134934
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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.
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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
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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 '~
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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;
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:,
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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'
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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
1340934
_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 ~~
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(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 '~
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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'~
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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.
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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 .
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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 '
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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
1340934
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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~ ~
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A number of references discuss plasmids
containing they promoter of a mouse metallothionein
05
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 '~
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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
1340934
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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;
134Q934
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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-
1340934
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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
- 21 -
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 .
- 22 -
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
1340934
- 23 -
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 '~
- 24 -
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
1 340 934
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 '
- 26 -
"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 ~
- 27 -
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 '~
- 28 -
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
1340934
29
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
- 30 -
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
- 31 -
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
32
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 '
- 33 -
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 .
- 34 -
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 ?
- 35 -
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
1340934
- 36 -
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 "~
- 37 -
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 _.
- 38 -
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 ,~,
- 39 -
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 '
- 40 -
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
1340934
- 41 -
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
1340934
- 42 -
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.'~
- 43 -
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 '
- 44 -
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
1340934
- 45 -
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
1340934
- 46 -
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;
- 47 -
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.
1340934
- 48 -
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 .
- 49 -
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.
- 50 -
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
134934
- 51 -
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
- 52 -
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
- 53 -
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 ~
- 54 -
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
134Q934
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.
05
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'
- 56 -
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 '
- 57 -
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
X340934
- 58 -
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'.
59
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
05
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
1340934
- 60 -
"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:
05 121
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 ~,
- 61 -
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.
1340934
- 62 -
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
1340934
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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 '
- 64 -
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
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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
1340934
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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 ~
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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 :.
- 68 -
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
134034
- ~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.
- 71 -
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).
12. Remove aqueous layer; add podium
acetate to 300mM; add 2 volumes cold 100s
etr~anol; mix thoroughly. Hold at -20C
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.
17. Remove ethidium bromide from the
plasmid :Fraction by 3 successive extractions
with equal volumes of isobutanol.
*Trade mark
1340934 ~~
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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.
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
134093
4
- 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.
1340934
_ 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 '
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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
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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
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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
30
134934
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Example 1
E:K ression of Particles Comprising
PreSl-Pr~sS2-S Protein Coding Region Poly~peptides
05
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 ~,
- 85 -
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
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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 -,
87
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).
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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
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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.
1340934
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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
1340934
92
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
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~ ~ 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.
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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).
1340934
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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.
1340934
96
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,
1340934
_ 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
1340934
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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.
1340934
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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.
1340934
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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
1 101 101 100 100
2 99 99 99 100
3 100 100 99 101
4 99 100 101 99
5 102 99 99 98
E> 100 101 100 100
7 99 98 98 101
___ 1 3 4 0 9 3 4
- 102 -
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.
35
X
__ 1340934 ~
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: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.
X340934
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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.
- 106 -
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.
1340934
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~'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
X340934
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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
1340934
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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.
25
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 ,
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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 .
- 112 -
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.
1340934
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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
1340934
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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
1 340 93 4
- 115 -
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 .
- 116 -
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
1340934
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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
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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.
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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 :~'~
- 120 -
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.
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