Note: Descriptions are shown in the official language in which they were submitted.
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BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a preparation cOntaining
antigens possessing immunological properties, notably anti-
= genic properties, characteristic ofviruses of various forms
of hepatitis, such asB viral hepatitis or various other forms
of hepatitis. such a. those which are known to be developed in
certain patients following blood transfusions, for example
hepatites called "non A" or "non B". The invention relates
more particularly to preparations of this type, which are
characterised by high purity, the absence of proteins of human
origin and, when it concerns preparations having antigen pro-
perties similar to those of the HBs antigens, free of Dane
particles. It also relates to a method for the production of
these antigens. . .
2. Description of the Prior Art
It is known that certain of the specific antigen pro-
perties of the virus of hepatitis must be attributed to an
antigen called "HBs Antigen" oriliBsAg", essentially formed
a) . by the envelope of the virus of viral hepatitis of the B type (HBV)
. .
,
11
CA 1341641 1981-04-22 =
..
Ill/
2
or Dane particle. This antigen, which possesses vaccina-
ting properties with respect to viral hepatitis of type B,
is, at the present time, essentially obtained from human
serum specimens. However, no other sources for the supply
of HBsAg, are by now available by reason of the particular
characteristics of the virus of B hepatitis (HBV) . It
seems only capable of infecting man, the chimpanzee and,
perhaps, a small number of other primates. It has not been
possible hitherto to propagate it in vitro in cell cultures.
Certainly lines of hepatocarcinomas which synthesize HBsAg
are known. The use in man of cancerous cells for the produc-
tion of particles with a vaccinating character runs up
against quite comprehensible objections. The use in preven-
tive therapy of HBs antigens of human origin is not however
devoid of serious risks. In fact, they arise generally from
persons who have been exposed to the virus of B viral hepa-
titis, so that the presence of Dane particles, sometimes
also highly infectious, in preparations of HBsAg antigens of
seric origin cannot always be completely excluded, even in
the case of extremely pure preparations. The contents of the
even highly purified preparations of HBsAg antigen from the
state of the art in serum proteins or other possibly antige-
nic components, capable of inducing in the treated subjects
troublesome immunitary reactions, are not negligible, by
reason of their very low initial content of HBsAg with res-
pect to the proteins of the initial serum (for example, of
the order of 50 pg of HBsAg with respect to 80 mg of protein
in a 1 ml of serum).
=
it
CA 1341641 1981-04-22
. .
.
.
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.
Figure 1 comprises a diagrammatic map of the DNA
of the genome of the Dane particle. =
.
-
In spite of the difficulties encountered to have
available sufficient amounts of virus, it has however been
5. established that the genome of the virus of B hepatitis is
- formed from a partially singlestranded circular DNA molecule,
of which the longest strand includes of the order of 3200
nucleotides (SUnmERS J. and coll.. (1975) Proc. Nat. Sci.
U.S.A. 72, 4597-4601). Atthe most, it has been possible to
localise the gene coding the portion of the protein of the '
HilsAg antigen, which is responsible for the immunological
properties of the envelope of the virus of B hepatitis.
The position of this gene, named "S gene" results notably
from the diagrammatic mai) of the genothe of the Dane particle,
. 15 which is given in Figurel of the drawings.
This DNA comprises two strands b1 and b2,
the shortest of them (bd being normally devoid of the
portion shown by a dashed line in the drawing.
=
It is known that this DNA only includes a single
EcoRI site.
' The arrow El gives the direction of the numbering of
the nucleotides from which the longest strand 1)1 is composed,
.and the arrow f2 gives the direction of the transcription of
the S gene by the cellular machinery of the cells invaded by '
the ViTUS of B hepatitis.
The EcoRI site can hence be numbered 0, or 3182 (in the
case of B hepatitis viruses belonging to serotype 3182)
(NATURE, 1979, vol. 281, p. 646-650).
. .
ja .
CA 1341641 1981-04-22 .
411/ 4
The inner concentric circle e1 gives the scale in
numbers of nucleotides. This circle enables the positions
of certain of the parts of this DNA to be specified. The
= numbers 3', 5' and 5', 3', at the lower part of the map
indicate the terminal ends bearing the same numbers in conven-
tional representations of the ends of the nucleic acid chains.
At the most, the coding gene for the part of the protein
of the HBsAg antigen has been localised. The position of this
gene, named "S gene", is shown diagrammatically by the arrow S
. in the Figure. The "S gene" is essentially borne by the frayment
of the longest strand bl situated between the nucleotide posi-
tions 155 and 833 of the diagrammatic map of. figure 1 in the
direction of the transcription of the S gene.
The shortest strand (b2) of the genome of the Dane
particle can be "repaired" in vitro in the presence of pre-
cursor nucleotides and of a polymerase, for example by the
technique of T.A. LANDERS and coll., J. VIRUL., 23, 1977,
p. 368-376.The genomes so-repaired of the viral hepatitis
viruses (or any DNA capable of coding for the same aminoacid
2D sequences) will be denoted below by the abbreviation DNA HBV.
It is therefore an object of the invention to provide
processes such as have been specified above, which are appli-
cable generally to the study and to the expression in eukaryotic
cells of all or part of the whole genomes of the viruses
=
responsible for the various viral hepatites, more particularly
of DNA HBV. It is also an object to provide for the production
of modified vectors enabling the practising of these methods.
Lastly, it is more particularly an object of the
invention to provide for the production of preparations
=
1
1 it
CA 1341641 1981-04-22
5
111/-
containing antigens having the same immunological specificity
as HBsAg or of a similar antigen of high purity, as has been
indicated above, from a system other than human serum, which
system is both reproducible and stable (or having a certain
number of particular characteristics which can be made the
subject of constant surveillance).
surovunruF THE INVENTION
According to the invention there is provided a method
for determining the eventual possibility of transformation
of a culture of particular cells by a DNA normally circular
or a whole genome of the corresponding virus, which method
comprises the operation which consists of transforming this
cell culture with a vector, more particularly a plasmid,
itself containing an insertion sequence including itself at
least the portion of the viral DNA coding for the immunogen
protein, capable of inducing in vivo the production of active
antibodies with respect to the whole virus, as well as the
viral promoter under the control of which the transcription
and the translation of the above¨said portion of viral DNA
is normally carried out, in particular in a host infected by
* the corresponding virus.
BRIEF DESCRIPTION OF THE DRAWINGS
In this description, reference will again be made to
Figure 1 already mentioned and to Figures 2a, 2b and 2c, which
represent diagrammatic maps of preferred plaSmids which have
been utilised according to the invention.
According to a first embodiment of the invention, the
above¨said insertion sequence is formed by at least two of
these DNAstsuch as two DNA HBVst oriented in the same direction
of transcription and in which the tail of one is connected
to the head of the other. In the following, there will often
be used, for convenience of language, the expression "tandeu
sequence", to denote insertion sequences comprising two DNAs
of the type concerned, except when otherwise specified.
CA 1341641 1981-04-22
:-
. .
,
, =
. .
6
In a preferred embodiment of the method according to
the invention, the above,said insertion sequence is formed
. from at least two of these DNA HBVs or from two fragments of
DNA HBV, .normally contained in DNA HBV on each side of its
EcoRI site, oriented in the same direction of transcription', .
one of these fragments of DNA HBV containing the sequence of
nucleotides of the.S gene, which is adapted to code for the
polypeptide responsible for the immunological properties of .
the HBs antigen and the other of said fragments being suffi-
,
ciently long for it to be capable of including the promoter
of the S gene. =
According to a first preferred embodiment of the
,
,
process according to the invention, the oriented DNAs of the .
insertion sequence are constituted by fragments of DNA HBV,
,
the tail of one of these :fragments being connected to the head
- of the other fragment at the level of ah EcoRI site.
.
,
It is self-evident that certain non essential parts
,
of the latter insertion sequences can be deleted. Such an .
,
=
) insertion sequence contains therefore advantageously, on the
k
j
23 one hand, the DNA HBV fragment which contains the nucleotide
f sequence of the coding S gene for the polypeptide
responsible
f
for the immunological properties of the HBs antigen and, on
. the other hand, a DNA HBV fragment being sufficiently long for
it to be capable of including the promoter of the S genetup-
,stream of the preceding fragment in the direction of transcription
II,
,
,
these two fragments being noimally situated on each side of
the DNA HBV ElcoRI site.
In other words, this insertion sequence comprises
_ notably, if one refers to Figure 1, on the one hand, the
-.A
111 .
, .
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.
I . . .
=
CA 1341641 1981-04-22
,
7
sub-fragment comprised between the nucleotide positions 833 to 0
(direction reverse to fi), when this sub-fragment comprises the S
gene as a whole and, on the other hand, a sub-fragment comprised
between the position 3182 and a position corresponding to a number
,5 sufficiently distant from this 0 position, in the reverse direction
of the transcription, so that the above-said promoter can be included
therein.
It is observed that the last sub-fragment must normally comprise
a TATATAA sequence, which is normally found upstream of the beginning
of the S gene, at a distance corresponding to a number of nucleotides
of the order of 235. More particularly again, the insertion sequence
will comprise an additional portion of the DNA HBV situated upstream
of the BglII site, itself at the level of the nucleotide 2840.
= 15
According to an additional feature of the invention, the
insertion sequence comprises also in addition a sub-fragment
downstream of the S gene in the direction of transcription, this
fragment extending normally in the direction of the transcription
beyond the DNA HBV BamHI site.
A preferred insertion sequence comprises the whole of the coding
gene for .the messenger RNA corresponding to. the HBs antigen. The
relative position of this RNA messenger is represented in FIG. 1 by
the arrow RNA of which the beginning coincides substantially with
the position of the nucleotide 2800, if one refers to the map of
Figure 1, and which terminates between the BamHI and BG1II 1986
sites.
A preferred insertion sequence capable of being applied in the
method according to the invention is hence characterised
la
,S11'=.'CA 1341641 1981-04-22
- 8
in that it comprises both the S gene and the coding gene for
the messenger RNA corresponding to the HBs antigen. As is to
be concluded from the rest of this description, this sequence
is preferably devoid of any part of the coding gene for .the
HBc antigen, sufficing to be expressed in the form of a protein
having the immunogenic properties of the HBc antigen.
In the rest of this description, reference will be
'made more particularly to .DNA HBV. It is nonetheless under-
stood that all the techniques which are described are also
applicable to the DNA or corresponding whole genomes of other
viruses, responsible for other types of hepatitis.
Preferably, conditions are adopted which permit at
the same time labelling of the transformed cells, st) as to
=
render their detection easy, for example, by putting to use
cells or mutants of these cells rendered deficient, for select-
ion purposes (natural or induced deficiency), of a selectable
labelling gene normally necessary for their growth, when they
are placed in certain particular culture media, these cells
being none the less capable of developing again in
one
2D at least of these same culture media,' after introduction in
these cells of a gene or fragment of homologous DNA, although
of foreign origin (complementation DNA).,. capable of compensa-
ting or "complementing" said deficiency, the method consisting
then, either of attempting to carry out the transformation of
said cells :
- either with a single vector containing, on the one hand,. the
insertion sequence studied, notably derived from DNA HBV, and
on the other hand, such a "complementation DNA", respectively
inserted in its own genome,
11
=
CA 1341641 1981-04-22
=
=
lilt
= 9.
-or with two vectors (simultaneous transformations, or co-
transformation),
one of these vectors, preferably a plasmid, having previous-
ly been modified by the insertion in its genome of such a
complementation DNA ;
the other vector, preferably a plasmid also, having pre-
viously been modified by the insertion in its genome of the
abovesaid insertion sequence,
and of collecting, after their cultivation in the above-
defined medium, of the colonies which both developed and
. which were transformed by the insertion sequence..
To the extent that the DNA HBV is capable of being
.expressed in the cell of said culture, the tandem sequence
(or containing more than two DNA HBV units), or More general-
ly the insertion sequence such as has been defined above,
must normally behave as would the corresponding circular DNA
thus introduced into the cell, in the absence of any vector.
The use of such a vector, notably a plasmid, instead
and in place of the circular DNA itself is of very great
interest in that it is possible to have considerable amounts
of such a vector after amplification by cloning in a bacte-
rium of the vector previously constructed in vitro.
The process according to the invention, hence permit3testing
the capacity of a circular DNA or of a whole virus genome - in parti-
cular DNAs of hepatitis viruses other than DNA HBV - to be expressed
in eukaryotic cells, to he tested, without it being necessary to give
regard to the exact position of the promoter under the control of which
the transcription- is to be effected.
=
11 =
CA 1341641 1981-04-22
. .õ(,,
. .
The vectorS.modified by the above-said insertion . .
sequences, notably those derived from DNA HBV.can be used
to induce the expression of these insertion sequences. in
. eukaryotic cell =cultures, = notably of the mbuse or of human =
origin. .
' When there ' is used, for the labelling of transformed
cells, a distinct vector, notably a plasmid containing the
marker, it is preferable to resort to a plasmid containing
. a "complementation. DNA",. such as the gene of thymidine-kinase. =
.10 of the Herpes simplex HSV-1 virus, which can be excised from.
the genome of the virus by specific enzymes restrictions.
.
= such as Banda. To this gene of thymidine-kinase of viral
origin correspond, in numerous types of eukaryotic cells,
homologous genes adapted to direct the synthesis of the
15 thymidine-kinase (enzyme phosphorylating the exogenous thymidine
supplied by the culture medium). . .
..
=
The possiblity of overcoming certain genetic deficien-
cies of eukariotic cells, notably of the mouse, for example,
those relating to their gene of endogenous thymidine-kinase,
20 by reason of an induced or provoked mutation, has already been
described by ifie WIGLEk and coll. (Cell. vol. II, 223-232, 1977).
=
= The deficient cells, called TK-, are selected .by reason of .
=
their incapacity to synthesize- thymidine-kinase in a medium . õ . .. .
. .._ ....
, such as that known by the name "HAT medium" (containing . .
25 hypoxanthirce and aminopterine in addition to thymidine). This
medium is known Bor only penaitting the possible = synthesis of .
thymidine phosphate via a metabolic route called "salvation
.
pathway", this route implies however that, the integrity. of = .
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CA 1341641 1981-04-22 .
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11
III
the TK gene of the cells capable of being developed therein be
preserved. TK- cells become nonetheless capable of development in
this same medium as soon as they have been modified by incorporation
of the TK gene of the virus of the herpes which is then genetically
transmissible to their descendants, as a result of successive cell
divisions. From "TK- " as they were previously, before the abovesaid
incorporation, these cells have then become "TK + ", due to the fact'
of the restoration then observed of their capacity to phosphorylate
thymidine in the abovesaid medium and consequently of developing
therein.
Of course, the DNA fragment containing the gene of thymidine-
kinase can be replaced by any other suitable complementation DNA. By
way of example of other complementation genes which can be used for
the constitution of the vectors according to the invention, will be
mentioned that containing that of dihydrofolate reductase (DHFR), or
in general, any complementation gene of which numerous examples exist
in nature.
The complementation DNA may naturally include a natural gene.
It may also be synthesized, notably enzymatically, by copying a
corresponding messenger RNA.
As regards the technique of co-transformation itself, it is
advantageous to use the vector in which the complementation gene is
inserted and the vector in which the derived DNA HBV fragment is
inserted in amounts that are in a high ratio above 100, for example
of the order of 1000. The higher this ratio, the greater than the
number of cells co-transformed by the two vectors at the same time.
j
CA 1341641 1981-04-22
- = 12
It is advantageous to resort to the same basic vectors'
to form the two types of modified vectors used for the co-
transformation. A particularly favorable basic vector is cons-
tituted by the plasmid pBR 322. It will be possible in parti-
cular to insert a "DNA HBV tandem" into its EcoRI site to form
- one of the modified co-transformation vectors (pCP10).
By the insertion of a tkHSV gene, either into a =
PvuII site, or into aBam HI site of the same PBR 322 plasmid
it is possible to obtain the second co-transformation vector
(pAG0). =
As regards the cultivation conditions, it is naturally
self-evident that the medium previously used to reach a sufficient
development of the cultures, must be replaced at the time of
the transformation or little after the latter by a.medium (such
as the HAT medium in the case where the complementation DNA =
selected is a thymidine-kinase gene) in whiCh. the non-
complemented cells are incapable of developing.
The, production of said co-transformations, particularly
when the above-said preferred modified plasmids are brought
2D into play in mouse fibroblasts, leads to the remarkable result
constituted by the excretion by the co-transformed cells into
the culture medium of particles having immunological properties
characteristic of' the envelopes of the hepatitis B virus,
notably as regards their agglutination by antibodies capable
of agglutinating the natural HBsAg antigens, as isolated from
human serums. The presence of excreted particles agglutinatable
by anti-HBsAg antibodies can be detected by conventional radio-
immunological tests, for example by indirect immunofluorescence
bringing into play fluorescent anti-HBsAg and anti-IgG rabbit
serums.
CA 1341641 1981-04-22
13
-
The amounts formed can be measured by direct passive
hemagglutination tests, in manner known in itself for natural
HBsAg.
It is also remarkable that the excreted cells, notably
in mouse cell cultures, do not contain detectable traces of
HBcAg and HBeAg antigens, detectable by direct immunoflurores-'
cence bringing into play fluorescent human antibodies anti-
HBc and anti HBe/1, 2, 3. By contacting the co-transformed
cells themselves .with an anti-HBsAg serum, individualised
cytoplasmic granules are revealed by indirect immunofluorescence
in the major part of the co-transformed cells. These particles
can also be spotted after lysis of the cells. In the majority
of cases the excreted particles form the major part of the
particles agglutinatable by anti-HBsAg antibodies which can be
produced.
The particles formed, whether excreted into the medium
. or retained in the cytoplasm of the co-transformed cells, have
still the properties which will again be mentioned below in
the description of the examples.
2D It was found however that the transformation of HeLa
human cells with a circulariscd cloned DNA of a hepatitis B
virus led to the excretion by these cells not only of HBs.
antigen, but also of complete viral particles containing HBc
antigen in the culture medium (9).
Also it is advantageous, according to an additional
improvement of the invention, to -resort to an insertion
sequence such as has been described above, derived from
DNA HBVI from which there has previously been excised certain
portions external to the S gene of the DNA HBV, so as to use
1
' 11
CA 1341641 1981-04-22
- 14
only effective genames which are no longer capable of coding for
whole Dane particles. In particular, recourse is advantageously
had to an insertion sequence from which a sufficient portion
- of .the .coding gene for the HBc antigen has been deleted, in _
order to prevent the production of the latter antigen by
the infected cells.
These insertion sequences can consequently be used
to modify vectors which will then be suitable for transforming
eukaryotic cells,- whether it relates to mouse fibroblasts or
human cells, but in the absence of any possibility of HBc
antigen production.
In particular, the DNA insertion is advantageously
cOnstituted by the DNA of the virus of viral hepatitis .13,
from which the fragment bounded by the BglII ends at the level
of the nucleotides 1986 and 2425-has been deleted, if reference
is made to the, diagrammatic map of Figurel,inSide the p gene,
whose relative position with respect to the whole DNA has been
=
symbolised.by the C arrow.:
It is also possible to resort to original insertion
2D sequences of DNA HBV, from which larger fragments have been
deleted, notably such as already defined above.
02 course, this insertion DNA can comprise additional
deletions as long as the latter affect, neither .the expression
capacity of the insertion sequence, nor the phases of its
translation. In the same way, the invention extends to the
use of any equivalent insertion sequence, whether it relates
to the sequence corresponding to a viral DNA belonging to
another sub-type than that which has been contemplated in
the present description and taken up in the examples, or
=
= =
= 11
CA 1341641 1981-04-22
. . / .
. . ..
.
. ,
_ , = .
.
, . .
. . . .
,
.
of a sequence .from:vrhich certain parts would have been modified . .
Without however having*altered.the Capacity of the expression
products to react immunological*. with antibodies against
HBs antigens. = =
=
,
5 = It has thus been possible to obtain, from cell
cultures
of mouse fibroblasts transformed with modified vectors by
= .
. .
insertion sequences, such as defined above, antigen preparations
. having the immunological characteristics of HBsAg antigen,
.
. devoid (within the limits 92 available detection methods) of.
, 10 any Dane particle, and of other characteristic antigens con-.
.
tained in the latter, in the absence of components of human
.
origin, notably of any serum prOtein. .
. .
. .
- In addition, it has been, verified that the proteins.
. excreted into the culture media possess in fact vaccinating
15 immunogenic properties, as.is witnessed by their capacity to,
induce the production in Vivo of antibodies, similar to the
human anti-HBsAg antibodies and active-against hepatitis virus, .
.more particularly B hepatitis, when they are administered- .
in vivo, notably to the-mouse or the rabbit, according to
currently used.experimental procedures to verify the immuno-
genitity of the natural HBs antigens, extracted from human
=
= . Serums. = .
.,_
..___ _ __ _
. The invention hence also relates to 'novel - '
preparations useful for the constitution of vaccines, charac-
.25 tensed in that they - consist of proteins having immunogenic
' and immunological properties characteristic of the HBsAg
. antigen , having a total purity level as regards the 'absence
= of any Dane particle, of HBo antigen and of any serum component
of human origin.
.
. .
, ..
. = =
,
. . .
. . . .
. . . , .
. .
= '
. =
,
. .
. . . .
.
di
. _ CA 1341641 1981-04-22 I . '
. = ' ' .
,
111V
16
The invention relates more particularly to vaccine
compositions containing said antigens (such as can be recovered
from culture media of said co-transformed cells or from lysates
of the latter), if necessary, associated with any suitable
pharmaceutical vehicle for the constitution of active vaccine
against viral hepatitis, capable of being administered by
the oral or parenteral route.
It relates also to laboratory reagents containing par-
ticular doses of 'these antigens, capable notably of being used
as standards or references with respect to which the degree
of purity of preparations containing HBsAg antigens, whether
of natural origin or not, can be evaluated as regards in parti-
= cular their relative content of serum proteins, or other
antigens, such as HBcAg or HBeAg, etc.
The invention will again be further illustrated by
the description of examples of the application of modified
plasmids (which themselves form part of the invention) for
the manufacture of antigens having immunological properties
of HBsAg.
In the description, the numbers between parentheses
refer to the bibliography added at the end of this specification.
D.
CA 1341641 1981-04-22
- 4101) 17
DETAILED DESCRIPTION OF PREFERRED EMBDDIMENTS
. EXAMPLE 'I Construction of a recombinant containing DNA of
the virus of B viral hepatitis.
200 ng of pBR 322 were subjected to digestion by
the endonuclease EcoRI and treated with 2.6 units of alkaline
phosphatase in 100 nM of Tris-HCl, pH 8, at 60 C for 60 minutes.
After two extractions with phenol, then three extractions
with ether, the DNA was precipitated by ethanol. The solid
residue was dissolved in water and to the solution 100 ng of
Eco HBV DNA added. The ligature was effected according to
. the method described in (1).
A culture of E. coli DP 50 followed in a culture medium
constituted by a broth L containing 100 11011 of diaminopimelic
acid and 23 pg/m1 of thymidine.
The bacteria were then transformed according to the
above-indicated method by mixing recombinants selected according
to their capacity of withstanding doses of 100 1.1g/m1 of
ampicilline and 15 jig/ml of tetracycline. 900 colonies were
obtained. They were all tested for the determination of the
presence or not of HBV DNA by hybridatiOn in situ. The test
was positive for 800 colonies. The 16 Colonies which induced
hybridation signal of higher intensity were collected. The
pIasmids . were extracted and their structure Was analysed by
digestion in the presence of EcoRI, Xho I, Hind III and Xba I.
The structure of the recombinant obtained results
from Figures 2a. and 2b in which have been shown diagrammatically,
one one hand, the structure of the Eco .HBV DNA gene and,
on the other hand, the structure of the modified PCP 10 plasmid
=
lr
CA 1341641 1981-04-22
=
= Ili= 18
and such as results from the insertion in the plasmid
pBR 322 of a DNA fragment constituted by two successive
Eco HBV DNA fragments, the head of one being connected to
the tail of the other, at the level of an EcoRI site (inser-
tion in tandem head to tail).
It is observed that among the 16 above colonies which
were retained, 14 among them harbored plasmids of the pCP 10
type, in which two Eco HBV DNA occured inserted according
to the head to tail tandem arrangement in the two directions
of orientation possible.
It is in particular due to the digestions with
Hind III and Xba. I endonucleases that it has been possible
to determine the insertion directions of the DNA fragments Eco
HBV DNA,
The integrations in the pCP 10 plasmid of two
Eco HBV DNA fragments were demonstrated by digestion in the
presence of Xho I endonuclease, which produced the excision
of an DNA fragment having a size similar to that of Eco
HBV DNA from the hybrid plasmid pCP 10. The latter includes
10,626 pairs of bases.
The respective positions of the S gene in Eco
HBV DNA (Figures 2a and 2h) and in the hybrid pla.smid
pCP 10 are respectively represented by the arrows S drawn
in heavy lines. The small side arrows locate the relative
positions of certain of the restriction sites in the corres-
ponding DNA chains. The positions of the resistance factors
to ampicilline (ApR) and to tetracycline (TcR) are also
shown diagrammatically.
CA 1341641 1981-04-22
19
= 4101'
The same conventions apply to the pAGO plasmid
(Figure 20. which has..been obtained by insertion into the
Pvu II site of the pBR 322 plasmid of the gene of thymidine-
- kinase of the Herpes simplex virus HSV-1-(HSV-tk)
. Cultivation and transformation.by the above-said Elasmides of
thimidine-kinase deficient mouse cells iLtk-).
Mutants of LM mouse cells deficient in thymidine-
kinase (Ltk-) were cultivated in the minimum essential medium
MEM 0 111 Gibco, if necessary in the presence of 10 % calf
serum. Confluent monolayers of these cells (2 x.106 cells per
25 cm2) in Falcon flasks were inoculated with DNAs corres-
ponding to said plasmids y for their transformation, according
to the method of GRAHAM and VAN DER EB (3) modified by
STOW and WILKIE (4).
To carry out this transformation there was used at
the same time the pAGO plasmid (linearised by Hind III
endonuclease) and pCP 10 plasmid linearised or not by the
same enzyme. In all the tests the molecular ratio pAGO/pCP 10
has been of the order of 1/1,000. DNA salmon sperm was
2D used as a vehicle to adjust the concentration of DNA to
at least 10 1.1g/ml.
After the transformation, the cell cultures were
= kept in this medium containing in addition 15 lag/m1 hy-13o). anthine,*
0.1 1.1g/m1 of aminopterine and 5 1.1g/m1 of thymidine (selective
.25 HAT medium).
24 hours after the transformation, there was added
to the medium a: HAT. solution concentrated 100 times, which.
concentrated solution was changed .a week later, and then
every three days. .
= =
, __
. .
CA 1341641 1981-04-22
20
= I.
After 15 days of cultivation in the presence of
the two types of plasmides (co-transformation) in the selec-
tive HAT medium, the formation of colonies was observed.
= 20 days after the co-transformation, the HBsAg production
was detected in the culture medium by radio-immunological
' tests. Cultures formed Tinder the same conditions and trans-
formed for purposes of comparison with the single plasmid
pAGO did not induce any HBsAg production.
The tk'. colonies resistant to the HAT medium were
collected with a Pasteur pipette (20 days after the co-
transformation) and transferred to tissue cultures on micro-
plates. Passages of the colonies were carried out every
5 days and they were maintained under, continuous selective
pressure in the HAT culture medium.
Five of these colonies obtained from co-transfor-
mation in the presence of linearised pCP 10 and 10 colonies=
'
obtained from the co-transformation of the mouse cells
with the circular pCP 10 were sampled and cultivated in
HAT medium. All the cultures produced HBsAG which was released
in the external culture medium.
The amounts of HBsAg synthesizedwere variable from
one culture to the next (within limits in a
ratio 1 to 30). The amounts producedwere stable, even after
several passages of said cultures.
The HBsAg can be recovered by centrifugation 'of ,
the supernatant liquor of the cultures and purified by
centrifugation in a density gradient based on CsCl.
The HBsAg were collected in the zone of density 1.20 a/ml.
lt
CA 1341641 1981-04-22
= 21
. .
These HBsAg were completely neutralised by an anti-HBsAg
serum solution (in a ratio 10/1), after incubation at
37 C for one hour, as detectable by the tests of radio-
immunology. .
=
On examination under the electron microscope,
spherical particles were observed having sizes ranging
from 18 to 25 nm (on the average 22 nm). Their morphology
recalled that of the spherical particles of antigens of .
22 nm which can be isolated from human serum. Filamentous
structures such as those visible among the antigens extract-
able from human serum have not been observed, at least under
the conditions of the experiment. No Dane particle was .
= detectable.
= .
. .
, = The following Table illustratGsthe production
.
_
capacity of the mouse cells tk which had been co-transformed
. by the plasmids concerned.
The ratio P/N corresponds to the ratio of the
number of disintegrations per minute (dpm) measured on the
supernatant liquor to the number of dpm measured on a
controlled culture when the dosages of HBsAgviere done by
immunological tests 23 days after the co-transformation of . .
the cells. A ratio P/N higher than 2.1 Was considered as .
.
significant.
. ..,
' .
= .
,
! =
lr = .
,
CA 1341641 1981704-22
22
TABLE
Co-transformation of ,Ltk- cells of mice with
pCP 10 and pAGO plasmids,.
Flask =linearised linearised circular salmon number of P/N
pAGO pCP 10 pCP 10 sperm colonies
DNA tk+
b 0.010 10 140
24
0.005 5 15 200 16
0.010 - 15 26 1 After lysis of the
previously washed cells, diges-
tion of the proteins in the presence of proteinase, extract-
ion and purification of the cellular DNA, estimation of the
cellular DNA by the technique of SOUTHERN modified by WAHL
and coll. (5, 6) by resorting to a probe Eco HBV DNA prepared
by the technique of WEINSTOK and coll. (7), it has been
observed that the amount of intracellular HBsAg which had
been synthetisized corresponds approximately to a third
of the HBsAg which had been excreted into the culture medium.
Under the conditions of the experiment, the most
active clones produced up to 150 ng/ml of,HBsAg.
23= In addition to the absence already mentioned of
Dane particles, no
HBcAg and HBeAg antigens were
observed at least at a level detectable by direct immuno-
fluorescence utilising anti-HBc and anti-HBe/1,2,3 fluores-
cent antibodies of human origin (8). Moreover, the product-
ion of DNA-polymerase, by measurement of the DNA-polymerase
activity by the method of KAPLAN and coll.,has Piot been
observed (either in the centrifugation residue from the
11
CA 1341641 1981-04-22
'
IP 23
. , supernatant liquor or in that of the lysate of the previous¨
ly transformed cells).
By extraction of the DNAs of high molecular weight
of certain cloned products, digestion of these DNAs in the
presence of EcoRI, Hind III and Xho I, fractionation of the
fragments obtained by electrophoresis on agarose gel, transfer
to nitrocellulose filters, hybridation of these fragments
with Eco HBV DNA probes labelled with phosphorus 32, it
has been established that several copies of the plasmid
pCP 10 could be _ incorporated jilt the cellular .DNA. There has
also been noted the presence of HBV DNA diners both in
the plasmid copies contained in the transfamed
cells and among the fragments possibly 'incorporated into
the cellular DNAs.
It has also been determined by means of kinetic
studies that under the experimental conditions, which have
been described, HBsAg antigen particles can be excreted
into the medium by the cells, in the proportion of 2 x 104
to 4 x 104 particles/cells/24 hours, namely 2,000,000 to
. 4,000 ,000 molecules of polypeptide /cells/24 hours, if it .
is assumed that each particle contains about 100 molecules
of polypeptides.
EXAMPLE II : Expression into eukaryotic cells of vectors
modified by an insertion sequence derived from
DNA HBV, from which the major portion of the
C gene has previously been excised =
1 ) Cloning of PCP9 iresulting from the insertion of the
linearised EBR3 22 p2..asmid in the EcoRI site of DNA HBV.1.
The pBR3 22 plasmid hydrolysed by EcoRI was
treated with alkaline phosphatase, then ligated in the
,
/
CA 1341641 1981-04-22
=
24
' lir- presence of an equimolecular amount of DNA HBV cut by
=
EcoRI. The arrow PCP9 symbolises, in Figure 1, the insertion
site of pBR322 into the DNA HBV.
2 ) Construction of clones of the PAC series icontainin2 DNA
HBV fra2ments icut by B21II1 and PANG icontainin2 fra2-
ments of DNA HBV cut by BamHI). =
The pAGO plasmid was hydrolysed by BamHI and
=
= treated with alkaline phosphatase.
The HBVsfragments were obtained from PCP10 plasmid
described above, after cleavage by Hind III and Pst I (which
have no site in the DNA HBV), and more particularly, from the
fragments obtained purified by electroelution on an agarose
gel, and containing a dimer - or a "tandem". sequence - of
DNA HBV. These fragments were partially hydrolysed, either by
the restriction enzyme BglII, or by the BamHI enzyme, under
the conditions where only two cleavagetwere Made. The hydro-
lysates were then ligated in. equimolecular amount with
pAGO plasmid previously cleaved by. BamHI (this ligation
taking advantage of the fact that the BamHI and BglII sites
2D are characterised by common cohesive end portions).
A collection of clones sensitive to tetracycline
was obtained and analysed in order to isolate each insertion.
possibility.
Clones PAC 12, PAC 14, PAC 16 and PANC 34 were
obtained. The arrows accompanying the corTesponding symbols
in Figure 1, locate the insertions of the pBR322).inearised
in the BamHI or BglII sites corresponding to the sequences
of DNA HBV contained in the recombinant plasmids obtained.
=
=
it
=
CA 1341641 1981-04-22
. ,
, 25
41106 . These clones had_ in addition the following characteristics :
PAC12 : deletion of the BglII fragment 1985-2840 ;
PAC 14: deletion of the BglII fragment 1986-2425 ;
PAC 16: insertion of the genome of pBR3 22 in the 19 86 site
of the DNA HBV ;
PANC 3 4: it containedthe pBR3 22 genome in the BamHI 1400
site of DNA HBV.
These clones have been used to transfonn LTC cells
in the proportions of 2 lag of plasmid- per 2.106 cells.
4 weeks later, the TIC+ colonies reached
confluence. The
culture medium was then analysed for the presence of HBs
by applying the above-said radio-immunological tests.
The capacities or non-capacities of the various
recombinant plasmids to express the S gene, in the form of
HBs antigen excreted into the culture medium, have been
expressed respectively in Figure 1 by the signs "+" and "-".
The expression of the S gene in the clones PAC 14
and PAC 16 and the failure of expression in the clone PAC 12
show that the transcription of the S gene is initiated in
viral DNA and more precisely in the restriction fragment
BglII 2425-2840. There exists a sequence TATATAA also
called "TATATAA Box" situated at 72 nucleotides upstream
of the beginning of the "pre-S" region which appears to
control the transcription of the S gene..
The fact that no expression was obtained with the
PANC 34 clone, under the conditions in which the experiment
was carried out, is perhaps connected with the fact that
the DNA encoding,for the mRNA stops after the BamHI site.
2
CA 1341641 1981-04-22
=
26 .
-
It is .particuiarlj most signiTidant that the
PAC 14 clone, which produces the HBs, possesses a deletion
in the C gene of the virus, which eliminates the risk of
. the production of viral particles for the transformed cell,
whatever its nature, and permits its use for the production
of a vaccine.
The invention hence provides a product capable of
a purity unattainable hitherto. It relates more particularly. .
. to the preparations p-roteiri'pai-ti6les having the immunological
.properties
,of HBsAg, essentially free of seric protein. They are totally free of
Dane particles -detectable by the usual methods of radio-
immunological measurement. Theyare in.addition totally free.
of proteins, notably serum proteins of human origin. They
are free of DNA polymerase.
.The invention relates also again to the novel
= DNA insertion sequences themselves, as they-haVe been defined
above, and the Vectors, notably plasmids , containing said
sequences which are more particularly characterised by the
fact that they contain the promoter of the transcription
of the S gene. These vectors are of very particular interest
in that they can if necessary be modified by the insertion of
a particular DNA sequence corresponding to a protein whose
expression into eukaryotic cells is sought. In fact, this
vector has a particular interest in not being toxic with
respect to the cells concerned. The promoter is not repressed,
since these cells permit the expression of the S gene. In
addition, this vector can enable the excretion of proteins
synthesized directly in the culture medium.
=
=
=
it
CA 1341641 1981-04-22
=
. . .
,
-,
II'.
-=
. . 27
One of the above-said novel insertion sequences
can again be defined as containing, in addition to the
=
S gene, the DNA of the "pre-S" region (which in the DNA
. of the virus of viral hepatitis is situated immediately
upstream of the S gene in the reverse direction of the .
transcription, this gene including of the order of 163
.
nucleotides), the gene of the messenger RNA of the S gene
of the virus of B hepatitis, the promoter of the DNA of
the virus of B hepatitis, notably a TATATAA sequence
. .
(T being abbreviation for thymine and'A abbreviation for ,
adenine). It comprises notably also a DNA sequence corres-
ponding to a sequence localised between the nucleotide
positions 2.425 and 2840 of the DNA of the virus of B.
hepatitis.
. There are obviously included within the scope of
the invention, sequences including several units, of the
above-described type, notably "tandem" sequences.
The capacity of the vectors modified according to
the invention of being expressed into eukaryotic cells seems
,
to ' . witness bi-' the fact that the transcription of the
gene of HBsAg is under the control of a viral promoter con-
tained in the fragment derived from HBV DNA. These vectors,
such as the plasmid pCP 10, PAC 14 or PAC 16, can themselves
= be used as vectors for effecting the expression into
eukaryotic cells of a foreign DNA previously inserted
into said vectors. It appears that the advantage of the
vectors modified according to the invention must reside in
the fact that they do not induce lysis of the host-cells.
,
.
.
.
=
. . . .
. .
,
11 =
CA 1341641 1981-04-22
,
=
= 28
Moreover the S gene can be considered as having a sequence
signal under the control of which the hybrid protein formed,
resulting from the expression into the host cells of foreign
DNA previously inserted into the above-said vector, would be
excreted into the culture medium. The recovery of this hybrid
protein would obviously be considerably facilitated
therefrom.
Although the method of the invention has been described in
the foregoing, principally in its application to DNA HBV, it
can be extended to any other circular DNA or whole genomes.
In particular it can be applied to the study of circular DNAs
of "non A", "non B" viruses of the corresponding viral
hepatitis.
***
In some aspects, embodiments of the present invention as
described herein include the following items:
1. Genetically engineered non human and non infectious
antigenic particles comprising a protein antigen which
contains a polypeptide encoded by the S gene of a hepatitis
B virus infectious in humans, said protein antigen being
totally free of human components and free of Dane particle
and of HB core antigens (HBc Ags) and having vaccinating
properties against hepatitis B, which particles are
agglutinated by antibodies which agglutinate HB surface
antigens (HBs Ags) present in human serum, are free
totally of seric proteins of human origin, and are not
CA 1341641 1981-04-22
= 29
detected by anti-HBc antibodies, wherein said particles
have sizes in the range of about 18 to 25 nm.
2. The genetically engineered particles according to item 1,
wherein said particles have an average size of 22 nm.
3. The genetically engineered particles according to item 1
or 2, wherein said particles are spherical and free of
detectable filamentous structures.
4. The genetically engineered particles according to any one
of items 1 to 3, wherein said particles have a density of
about 1.2 g/ml in a CsC1 gradient.
5. The genetically engineered particles according to any one
of items 1 to 4, wherein said particles are capable of
inducing in vivo the production of active antibodies with
respect to hepatitis B virus.
6. The genetically engineered antigenic particles according
to any one of items 1 to 5, wherein said particles are
free of DNA polymerase.
7. A method for the production of the genetically engineered
antigenic particles as defined in any one of items 1 to 6,
said method comprising:
(a) providing a mammalian host cell transformed with a
recombinant vector comprising a DNA sequence encoding at
least one portion of the hepatitis B envelope protein,
said DNA sequence being operatively linked to a promoter;
(b) causing said at least one portion of the envelope
protein to be expressed and formed into said particles,
CA 1341641 1981-04-22
= 30
said particles being capable of inducing in vivo the
production of active antibodies with respect to the
hepatitis B virus, and;
(c) recovering the genetically engineered antigenic
particles.
8. The method according to item 7, wherein said promoter is
the promoter of the S gene.
9. The method according to item 7 or 8, wherein said host
cell is of murine origin.
10. The method according to any one of items 7 to 9, wherein
said DNA sequence comprises the nucleotide sequence of the
S gene encoding the polypeptide to which is attributed the
immunological properties of the HBs antigen.
11. A mammalian cell line transformed with a recombinant
vector comprising a nucleotide sequence of the S gene
encoding the polypeptide to which is attributed the
immunological properties of the HBs antigen, operatively
linked to a promoter, said nucleotide sequence being
capable, when expressed, of providing the antigenic
particles as defined in any one of items 1 to 6.
12. The mammalian cell line according to item 11, wherein
said promoter is the viral promoter under the control of
which the transcription and the translation of said
nucleotide sequence is normally carried out.
, CA 1341641 1981-04-22
410 31
13. The mammalian cell line according to item 11 or 12,
wherein said mammalian cell is of murine origin or an HeLa
cell.
14. The mammalian cell line according to item 13, wherein
the mammalian cell is a mouse cell.
15. The mammalian cell line according to item 13 or 14,
wherein the mammalian cell is a mouse cell deficient in
thymidine kinase.
16. The mammalian cell line according to item 11 or 12,
wherein said mammalian cell is an HeLa cell.
17. A composition of matter comprising the genetically
engineered antigenic particles as defined in any one of
items 1 to 6 and an excipient.
18. A vaccine composition which comprises in an amount
sufficient to confer immunity to HBV infection, the
genetically engineered particles as defined in any one of
items 1 to 6 and a pharmaceutically acceptable carrier.
19. An isolated recombinant surface antigen of a Hepatitis
B virus encoded by a recombinant DNA expressed in mammalian
cells, wherein a sequence localized between the nucleotide
positions 2425 and 2840 of the DNA of the hepatitis B virus
and a fragment bounded by the BglII ends at the level of
the nucleotides 1986 and 2425 of the DNA of the hepatitis
B virus is deleted, in reference to Figure 1.
CA 1341641 1981-04-22
111 32
20. The recombinant surface antigen of the Hepatitis B virus
according to item 19, comprising the pre-S and S sequence
of the Hepatitis B virus.
21. The recombinant surface antigen of the Hepatitis B virus
according to item 19 or 20, which is glycosylated.
22. A method of detecting the presence of an HBV antigen in
a medium comprising:
- contacting antibodies recognizing HBsAg or pre-S HBsAg
produced in mammalian cells with a medium, wherein said
HBsAg is totally free of human components, Dane
particles, and HE core Ag or HBe Ag; and
- detecting the immunocomplexes formed,
wherein the formation of immunocomplexes indicates the
presence of the REV antigen.
23. A method of detecting the presence of antibodies binding
to HE surface antigens (HBsAg) or pre-S HBsAg in a medium
comprising: contacting HBsAg or pre-S HBsAg produced in
mammalian cells with said medium, wherein said HBsAg is
totally free of human components, Dane particles, HBc Ag
and HBe Ag; and
- detecting the immunocomplexes formed, wherein the
formation of immunocomplexes indicates the presence of
the HBsAg or pre-S HBsAg.
24. A recombinant DNA molecule encoding a hepatitis B antigen.
25. The recombinant DNA molecule according to item 24, wherein
said recombinant DNA molecule comprises more than two
Hepatitis B virus (REV) DNA sequences recombined with a
CA 1341641 1981-04-22
33
vector, wherein said vector comprises sequences of E.
coli plasmid pBR322 and has an antibiotic resistance gene
as a selective marker, wherein each of said HBV DNA
sequences is comprised of full-length HBV DNA, and wherein
said HBV DNA sequences are arranged in a head-to-tail
tandem relationship.
26. A vaccine capable of producing protective immunity in a
mammal against Hepatitis B infection, said vaccine
containing a pharmaceutically acceptable excipient and
non-infectious 22 nm spherical particles of hepatitis B
surface antigen having a mammalian pattern of post-
translational modification, said vaccine totally lacking
primate seric proteins, Dane particles or hepatitis B
core antigen.
Below the bibliography relating to the state of the art and
to which reference is made in the description of the examples,
is given:
(1) TIOLLAIS, P., PERRICAUDET, M., PETTERSON, U. & PHILIPSON,
L. (1976), Gene 1, 49-63.
(2) COLBERE-GARAPIN, F., CHOUSTERMAN, S., HORODNICEANU, F.,
KOURILSKY, P. & GARAPIN, A. C. (1979), Proc. Natl. Acad.
Sci. USA, 76, 3755-3759.
(3) GRAHAM, F. L. & VAN DER EB, A. J. (1973), Virology 52,
456-458.
(4) STOW, N. D. & WILKIE, N. M. (1976), J. Gen. Virol. 33,
447-458.
(5) SOUTHERN, E. M. (1975), J. Mol. Biol. 98, 503-517.
riA
CA 1341641 1981-04-22
34
(6) WAHL, G. M., STERN, M. & STARK, G. R. (1979), Proc.
Natl. Acad. Sci. USA 76, 3683-3687.
(7) WEINSTOCK, R., SWEET, R., WEISS, M., CEDAR, H. & AXEL,
R. (1978), Proc. Natl. Acad. Sci. USA 75, 1299-1303.
.. (8) TREPO, C., HANTZ, L., VITVITSKI, L., CHEVALLIER, P.,
WILLIAMS, A., LEMAIRE, J. M. & SEPTJIAN, M. (1978) in Viral
Hepatitis, eds. VYAS, G. N., COHEN, S. N. & SCHMID, R. (The
Franklin Institute Press) pp. 203-209.
(9) SHALOMZ. HIRSCHMAN and Coll., Proc. Natl. Acad. Sci.
USA, vol. 77, No. 9, pp. 5507-5511, September 1980,
"Expression of cloned hepatitis B virus DNA in human cell
cultures".
CA 1341641 1981-04-22
