 8.0 7.9 7.8 SER 12.5 11.3 12.0 10.8 GLN + GLU 7.5 4.2 4.5 5.1 1'RO 12..5 10.8 10.5 12.6 GLY 8.6 6.6 7.1 8.3 ALF~ 5 . 0 ? . 8 4 . 5 5 . 4 VAL 4.2 5.2 5.2 4.8 CYS 3.2 6.6 5.2 3.8 r2ET 1.9 2.3 2.2 1.9 ILE 4.9 7.5 7.5 6.7 LEU 12.8 15.5 13.9 12.1 'iYR 2.0 2.8 2.6 1.9 PHE 5.6 7.5 6.0 5.4 LYS 2.1 1.9 1.5 1.3 HIS 0.9 0.5 1.1 1.9 ARG 2.1 2.4 3.0 3.0 !a8.8o 100.1s 99.90 100.10 *Tryptophan is lost in the acid hydrolysis procedure.
- 1C>0 -I. Electro:a .~i::roscony of Purified Hepatitis B
Particl~=s of this Invention_ In the= electron microscope, the purified particles of t7zis invention were visualized as 05 spherical 22 nm particles which are similar to those typically Formed by the assembly of hepatitis B
S protein polypeptides.
J. Preparation of the Adjuvant of Purified Particles of this Invention and Stability Testing.
In order to prepare an adjuvant of the vaccine of this invention, :1/10,000 volume Thimerosol, and 1/10 volume of filter-sterilized 0.2 M Al K(S04)2:12 Fi20 are added to the desired concentration of antigen in sterile saline. The pH is adjusted to 5.0 wlth Sterile 1L~1 P~;faOH and the suspension is stirred at room temperature for 3 hours. The alum-precipitated antigen is recovered by centrifugation for 10 minutes at 2000 rpm, resuspended in sterile normal saline containing 1:10,()00 Thimerosol, and aliquoted under sterile con~3itions.
To determine the stability of the particles of this i.nvent:ion absorbed to alum, the antigen was recovered by re:~olubilizing the alum in 3% sodium citrate followed by successive dialyses against 3%
sodium citrate and PBS. The quantity of particles of this invention was then determined by parallel-line radioimmune assay against dilutions of a purified standard in PBS-~50% newborn calf serum. Stability testing results (Table 4) show the purified particles (either bulk or alum-absorbed) to be stable at 2°-8°C
for at least 7 months.
Table 4 Stability ~l~'eSting SDS-PAGE:
BandingPattern Results Bulk Sample Alum Absorbed Sample S torage i~9on 1 2 1 2 th Start normal normal normal normal 1 " " " "
" " " "
" " " "
" ~, " "
5 " " " "
" " "
" " " "
Quantitative Determination by RIA:
Percent (s) Activity Bulk Sample Alum Absorbed Sample Storage Month 1 2 1 2 Start 101 101 E> 100 101 100 100 ___ 1 3 4 0 9 3 4 K. Seroconversion and EDso of the Particles of this Invention.
The seroconversic>n testis are carried out on five groups of ten adult white r~iice (F?igures 8A, 8B and 8C) . Gm female 1CR strain swiss mice are injected subcutaneously with 1 ml of alum adjuva.nt vaccine containing particles of this invention at the following dilutions: 1/1 (5.0 ,ug), 1/4 (1.3 ,ug) , 1/16 (0.31 ,ug) , 1/64 (0. 16 ,ug) , and 1/256 (0.078 ,ug). Mice are bled after 28 days, and anti-body titer is quantitated by the AUSAB test in comparison with the HBIG standard.. The serum samples from each animal were diluted four-fold serially and tested in triplicate.
The percentage of mice exhibiting a positive serotype declined with increasing dilution of particles of this invention. The dose re~~ulting in 50% of the animals exhibiting a seropositi.ve response (EDso) is about 0.05-0.25 ~cg of vaccine containing particles of this invention. In general., the seroconversion results obtained with the vaccine of this invention are indistinguishable from those obtained with the naturally derived vaccine.
Example 2 Expression cf Part:icles Comprising PreSl-PreS2-S
Protein. Codina Recrion Polypeptides A. Preparation of Recombinant Plasmid pENDO-1.
Gene sequences from recombinant plasmids, pBglII2.8, pBR322.(3G2 (a plasmid made by subcloning into the EcoRI
site of pBR322 the 7 kb EcoRI (3G2 fragment from plasmid pMB9.(3G2 described by Tilghman, x - l03 - 1340934 S.M. et al, Proceeding Natl. Acad. Sci., Vol. 75, p. 725, 1978), pDM2, POLI:NK 23456 (see infra), and the pMMT-neo plasmid, are used in the steps to prepare vector constructions to produce recombinant expression vectors having no non-HPV vira7L genomic portions.
Referring to Figures 9A and 9B, the pBglII2.8 plasmid contains a gene segment: (1.34 kb BstEII-HpaI fragment) containing the PreSl-PreS2-S protein coding region and carries the natural strong promoter for transcription of the PreS2-S protein coding region, but lacks the natural (weak) promoter for transcription of the PreSl-PreS2-S
protein coding region, as well as the transcription termination functions and the polyadenylation signal.
Plasmid pBglII2.8 was ~;ubjected to BstEII and HpaI
digestion to produce two DNA fragments. Following separation by 3.5% polyacrylamide gel electrophoresis of the fragments, one of them (1.34 kb), which contains the PreSl-PreSz-S protein coding region, was purified and retained. The H~aI end. is blunt. The BstEII end was made blunt by filling in (ma.de double-stranded) using DNA
polymerase I (Klenow fragment) and the four dNTP's using conventional techniques to give rise to a DNA fragment with two blunt ends.
A polylinker plasmid, POLINK 23456, having a number of restriction sites contained within the polylinker region (below) was employed.
X
__ 1340934 ~
:C~aI Bc'_I foal C:laI _3in3III hen= _3am:~Il S_ohI _Sac. SaII
G=~:.TTCT=..G?.TC'_"GnTC~GTTA_aCA'rC:=:
T~~GCT'='GGiACCGGaTCCCGGGC~TGCG~GCTCGaG'~'~
Scoa_T 3ylII Smal Xhol Xnal waI
?.val The polylinker adapter is carried within the EcoRI-SalI
back-bone fragme:zt (3.1 kb) of pBR328. POLINK 23456 is digested with H~~I to linearize the plasmid. Such HpaI
linearized plasm.id bears two blunt ends. The linear plasmid is then blunt-end ligated with the 1.34 kb DNA
fragment containing the PreSl-PreS2-S protein coding region obtained :From the pBglII2.8 plasmid (above). Since the 1.34 kb fra<~ment can be incorporated into the polylinker plasm:id irm~ither orientation, the correct orientation (cloc=kwise with respect to the natural direction of transcription) is ascertained by digestion with EcoRI, followed by size analysis on to agarose gels.
EcoRI digestion of the plasmid carrying the PreSl-PreS2-S
protein coding region .in the correct orientation gives rise to two fragrnents of 0.4 kb and 4.1 kb; the incorrect orientation giver rise to two fragments of 1.0 kb and 3.5 kb. The new plasrlid containing the PreS1-PreS2-S protein coding region is then digested with HpaI and "partially"
digested with BaryHI. The short (24 b.p.) sequence between the H~aI and BamFiI restriction sites is separated from the HpaI-BamHI "partial" back-bone (5.0 kb) and discarded along with the fz-agmeni~s in the reaction mixture resulting from complete dic~estlOIl.
The pE3R322.~G2 plasmici is digested with Ball (blunt end) and BglII ("sticky" end) to obtain a gene secsuence (1.E. kb) containing the mg-PAS-t polyadenylation-termination sequence from the mouse 05 globin gene. The mg-PAS-t fragment is then ligated into the opened HpaI-BamHI linearized plasmid from above, Ball-to-HpaI and BglII-to-BamHI (BglII and BamF~I "sticky" ends are identicle and therefore are compatible, and suitable for ligation to each other), to form a new intermediate recombinant plasmid (6.0 kb) containing both the PreSl-PreS2-S protein coding region and the mg-PAS-t sequence in the correct orientations and order. The PreSl-PreS2-S protein coding region/mg-PAS-t-containing plasmi~:i is then digested with BglII and SalI, splitting the plasmid into two DNA fragments (2.95 kb and 3.05 kb), as ascertained by 1% agarose gel electrophori=sis. Following digestion of the 3.05 kb fragment with PvuI to produce two smaller fragments (1.08 kb and 1.98 kb), the remaining 2.95 kb BglII-SalI DNA fragment (containing no PvuI site) containing the fused PreSl-PreS2-S protein coding region/mg-P~~S-t segments is purified and retained.
'I'iie pMI~'IT-neo plasmid, the plasmid formed from ligation of the 6.65 fragment obtained in Example 1, Section A (see Figure 9) is then digested with BglII
and SalI resulting in two DNA fragments (4.23 kb and 2.42 kb). The 2.42 kb fragment containing the neomycine :election marker and the SV40 PAS-t is discarded. The remaining 4.23 kb fragment is purified a sing 0.8°s agarose gel electrophoresis and X340934.
is then ligated with the BglII-SalI 2.95 kb PreSl-PreS2-S protein coding region/mg-PAS-t DNA fragment obtained move to form a 7.15 kb recombinant plasmid containing in order: the MMT-promoter, the 05 PreSl-)?reS2-S protein coding region and the mg-PAS-t pol yadenylation and termination sequence, the plasmi3 being referred to as pENDO-1. The pENDO-1 plasmid contains no non-HBV
viral genomic portions ( s~=e Figure 9) .
B. Preparation of Recombinant Plasmid pELTDO-2.
F,eferri:zg to Figure 10, pDM2, which includes the MMT-promoter, thE= PreS2-S protein coding region and the protein X coding region, is digestecz with restriction enzymes SpeI and SalI. The SpeI
restriction the site is in the S protein rection of PreS2-S protein coding region and is unique in pDl~2, as i;s the SalI site downstream of the gene.
Using these two enzymes, the plasmid is split into two DNA fragment;> (1.58 kb and 4.88 kb) and the 1.58 kb fragment, containing the carboxy end of the PreS2-S protein coding region and the protein X
coding region, is discarded, while the 4.88 kb fragment is purified and retained.
The pEN:~O-1 plasmid is also subjected to SpeI and SalI digestion, splitting the plasmid into two DNA
fragments, 1.9 kb and 5.25 kb. The 1.9 kb fragment containing the carboxy end of the S protein region of the PreS2-~S protein coding region, plus the mg-PAS-t rec3ion is purified and retained.
~'lne two retained DLTA fragments (1.° kb and 4.88 kb) are then ligated to produce a new recombinant plasmi:3 (6,.8 kb,) designated pENDO-2 which contains the MMT-promoter, the PreS2-S protein coding region 05 and the mg--PAS-t sequence. Plasmid pENDO-2 contains no non-HBV viral genomic portions (see Figure 10).
C. Preparation of Recombinant Plasmid pENDO-0.
A third new plasmid, referred to as pENDO-0 (see Figure 11), containing a neomycine selection marker, is formed by digesting pLMiMT-neo, the plasmid formed from ligation of the 6.65 kb fragment obtained in Lxample 1,. Section A (see Figure 11), with restriction enzymes SmaI (blunt end) and BamHI. The plasmid is split into two DNA fragments (5.5 kb and 1.15 kb). The 5.5 kb fragment containing the PMMT-promoter and the neomycine selection marker from tile pl,lrlT-neo p7_asmid backbone is retained. The latter fragment is then ligated with the DNA (1.6 kb) fragment ccntaini.ng the Ball-BglII mg-PAS-t sequence as described with reference to the formation of pENDO-1. The resulting 7.2 kb recombinant plasmid, pELlDO-;), now contains the MMT-promoter, the neomycine selection marker and the mg-PAS-t sequence and no viral I)NA sequences (see Figure 11).
D. Introdu~~tion of Plasmid Vectors pENDO-0, pENDO-1 and wENDO-2 into r?ammalian Cells by Co-Transfection.
The recombinant plasmid vectors pENDO-0, pENDO-1 and pENDO-2 are co-transfected into mammalian cells, such as mouse L~-cells, Vero (African green monkey) cells, CHO cell; and 3T3 mouse fibroblasts, using standard co-transfection procedures. 6418-resistant transfectants are isolated and screened for production of the particles of this invention using the RIA method.
E. Preparation of Mixed Concatamers of pENDO-0, 05 pElvlDO-1, pECdL>O-2 Plasmid DNAs for Transfection of Cell Lines.
9Jach of t:he plasmids, pENDO-1, pENDO-2 and pEPTDO-0, contain a unique restriction site, PvuI, which exists within the Amp gene, counterclockwise to the EcorI restriction site (see Figures 9, 10 & 11).
Each o.f the plasmids is digested separately with PvuI
to linearize. Then such plasmids are polymerized to form mixed concatamers with various ratios of the different plasmids. For example, polymerization of the pFNDO-:L and pENDO-0 linearized plasmids using ligase, such as T4 DLdA ligase, can form a mixed concatamer with a ratio of 10:1 pENDO-l:pENDO-0, if the input ratio of the component linearized plasmids is lU:l pENDO-l:pENDO-0. The resulting pEivDO-1/pEL~rDO-o mixed concatamer is transfected into a eukaryotic cel7_, such as a mammalian cell, and the cell subjected to 6418 selection. It is believed, on the average, that every G47_8-resistant (transfected) cell shou7.d cc>ntain about ten copies of the PreSl-PreS2-S protein coding region per one copy of the neo gene.
Similarly, the pENDO-0 fragment is polymerized with the pENDO-2 fragment by ligase, such as T4 DNA
ligase, at a ratio of 1.0:1 pENDO-2:pENDO-0, for example, tc> form a pENDO-2/pENDO-0 mixed concatamer. ' The concatamer i;s then transfected into a eukaryotic cell, such as a mammalian cell. The cells will then be subjected to 6418 selection and on the average each 6418-resistant (transfected) cell will contain about ten copi.e;s of pEIdDO-1 to pENDO-0, in this example.
05 Similarly, pENDO-1, pENDO-2 and pENDO-0 are polymerized by ligase, such as T4 DNA ligase, to form a mixed concatamer at a ratio of 10:10:1, pENDO-l:pENDO-2:pENDO-0, for example. The resulting mixed concatamer is transfected into a eukaryotic cell, such as ma:~,~malian cell, and the cell subjected to 6418 selectic>n. On the average, the resistant (transfected) cells should contain ten copies of pEi;DO-1. to ~~en copies pELdDO-2 to one copy pENDO-0.
Fach of the resulting resistant cells should result in higl-ier yields of the particles containing the proteins encoded by the PreSl-PreS2-S protein coding region.
1340934 ~' - llo -Applications of Non~Re~li.cating_Vectors Comprising the Mouse bletallothionein Promoter to the Expression of Other Proteins in TransfEected Fukaryotic f-lost Cells 0~
The invention also comprises recombinant DNA
transfer vectors comprising the M.MT promoter, any functional DNA sc~quenc~= and additional DNA other than viral DNA.
By the term functional DNA sequence is meant any discrete region of DNA derived directly or indirectly from any source which functions in a eukaryotic host cell transfected with the vector of this invention as a complete gene .expression module, a structural gene, 1~ a promoter or regulatory region.
By complete gene expression unit is meant a structural gene and the promoter and other regulatory regions required for its transcription and translation.
By promoter is meant any region upstream of a structural gene which permits binding of RNA
polymerase and initiation of transcription.
By regulatory region is meant any region which regulates the rate or extent of transcription of the 2~~ structural gene.
By structural gene is meant a coding sequence which serves the template for the synthesis of messenger RNA.
Preferred structural genes include those coding ?0 for polypeptides of pharmaceutical importance such a.s viral antigens, insulin, interferon or other l.ynphokines, rat, human or other growth hormones, Tissue Plasminoge~n Activator, alpha-1-antiprypsin anc).
1340934 , the like. Most preferred is rat or human growth hormone.
This invention relates to a recombinant DNA
vector comprisin~~ the functional DNA sequence and an 0~~ MMT-promoter. The MM'r-promoter may be incorporated into the DNA vec~_or either in addition to the natural promoter for th~~ DNA sequence or in place of the natural promoter. Preferably, the MMT-promoter is located in the D1~1A vector immediately upstream of the 1C~ DNA sequence protein coding regian. Preferably, such vector also comprises a transcription termination sequence and a selection marker. Preferably, such selection marker is a drug-resistance marker, such as the neomycine gene. Preferably, such transcription 1~ termination sequence is an SV40 termination site (sv-PAS-t) or more preferably the DEF region (mg-PAS-t) of th<~ mouse globin gene. Such vector may be prepared by conventional recombinant DNA and other molecular biological techniques. In the most 20 preferable case, using the mg-PAS-t region, the vector does not <~ontain any viral DNA segments and is not oncogenic, i.e., it will not transform (make cancerous) any host. cell into which it is introduced. Such vector, upon transfection into a 25 host, if it does not contain an autonomous replication sequenc<a (replicon) capable of functioning in a host transfected therewith, integrates into the host chromosome and replicates passively with the hash genome.
3C~ Preferably, the recombinant DNA vector of this invention should comprise the following characteristics:
134pg34 .
1) The DNA sequence of interest.
2) An MMT-promoter located immediately upstream of the DNA sequence of interest.
3) The vector should be able to replicate in 0.'i bacteria, or other procaryotic host into which it is transformed, For growth, amplification and preparation of large quantities of the recombinant vector. Thus, such vector should include a bacterial or other procaryotic replicon, i.e., a DNA segment bearing all the functions required for autonomous replication arid maintenance of the vector extrachromosomally in a procaryotic host cell, such as a bacterial host cell, transformed therewith.
Such replicons are well known in the art.
1.'i 4) The vector replicon should be small (i.e., probably smaller than 6-8 kilobase pairs) to enable easy genetic arid molecular biological manipulation thereof.
5) The vector should carry a selection marker, preferably a drug-resistance marker such as ampicillin, fog usc~ in bacterial host cells transformed therewith.
6) The vector should carry a second selection marker, preferably a drug-resistance marker such as 2!5 neomycine, for use as such in eukaryotic host cells transfected therewith.
7) The vector should contain convenient endonuclease restriction sites for cloning.
8) The vector ;should contain a transcription termination and polyadenylation sequence.
9) The gene expression module does not contain any viral segments and is not oncongenic.
Most preferably the vector of this invention does not comprise an autonomous replicating sequence (replicon) capable oi= functioning in a eukaryotic host cell transfected therewith. A primary reason 05 forusing a non-replicating vector system in eukaryotic host cells is that. all known vector systems capable of autonomous and extrachromosomal replication in a mammalion eukaryotic host cell transfected therewith comprise replicons which are derived from onc:ogenic viruses. It is desirable to employ vectors comprising DNA not derived from oncogenic viruses for expressing DNA sequences encoding po7.ypept:ides of pharmaceutical importance.
This invention also relates to a transfected host 1'. eukaryotic cell transformed with a recombinant DNA
vector containin<~ the functional DNA sequence of this invention. Prei=erably, such host is a mammalian cell, most preferably a Chinese Hamster ovary (CHO) cell line, a vero cell line, an L-cell line, or a mouse or rat fiY~robla:~tic cell line. Such host may be prepared by transfe~cting a eukaryotic cell with a recombinant DNP, vector of this invention by conventional techniques.
This invention also relates to a method for preparing the proteins coded for by the DNA sequence of interest. which comprises: a) cultivating the transfected host: of this invention under culture medium condition; which enable such host to express such proteins, and b) isolating such proteins.
Preferably, such transfected host is able to secrete such proteins into the medium. Preferably, heavy metal ions or steroid hormones, such as dexamethasone, are added to such culture medium to induce the MHT-promoter and thereby enhance expression of such coding region. Heavy metal ions such as cadmium or ::inc are most preferred. The OF~ optimal concentration of heavy metal ions or steroid hormone contained in t:he medium can be determined by conventional_ techniques.
This invention also relates to the proteins prepared by suc:z method. If the transfected host cell of this invention secretes such proteins directly into tl~e culture medium, such proteins can then be isolated from the culture medium of the transfected host of this invention by conventional protein isalation techniques. If the transfected 1~ host cell of tr.is invention does not secrete such proteins, they are obtained from a culture lysate of such host by con~~entional culture lysate techniques.
Example 3 2C~ Expression of Human Growth Hormone Preparation of a Recombinant DNA Vector Encoding Human Growth Hormone, Said Vector Comprising the Metallothionein Promoter 2c A. Isolation of a DNP, Fragment Containing the Human Growth Hormone Gene Without the Natural Promoter For Transcription.
The recombinant plasmid pBR322 containing a 2 kb 30 EcoRI DNA fragment insert encoding the human growth hormone gene (in a counterclockwise orientation) is digested with the restriction endonuclease BamHI in a total volume of 500 ;ul containing 100 ~g of plasmid DNA, 240 units BamHI, in BamHI (high salt) buffer.
Two fragments are generated, one being about 5.6 kb and _another fragment. 0.8 kb. The fragments are 05 separated by e:~ectrophoresis in a 0.8$ preparative agarose ge.l. 'fhe larger fragment, containing the structural gene for human growth hormone without the natural promoter for gene, is electro-eluted from the gel and purified by two cycles of phenol/chloroform extraction, two cycles of chloroform extraction and two cycles of ethanol precipitation. The pelleted DNA is resuspended in 20 ~1 10 mM Tris, pH 8Ø
B. Isolation of a DNA,Fr_agment Containing the Metal-1-'~ lothionein Promoter and the pML2 Vector Backbone.
Plasmid pMMT-neo (see Figure 5) is digested with restriction endonucleases BglII and BamHI to produce two fragments, one being 4.5 kb and containing the MMT-promoter and the pML2 vector backbone, and the other being 2.15 kb and containing the neo gene coupled to the s'r-PAS-t segment. In order to prevent self-ligation, the .Linear DNA is treated with alkaline phosphatase in a total volume of 100 ~ul containing 25 dug of the DNA fragment, and, 28 U of alkaline phosphatase. The mixture is incubated 20 minutes at 37°C, and the reaction is stopped by adding 10 ~ul 50 mM EI)T,A and heating for 10 minutes at 68°C. The fragments are separated by electrophoresis in a 0.8$ agarose gel. The 4.5 kb fragment is electro-eluted from the gel as described and is purified by t:wo cycles of phenol/chloroform extraction, two cycles of chloroform extraction and 134093 .
two cycles of e~thano~L precipitation. The pelleted DNA is resuspende~d in 2.0 ~ul 10 mM Tris, pH 8Ø
C. Lilgation of the DL~fA Fragments Isolated _in A. and B. Above.
05 A 1.5 dug quantity of the 5.6 kb fragment from A.
above is taken a;zd mixed with 0.3 dug of the fragment from B. above in a total volume of 10 ~ul containing 0.9 units of T4 DNA-ligase and 1X ligation buffer, plus 100 mM ATP. The' mixture is incubated for 1 hour at 15°C and overnight at 4°C. The ligation mixture is then introduc~sd into the bacterial strain, HB101, using the procedure described in Materials and Procedures. Cells from the transformation mixture are spread onto LB-ampicillin agar plates and isolated colonies are screened for those containing plasmids of 10.1 kb in size that can be linearized with restriction endonuclease BamHI. The orientation is determined by EcoRI digestion. Plasmids containing the insert: in the correct orientation yield two EcoRI fragments, one of 3.5 kb and the other of 6.6 kb. Such plasmids, designated pMMT-hGH, are subjected to large scale growth and purification as described.
D. Introduction of the Recombinant DNA Vector Obtained in C. Af>ove into Mammalian Cells and Establishment of Cell Lines Producing Human Growth Hormone.
The vector, pMMT-hGH, is then transfected into eukaryotic host cells (mammalian) by standard co-transfection techniques to yield transfectants that synthesize the human growth hormone polypeptide. Such transfectants are identified by the RIA technique using antibodies to human growth hormone. Transfectants showing secretion of human growth hormone are established as cell lines using 05 conventional techniques.
Example 4 Expre=~sion of Rat Growth Hormone 1~ Preparation of a Recombinant DNA Vector Comprising the Rat Growth Hormone, Said Vector Containing the Metallothionein Promoter.
A. Conversion c~f the- BglII- Restriction Endonuclease l.'~ Site in pMMT-neo to XhoI.
A 200 fag quantity of pMMT-neo is digested with 500 U of restriction endonuclease BglII in medium salt buffer in ,~ total reaction volume of 1,000 ~ul.
The ends of the 6.65 hb linear plasmid DNA are first 20 made blunt by filling in the BglII ends using DNA
polymerase I (Kl_enow fragment) and the four dNTP's, as described previously, followed by attachment of the following synthetic DNA linker fragment containing the XhoI restriction endonuclease recognition and 2~~ cleavage site (from PL Biochemicals, Milwaukee, Wisconsin 53205):.
5'-CCTCGAGG-3' 3'-GGAGCTCC-5' 3C The filling in reaction is performed in a total volume of 30 ~ul cont,aining 2.5 dug of the 6.65 kb fragment, 200 mM of the four dNTP's, 3 dal NT-buffer, l~ul Klenow fragment. The mixture is incubated at room temperature for 30 minutes. The reaction is stopped by the addition of 2 N1 0.25 M EDTA and the DNA is washed once with an equal volume of phenol/chloroform and then with an equal volume of 0'.i chloroform alone. The DNA in the aqueous phase is further purified by two ethanol precipitations. The precipitated DNA is resuspended in 10 ~..il 10 mM Tris, pH 8.0, at a con~~entration of 50 ng//ul.
The linker fragment is dissolved in 50 ~ul of 10 mM Tris, pH 8.1, at a concentration of 1 pmole/J.zl.
The blunt-ended 6.65 kb fragmen t is ligated to the linker fragment in a total volume of 20 ~ul containing 2 pmoles of the linker fragment, 0.5 dug of the 6.65 kb fragment., 9 units of T4 DNA-ligase (Boehringer 1_'i Mannheim), 30 ~u:L 2X blunt-end ligation buffer, plus 100 mM ATP. The ligation is performed at 15°C for one hour and at ~~°C overnight.
This ligation mixture is used to transform the bacterial strain, HB101, which is made competent as described in Materials and Procedures. Cells from the transformation mixture were spread on LB-ampicillin agar plates. Bacterial colonies able to grow on ampicillin plates are picked and grown up in 2 ml cultures and subjected to plasmid preparations as descrilbed above.
From 12 bacterial clones, four (OK 3, OK 4, OK 11, OK 12) were identified as having a plasmid showing the expected size after digestion with XhoI
alone or together with BamHI and EcoRI. The plasmid DNA from OK 3 and OK 7L1 were propagated in HB101 for large scale plasrnid preparations.
B. Isolation of a DNA Fragment Containing the Metal-lothionein Promoter and the pML2 Vector Backbone.
A 100 dug quantity of each of OK 3 and OK 11 was digested with th<~ restriction endonucleases BamHI and 05 Xhol which yield two fragments, one (5.8 kb) with the MMT-promoter located close to the XhoI end and the pML2-part at the BamHI end, a.nd the other (0.85 kb) containing the sv--PAS-t sequence. The fragments were separated by electrophoresis in 0.8$ agarose.
The 5.8 kb fragment was purified by electro-elution, followed by two cycles of phenol/chl_oroform extraction, two cyclea of chloroform extraction and two cycles of Ethanol precipitation. The pelleted DNA was resuspended i.n 50 ~.zl 10 mM Tris, gH 8Ø
1 '~
C. Isolation of a DNA Fragment Containing the Rat Growth Hormone Ge_n~e_.
Plasmid pBR~s22.rGH, containing a genomic BamHI
insert encoding the rat growth hormone gene (in a counterclockwise orientation), was digested with BamHI and XhoI. The genomic BamHI-XhoI fragment containing the structural gene is isolated from a 0.8$ preparative agarose gel as above.
2~ D. Ligation of the Fragments Isolated as in B. and C_ Ahnve_ The fragments are ligated to each other such that the MMT-promoter will be linked, via the Xhol "sticky" end dii:ectly to the structural gene via its XhoI sticky end, in the correct orientation.
The ligation mixture is used to transform the bacterial strain, HB101, as described above. Trans-1 3 40 9 :~'~
formants forming colonies on LB-ampicillin agar plates are picked and grown up in 2 ml cultures for small scale plasmid preparation. Plasmids yielding the two expected fragments upon digestion with BamHI
0!p and XhoI, designated pMMT-rGH, are then grown up in large scale for transfection into mammalian cells.
E. Introduction of the Re cc>mbinant DNA Vector Obtained in D. Above into Mammalian Cells and Establishment of Cell Lines Producing Rat Growth Hormone.
The vector, pMMT-rGH, is then transfected into eukaryotic host: cells (mammalian) by standard co-transfection techniques to yield transfectants that synthesize the rat growth hormone polypeptide.
1_'. Such transfectamts are identified by the RIA
technique using antibodies to rat growth hormone.
Transfectants sh~~wing secretion of rat growth hormone are established as cell lines using conventional techniques.
2C~ Although the present invention has been described with reference to preferred embodiments, workers skilled in the a.rt will recognize that changes may be made in form and detail without departing from the spirit and scope of thc~ invention.
Claims (107)
1. A particle prepared by recombinant DNA
techniques which comprise at least one protein coded for by the entire PreS1-PreS2-S protein coding region of an HBV genome.
techniques which comprise at least one protein coded for by the entire PreS1-PreS2-S protein coding region of an HBV genome.
2. A recombinant DNA vector comprising:
a DNA sequence encoding the PreS1-PreS2-S
protein coding region of an HBV genome; anon mousse metallothionein (MMT) promoter operatively linked to the protein encoding sequence.
a DNA sequence encoding the PreS1-PreS2-S
protein coding region of an HBV genome; anon mousse metallothionein (MMT) promoter operatively linked to the protein encoding sequence.
3. The vector of claim 2 which additionally comprises the natural promoter for the DNA sequence.
4. The vector of claim 2 wherein the mouse metallothionein promoter is incorporated into the DNA
vector immediately upstream of the PreS1-PreS2-S
protein coding region.
vector immediately upstream of the PreS1-PreS2-S
protein coding region.
5. The vector of claim 2 which additionally comprises a selection marker.
6. The vector of claim 5 wherein the selection marker is a drug resistance marker.
7. The vector of claim 6 wherein the drug resistance marker is a neomycine gene.
8. The vector of claim 2 which additional comprises a transcription termination sequence.
9. The vector of claim 8 wherein the transcription termination sequence is an SV40 termination site.
10. The vector of claim 8 wherein the transcription termination sequence is a DEF region of the mouse globin gene.
11. The vector of claim 2 wherein the vector additionally comprises DNA other than viral DNA.
12. The vector of claim 2 which is further characterized by passive replication in a host eukaryotic genome upon transfection into the host.
13. The vector of claim 2 which additionally comprises an autonomous replicating sequence capable of functioning in a prokaryotic host cell.
14. They vector of claim 5 which additionally comprises a second selection marker.
15. The vector of claim 14 wherein the second marker is a drug resistance marker.
16. The vector of claim 1 which additionally comprises a polyadenylation sequence.
17. A eukaryotic cell transfected with the recombinant DNA vector of claim 2.
18. The cell of claim 17 wherein the eukaryotic cell is a mammalian cell.
19. The cell of claim 18 wherein the mammalian cell is a Chinese hamster ovary cell.
20. The cell of claim 18 wherein the mammalian cell is a vero cell.
21. The vector of claim 18 wherein the mammalian cell is an L-cell.
22. The cell of claim 18 wherein the mammalian cell is a mouse fibroblastic cell.
23. The cell of claim 18 wherein the mammalian cell is a rat fibroblastic cell.
24. The cell of claim 17 wherein the vector additionally comprises the natural promoter for the DNA sequence.
25. The cell of claim 17 wherein the metallothionein promoter is incorporated into the DNA vector immediately upstream of the PreS1-PreS2-S protein coding region.
26. The cell. of claim 17 wherein the vector additionally comprises a selection marker.
27. The cell of claim 26 wherein the selection marker is a drug resistance marker.
28. The cell of claim 27 wherein the drug resistance marker is a neomycine gene.
29. The cell of claim 17 wherein the vector additionally comprises a transcription termination sequence.
30. The cell of claim 29 wherein the transcription termination sequence is an SV40 termination site.
31. The cell of claim 29 wherein the transcription termination sequence is a DEF region of the mouse globin gene.
32. The cell of claim 17 wherein the vector additionally comprises DNA other than viral DNA.
33. The cell of claim 17 wherein the vector additionally comprises an autonomous replicating sequence capable of functioning in a prokaryotic host cell.
34. The cell of claim 26 wherein the vector additionally comprises a second selection marker.
35. A method of preparing a transfected host eukaryotic cell containing the PreS1-PreS2-S
protein coding region which comprises transfecting a eukaryotic cell with the vector of claim 2.
protein coding region which comprises transfecting a eukaryotic cell with the vector of claim 2.
36. A method of preparing a particle comprising proteins coded for by the PreS1-PreS2-S protein coding region wherein at least one of such proteins corresponds to a polypeptide coded for by the entire PreS1-PreS2-S protein coding region, the method comprising:
a. transfecting a host eukaryotic cell with the recombinant DNA vector of claim 2 to produce a transfected host cell containing the PreS1-PreS2-S
protein coding region;
b. cultivating such transfected host cell under culture medium conditions which enable the host cell to express such proteins; and c. isolating such particle.
a. transfecting a host eukaryotic cell with the recombinant DNA vector of claim 2 to produce a transfected host cell containing the PreS1-PreS2-S
protein coding region;
b. cultivating such transfected host cell under culture medium conditions which enable the host cell to express such proteins; and c. isolating such particle.
37. The method of claim 36 which additionally comprises adding heavy metal ions to the culture medium.
38. The method of claim 37 wherein the heavy metal ion is cadmium.
39. The method of claim 37 wherein the heavy metal ion is zinc.
40. The method of claim 36 which additionally comprises adding steroid hormones to the culture medium.
41. The method of claim 40 wherein the steroid hormone is dexamethasone.
42. The method of claim 36 wherein the metallothionein promoter is incorporated into the DNA
vector immediatelly upstream of the PreS1-PreS2-S
protein coding region.
vector immediatelly upstream of the PreS1-PreS2-S
protein coding region.
43. The method of claim 36 wherein the DNA
vector additionally comprises a selection marker.
vector additionally comprises a selection marker.
44. The method of claim 43 wherein the selection marker is a drug resistance marker.
45. The method of claim 44 wherein the drug resistance marker is a neomycine gene.
46. The method of claim 36 and wherein the DNA
vector additionally comprises a transcription termination sequence.
vector additionally comprises a transcription termination sequence.
47. The method of claim 46 wherein the transcription termination sequence is an SV40 termination site.
48. The method of claim 46 wherein the transcription termination sequence is a DEF region of the mouse globin gene.
49. The method of claim 36 wherein the DNA vector additionally comprises DNA other than viral DNA.
50. The method of claim 36 wherein the eukaryotic cell is a mammalian cell.
51. The method of claim 50 wherein the mammalian cell is a Chinese hamster ovary cell.
52. The method of claim 50 wherein the mammalian cell is a veto cell.
53. The method of claim 50 wherein the mammalian cell is an L-cell.
54. The method of claim 50 wherein the mammalian cell is a mouse fibroblastic cell.
55. The method of claim 50 wherein the mammalian cell is a rat fibroblastic cell.
56. A particle produced by the method of claim 36 which is composed of at least one protein coded for by the entire PreS1-PreS2-S protein coding region.
57. A vaccine comprising an immunoprotective amount of the particle of claim 56.
58. The vaccine of claim 57 wherein the amount is 1-20 ug.
59. A method for detecting the presence of antibodies to proteins coded for by the PreS1-PreS2-S protein coding region in a sample of mammalian blood sera, the method comprising:
a. contacting the sample with a solid substrate coated with non-labeled particles of claim 56;
b, incubating and washing said contacted sample;
c. contacting said contacted sample with labeled particles of claim 56, thereby producing a labeled contacted sample;
d. incubating and washing said labeled contacted sample; and e. determining the extent of labeled particle in the labeled contacted sample.
a. contacting the sample with a solid substrate coated with non-labeled particles of claim 56;
b, incubating and washing said contacted sample;
c. contacting said contacted sample with labeled particles of claim 56, thereby producing a labeled contacted sample;
d. incubating and washing said labeled contacted sample; and e. determining the extent of labeled particle in the labeled contacted sample.
60. A method for detecting the presence of proteins coded for by the PreS1-PreS2-S protein coding region in a sample of mammalian blood sera, the method comprising:
a. producing a composition containing an antibody which binds specifically to an immunogen comprising the particles of Claim 56;
b. contacting the sample with a first portion of toe composition anal the immunogen which has been labeled, incubating and washing the first portion;
c. contacting an antigen-free control with a second portion of the composition and the immunogen which has been labeled, incubating and washing the second portion;
d. adding the same amount of staphylococci bearing protein A to the composition of steps b. and c. above, incubating both compositions and separating liquid from cells; and e. determining the extent of labeled immunogen in each of the resultant compositions from step d. above.
a. producing a composition containing an antibody which binds specifically to an immunogen comprising the particles of Claim 56;
b. contacting the sample with a first portion of toe composition anal the immunogen which has been labeled, incubating and washing the first portion;
c. contacting an antigen-free control with a second portion of the composition and the immunogen which has been labeled, incubating and washing the second portion;
d. adding the same amount of staphylococci bearing protein A to the composition of steps b. and c. above, incubating both compositions and separating liquid from cells; and e. determining the extent of labeled immunogen in each of the resultant compositions from step d. above.
61. A diagnostic kit for detecting the presence of antibodies to proteins coded for by the PreS1-PreS2-S protein coding region in a sample of mammalian blood sera comprising:
a) unlabeled proteins comprising the particle of claim 56 attached to a solid support; and b) labeled antibodies to human IgG or IgM
which bind specifically to protein coded for the by the PreS1-PreS2-S protein coding region.
a) unlabeled proteins comprising the particle of claim 56 attached to a solid support; and b) labeled antibodies to human IgG or IgM
which bind specifically to protein coded for the by the PreS1-PreS2-S protein coding region.
62. A diagnostic kit for detecting the presence of proteins coded for by the PreS1-PreS2-S
protein coding region in a sample of mammalian blood sera comprising:
a) antibodies, produced by the particle of claim 56, attached to a solid support;
and b) labeled antibodies, produced by the particle of claim 56.
protein coding region in a sample of mammalian blood sera comprising:
a) antibodies, produced by the particle of claim 56, attached to a solid support;
and b) labeled antibodies, produced by the particle of claim 56.
63. A method for detecting HBV antigens contain-ing proteins coded for by the PreS1-PreS2-S
protein coding region in sera of HBV infected animals, the method comprising:
a) providing a solid substrate in bead form containing binding sites thereon coated with antibodies to the particle of claim 56;
b) washing the coated beads to remove excess antibodies;
c) contacting the beads with a protein-containing solution to reduce non-specific binding;
d) washing the beads to remove excess protein-containing solution;
e) incubating the beads with serum samples suspected of containing HBV or HBV surface antigens;
f) washing the beads with a solution mixed with a labeled antibody which binds specifically to protein encoding for by the PreS1-PreS2-S protein encoding region; and g) determining the extent of labeled antibody.
protein coding region in sera of HBV infected animals, the method comprising:
a) providing a solid substrate in bead form containing binding sites thereon coated with antibodies to the particle of claim 56;
b) washing the coated beads to remove excess antibodies;
c) contacting the beads with a protein-containing solution to reduce non-specific binding;
d) washing the beads to remove excess protein-containing solution;
e) incubating the beads with serum samples suspected of containing HBV or HBV surface antigens;
f) washing the beads with a solution mixed with a labeled antibody which binds specifically to protein encoding for by the PreS1-PreS2-S protein encoding region; and g) determining the extent of labeled antibody.
64. A method for detecting antibodies to proteins coded for by the PreS1-PreS2-S protein coding region in a given sample, the method comprising:
adsorbing particles of claim 56 on a solid substrate containing binding sites thereon;
contacting the substrate with a substance to saturate non-specific binding sites thereon;
washing the substrate with a buffered solution and removing the buffer;
adding the sample to the substrate;
incubating and washing the substrate-containing sample;
adding labeled antibodies to human IgG or IgM, which binds specifically to protein coded for by the PreS1-PreS2-S protein coding region, to the substrate;
incubating and washing the substrate; and determining the extent of labeled antibody.
adsorbing particles of claim 56 on a solid substrate containing binding sites thereon;
contacting the substrate with a substance to saturate non-specific binding sites thereon;
washing the substrate with a buffered solution and removing the buffer;
adding the sample to the substrate;
incubating and washing the substrate-containing sample;
adding labeled antibodies to human IgG or IgM, which binds specifically to protein coded for by the PreS1-PreS2-S protein coding region, to the substrate;
incubating and washing the substrate; and determining the extent of labeled antibody.
65. A eukaryotic cell co-transfected with the recombinant DNA vector of claim 2 and a second recombinant DNA vector comprising a PreS2-S protein coding region of an HBV genome; or a S-protein coding region of an HBV genome; and a MMT promoter operatively linked to the protein encoding sequence.
66. They cell of claim 65 wherein the second vector additionally comprises a transcription termination site.
67. The cell of claim 65 wherein the second vector additionally comprises a selection marker.
68. A eukaryotic cell co-transfected with the recombinant vector of claim 2, a second recombinant DNA vector comprising a PreS2-S protein coding region of an HBV genome and a MMT promoter, and a third recombinant DNA vector comprising a MMT promoter operatively linked to the protein encoding sequence and a selection marker.
69. The cell of claim 68 wherein the second vector additionally comprises a transcription termination site.
70. The cell of claim 68 wherein the second vector additionally comprises a selection marker.
71. The cell of claim 68 wherein the third vector additionally comprises a transcription termination site.
72. The cell of claim 68 wherein the second vector additionally comprises a replicon capable of functioning in a prokaryote.
73. The cell of claim 68 wherein the third vector additionally comprises a replicon capable of functioning in a prokaryote.
74. A method of preparing a co-transfected host eukaryotic cell containing a first recombinant DNA
vector comprising the PreS1-PreS2-S protein coding region and, a second recombinant DNA vector comprising the PreS2-S protein coding region, or the S protein coding region, the method comprising:
co-transfecting a eukaryotic cell with the vector of claim 2 as a first recombinant DNA vector and a second recombinant vector containing the PreS2-S protein coding region, or the S protein coding region, and a MMT
promoter, operatively linked to the protein encoding sequence, and, optionally, a third recombinant DNA vector comprising an MMT promoter and a selection marker.
vector comprising the PreS1-PreS2-S protein coding region and, a second recombinant DNA vector comprising the PreS2-S protein coding region, or the S protein coding region, the method comprising:
co-transfecting a eukaryotic cell with the vector of claim 2 as a first recombinant DNA vector and a second recombinant vector containing the PreS2-S protein coding region, or the S protein coding region, and a MMT
promoter, operatively linked to the protein encoding sequence, and, optionally, a third recombinant DNA vector comprising an MMT promoter and a selection marker.
75. A method of preparing a particle comprising proteins coded for by the PreS1-PreS2-S protein coding region wherein at least one of such proteins corresponds to a polypeptide coded for by the entire PreS1-PreS2-S protein coding region, the method comprising:
a) cultivating the co-transfected host cell of claim 65 under culture medium conditions which enable the host cell to express such proteins; and b) isolating such particle.
a) cultivating the co-transfected host cell of claim 65 under culture medium conditions which enable the host cell to express such proteins; and b) isolating such particle.
76. A particle produced by the method of claim 75 which is composed of at least one protein coded for by they entire PreS1-PreS2-S protein coding region.
77. A hepatitis B virus vaccine comprising an immunoprotective amount of the particle of claim 76.
78. The vaccine of claim 77 wherein the amount is 1-20 ug.
79. A method for detecting the presence of antibodies t:o proteins coded for by the PreS1-PreS2-S protein coding region in a sample of mammalian blood sera, the method comprising:
a. contacting the sample with a solid substrate coated with non-labeled particles of claim 76;
b. incubating and washing said contacted sample;
c. contacting said contacted sample with labeled particles of claim 76 , thereby producing a labeled contacted sample;
d. incubating and washing said labeled contacted sample; and e. determining the extent of labeled particle in the labeled contacted sample.
a. contacting the sample with a solid substrate coated with non-labeled particles of claim 76;
b. incubating and washing said contacted sample;
c. contacting said contacted sample with labeled particles of claim 76 , thereby producing a labeled contacted sample;
d. incubating and washing said labeled contacted sample; and e. determining the extent of labeled particle in the labeled contacted sample.
80. A method for detecting the presence of proteins coded for by the PreS1-PreS2-S protein coding region in a sample of mammalian blood sera, the method comprising:
a. producing a composition containing an antibody which binds specifically to an immunogen comprising the particles of claim 76;
b. contacting the sample with a first portion of the composition and the immunogen which has been labeled, incubating and washing the first portion;
c. contacting an antigen-free control with a second portion of the composition and the immunogen which has been labeled, incubating and washing the second portion;
d. adding the same amount of staphylococci bearing protein A to the composition of steps b. and c. above, incubating both compositions and separating liquid from cells; and e. determining the extent of labeled immunogen in each of the resultant compositions from step d. above.
a. producing a composition containing an antibody which binds specifically to an immunogen comprising the particles of claim 76;
b. contacting the sample with a first portion of the composition and the immunogen which has been labeled, incubating and washing the first portion;
c. contacting an antigen-free control with a second portion of the composition and the immunogen which has been labeled, incubating and washing the second portion;
d. adding the same amount of staphylococci bearing protein A to the composition of steps b. and c. above, incubating both compositions and separating liquid from cells; and e. determining the extent of labeled immunogen in each of the resultant compositions from step d. above.
81. A diagnostic kit for detecting the presence of antibodies to proteins coded for by the PreS1-PreS2-S protein coding region in a sample of mammalian blood sera comprising:
a. unlabeled proteins comprising the particle of claim 76 attached to a solid support; and b. labeled antibodies to human IgG or IgM
which binds specifically to protein coded for by the PreS1-PreS2-S protein coding region.
a. unlabeled proteins comprising the particle of claim 76 attached to a solid support; and b. labeled antibodies to human IgG or IgM
which binds specifically to protein coded for by the PreS1-PreS2-S protein coding region.
82. A diagnostic kit for detecting the presence of proteins coded for by the PreS1-PreS2-S
protein coding region in a sample of mammalian blood sera comprising:
a. antibodies, produced by the particle of claim 76 attached to a solid support;
and b. labeled antibodies, produced by the particle of claim 76.
protein coding region in a sample of mammalian blood sera comprising:
a. antibodies, produced by the particle of claim 76 attached to a solid support;
and b. labeled antibodies, produced by the particle of claim 76.
83. A method for detecting HBV antigens containing proteins coded for by the PreS1-PreS2-S
protein coding regions in sera of HBV infected animals, the method comprising:
a, providing a solid substrate in bead form containing binding sites thereon coated with antibodies to the particles of claim 76 b. washing the coated beads to remove excess antibodies;
c. contacting the beads with a protein-containing solution to reduce non-specific binding;
d. washing the beads to remove excess protein-containing solution;
e. incubating the beads with serum samples suspected of containing HBV or HBV surface antigens;
f. washing the beads with a solution mixed with a labeled antibody;
which binds specifically to protein encoding for by the PresS1-PreS2-S protein encoding region; and g. determining the extent of labeled antibody.
protein coding regions in sera of HBV infected animals, the method comprising:
a, providing a solid substrate in bead form containing binding sites thereon coated with antibodies to the particles of claim 76 b. washing the coated beads to remove excess antibodies;
c. contacting the beads with a protein-containing solution to reduce non-specific binding;
d. washing the beads to remove excess protein-containing solution;
e. incubating the beads with serum samples suspected of containing HBV or HBV surface antigens;
f. washing the beads with a solution mixed with a labeled antibody;
which binds specifically to protein encoding for by the PresS1-PreS2-S protein encoding region; and g. determining the extent of labeled antibody.
84. A recombinant DNA mixture comprising (a) a plasmid vector comprising the PreS1-PreS2-S protein coding region of an HBV genome and at least one additional plasmid vector comprising one of the following coding regions:
PresS1-PreS2-S protein coding region of an HBV genome, PreS2-S protein coding region of an HBV genome or S protein coding region of an HBV genome, and (b) a MMT promoter operatively linked to the protein encoding sequences.
PresS1-PreS2-S protein coding region of an HBV genome, PreS2-S protein coding region of an HBV genome or S protein coding region of an HBV genome, and (b) a MMT promoter operatively linked to the protein encoding sequences.
85. The mixture of claim 84 which additionally comprises a transcription termination site.
86. A eukaryotic cell transfected with the mixture of claim 84.
87. The cell of claim 86 wherein the eukaryotic cell is a mammalian cell.
88. The cell of claim 87 wherein the mammalian cell is a Chinese hamster ovary cell.
89. The cell of claim 87 wherein the mammalian cell is a vero cell.
90. The cell of claim 87 wherein the mammalian cell is an L-cell.
91. The cell of claim 87 wherein the mammalian cell is a mouse fibroblastic cell.
92. The cell of claim 87 wherein the mammalian cell is a rat fibroblastic cell.
93. A method of preparing a host eukaryotic cell transfected with the PreS1-PreS2-S protein coding region and at least one additional coding region selected from the PreS1-PreS2-S protein coding region, the PreS2-S protein coding region or the S
protein coding region which comprises transfecting a eukaryotic cell with the concatamer of claim 84.
protein coding region which comprises transfecting a eukaryotic cell with the concatamer of claim 84.
94. A method of preparing a particle comprising at least one protein coded for by the entire PreS1-PreS2-S protein coding region comprising:
a) cultivating the transfected host cell of claim 86 under culture medium conditions which enable the host cell to express such proteins; and b) isolating such particle.
a) cultivating the transfected host cell of claim 86 under culture medium conditions which enable the host cell to express such proteins; and b) isolating such particle.
95. The method of claim 94 which additionally comprises adding heavy metal ions to the culture medium.
96. The method of claim 95 which additionally comprises adding steroid hormones to the culture medium.
97. A particle produced by the method of claim 94 which is composed of at least one protein coded for by the entire PreS1-PreS2-S protein coding region.
98. A hepatitis B virus vaccine comprising an immunoprotective amount of the particle of claim 97.
99. The vaccine of claim 98 wherein the amount is 1-20 ug.
100. A method for detecting the presence of antibodies to proteins coded for by the PreS1-PreS2-S
protein coding region in a sample of mammalian blood sera, the method comprising:
a. contacting the sample with a solid substrate coated with non-labeled particles of claim 97;
b. incubating and washing said contacted sample;
c. contacting said contacted sample with labeled particles of claim 97 thereby producing a labeled contacted sample;
d. incubating and washing said labeled contacted sample; and e. determining the extent of labeled particle in the labeled contacted sample.
protein coding region in a sample of mammalian blood sera, the method comprising:
a. contacting the sample with a solid substrate coated with non-labeled particles of claim 97;
b. incubating and washing said contacted sample;
c. contacting said contacted sample with labeled particles of claim 97 thereby producing a labeled contacted sample;
d. incubating and washing said labeled contacted sample; and e. determining the extent of labeled particle in the labeled contacted sample.
101. A method for detecting the presence of proteins coded for by the PreS1-PreS2-S protein coding region in a sample of mammalian blood sera, the method comprising:
a. producing a composition containing an antibody which binds specifically to an immunogen comprising the particles of Claim 97;
b. contacting the sample with a first portion of the composition and the immunogen which has been labeled, incubating and washing the first portion;
c. contacting an antigen-free control with a second portion of the composition and the immunogen which has been labeled, incubating and washing the second portion;
d. adding the same amount of staphylococci bearing protein A to the composition of steps b. and c. above, incubating both compositions and separating liquid from cells; and e. determining the extent of labeled immunogen in each of the resultant compositions from step d. above.
a. producing a composition containing an antibody which binds specifically to an immunogen comprising the particles of Claim 97;
b. contacting the sample with a first portion of the composition and the immunogen which has been labeled, incubating and washing the first portion;
c. contacting an antigen-free control with a second portion of the composition and the immunogen which has been labeled, incubating and washing the second portion;
d. adding the same amount of staphylococci bearing protein A to the composition of steps b. and c. above, incubating both compositions and separating liquid from cells; and e. determining the extent of labeled immunogen in each of the resultant compositions from step d. above.
102. A diagnostic kit for detecting the presence of antibodies to proteins coded for by the PreS1-PreS2-S protein coding region in a sample of mammalian blood sera comprising:
a) unlabeled proteins comprising the particle of claim 97 attached to a solid support; and b) labeled antibodies to human IgG or IgM
which binds specifically to protein coded for by the PreS1-PreS2-S protein coding region.
a) unlabeled proteins comprising the particle of claim 97 attached to a solid support; and b) labeled antibodies to human IgG or IgM
which binds specifically to protein coded for by the PreS1-PreS2-S protein coding region.
103. A diagnostic kit for detecting the presence of antibodies to proteins coded for the by PreS1-PreS2-S protein coding region in a sample of mammalian blood sera comprising:
a) unlabeled proteins comprising the particle of claim 97 attached to a solid support; and b) labeled antibodies to human IgG or IgM
which binds specifically to protein coded for by the PreS1-PreS2-S protein coding region.
a) unlabeled proteins comprising the particle of claim 97 attached to a solid support; and b) labeled antibodies to human IgG or IgM
which binds specifically to protein coded for by the PreS1-PreS2-S protein coding region.
104. A diagnostic kit for detecting the presence of proteins coded for by the PreS1-PreS2-S
protein coding region in a sample of mammalian blood sera comprising:
a) antibodies, produced by the particles of claim 97 attached to a solid support; and b) labeled antibodies, produced by the particle of claim 97.
protein coding region in a sample of mammalian blood sera comprising:
a) antibodies, produced by the particles of claim 97 attached to a solid support; and b) labeled antibodies, produced by the particle of claim 97.
105. A method for detecting HBV antigens containing proteins coded for by the PreS1-PreS2-S
protein coding region in sera of HBV infected animals, the method comprising:
a) providing a solid substrate in bead form containing binding sites thereon coated with antibodies to the particle of claim 97;
b) washing the coated beads to remove excess antibodies;
c) contacting the beads with a protein-containing solution to reduce non-specific binding;
d) washing the beads to remove excess protein-containing solution;
e) incubating the beads with serum samples suspected of containing HBV or HBV surface antigens;
f) washing the beads with a solution mixed with a labeled antibody which binds specifically to protein encoding for by the PreS1-PreS2-S
protein encoding region; and g) determining the extent of labeled antibody.
protein coding region in sera of HBV infected animals, the method comprising:
a) providing a solid substrate in bead form containing binding sites thereon coated with antibodies to the particle of claim 97;
b) washing the coated beads to remove excess antibodies;
c) contacting the beads with a protein-containing solution to reduce non-specific binding;
d) washing the beads to remove excess protein-containing solution;
e) incubating the beads with serum samples suspected of containing HBV or HBV surface antigens;
f) washing the beads with a solution mixed with a labeled antibody which binds specifically to protein encoding for by the PreS1-PreS2-S
protein encoding region; and g) determining the extent of labeled antibody.
106. A diagnostic kit for detecting the presence of antibodies to proteins coded for by the PreS1-PreS2-S protein coding region in a sample of mammalian blood sera, the diagnostic kit comprising:
a. unlabeled proteins comprising the particle of claim 97 attached to a solid support; and b. labeled antibodies to IgG or IgM
which binds specifically to protein coded for by the PreS1-PreS2-S protein coding region,
a. unlabeled proteins comprising the particle of claim 97 attached to a solid support; and b. labeled antibodies to IgG or IgM
which binds specifically to protein coded for by the PreS1-PreS2-S protein coding region,
107. A diagnostic kit for detecting the presence of proteins coded for by the PreS1-PreS2-S
protein coding region in a sample of mammalian blood sera, the kit comprising:
a. antibodies produced by the particle of claim 97 attached to a solid support;
and b. labeled antibodies produced by the particle of claim 97.
protein coding region in a sample of mammalian blood sera, the kit comprising:
a. antibodies produced by the particle of claim 97 attached to a solid support;
and b. labeled antibodies produced by the particle of claim 97.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US85056786A | 1986-04-11 | 1986-04-11 | |
US850,567 | 1986-04-11 |
Publications (1)
Publication Number | Publication Date |
---|---|
CA1340934C true CA1340934C (en) | 2000-03-28 |
Family
ID=33419032
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA000506686A Expired - Fee Related CA1340934C (en) | 1986-04-11 | 1986-04-15 | Hepatitis b surface antigen formed by recombinant dna techniques, vaccines, diagnostics, cell lines and method of forming same |
Country Status (1)
Country | Link |
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CA (1) | CA1340934C (en) |
-
1986
- 1986-04-15 CA CA000506686A patent/CA1340934C/en not_active Expired - Fee Related
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