Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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A METHOD OF PREPARING A VACCINE AGAINST HEPATITIS B AND
THE VACCINE THUS OBTAINED
The present invention relates to a method of
preparing a vaccine agains~ hepatitis B as well as the
vaccine obtained by putting this method into practice.
The difficulties of all kinds met with in the
industrial preparation of vaccines against hepatitis B by
purification of the antigen HBsAg from human blood have
led quite naturally to the search to obtain an~igenic
proteins from microorganisms transfected by gen~s
encoding the hepatitis B virus.
M. Michel et al. have expressed antigens from
the envelope of the hepatitis B virus from ovarian cells
of the chinese hamster (CHO) tsee reference 1),
transfected by a plasmid carrying the complete HBsAg
gene. These cells continuously excrete, in the cell
culture medium, ~BsAg particles containing both the
pre-S~ and S proteins.
However, during such expression, the HBsAg
particles are present in the cell culture medium
containing undesirable substances such as proteins of the
calf serum used for the cell growth, cellular proteins
and DNA and retroviral particles. These retroviral
particles come from the original cell before
transfection.
The invention proposes overcoming these
drawbacks by providing a method for pr~paring a vaccine
against viral hepatitis B by expression of the HBsAg gene
introduced into CHO cells, which makes it possible to
produce a vaccine against hepatitis ~ industrially.
Another object of the invention is to provide
such a method of preparation which makes it possible to
produce such a vaccine having an extremely high degree of
purity~ particularly in so ar as the cellular DNA or
DNA fragments are concerned as well as insofar as the
retrovirus contaminating the CHO cells is concerned.
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Another object of the invention is to produce
such a vaccine which has a particularly high protective
power.
~ he invention provides a method for preparing a
new recombining vaccine against hepati~is B, in which
surface antigenic particles of hepatitis B are produced
by expression from a culture of CHO cells transfected by
a plasmid carrying the HBsAg gene so as to release the
antigenic surface particles in the culture medium,
characterized in that
- the supernatant culture medium is recovered
from at least one culture, preferably effected with a low
animal serum content,
- the supernatant medium is subjected ~o
sterilizing filtration,
- it is subjected to concentration, preferably
about 50 times, preferably by ultrafiltration,
- the concentrate is precipitated by means of a
non degrading precipitating agent under conditions
precipitating the heavy DNA classes, sai~ precipitation
also entraining the retroviral particles, and proteins,
- a new concentration of the antigenic
preparation is carried out, preferably by means of a non
degrading precipitating agent under conditions
precipitating the surface antigenic HBsAg particles, then
red.issolving the precipitant in a small volume,
- a rate zonal centrifugation is carried out in
a non chaotropic density gradient or the retroviruses so
that their integrity is maintained so as to allow
separation thereof from the HBsAg particles, depending on
their size and density, preferably by also obtaining
discximination between the an~igenic HBsAg particles on
the one hand, and a separation between HBsAg, DNA and
proteins, on the other hand,
- an isopycnic zonal, of flotation type,
centrifugation is carried out eliminating the light and
heavy nucleic acids and the proteins,
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- chromatography is ef~ected on an anion ex-
changer so as to adsorb the traces of nucleic acids and
remaining non HBsAg proteins.
By non degrading precipitating agents is meant
agents which, of course, do not degrade the HBsAg
anti~ens and particularly those of type S and pre S, but
also which have no chaotropic or dissociating effect for
the retroviral particles. The preferred agent is
polyethylene glycol (PEG). Among other agents, may be
mentioned more particularly ammonium sulphate.
The purification steps therefore do not
denature the HBsAg particles and in particular do not
denature the pre S proteins which are the most exposed.
Among suitable precipitating agents, polyethylene glycol
(PEG) is to be preferred by choosing more especially a
molecular weight of the order of 4000 to 20000, which
makes it possible under suitable conditions, such more
particularly as a PEG concentration of the order of 5~,
to precipitate the retroviral particles while also making
possible in particular precipitation of the heavy DNA
classes while keeping the HBsAg particles in the
supernatant of the precipitate. These latter may be
recovered in a very small volume, prefera~ly after having
precipitated them by adding PEG to higher concentration.
The density gradient in which the rate zonal
centrifugation is carried out is also provided so as not
to adversely affect the HBsAg antigen, including S and
pre S antigeniciti~s. In addition, this gradient is
provided so as to have no chaotropic efect on the retro-
viral particles so that their integrity is maintained.
Preferably, this gradient, of low ionic force, is a su~-
rosa gradient. In a variant, o~her density gradients may
also be used, particularly glycerol.
The rate zonal centrifugation which is thus
carried out in such a density gradient thus makes it
possible to eliminate the retroviral particles extremely
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efficiently. At the same time, some DNA purification may
be obtained, par~icularly in heavy chains and in
proteins.
The isopycnic zonal centrifugation of
flotation type on a saline density gradient, such as KBr,
is thus effected on a preparation practically completely
free of retroviral particles. However, if retroviral
particles contaminated the sample to be purified, the
chaotropic effect of the salt forming the density
gradient, would destroy the viral structure and would
release the RNA viral genome in the dense zone of the
gradient where it would stop, thus making it possible to
separate it from the HBsAg antigen.
This step makes possible the elimination of
practically all the proteins as well as existing light or
heavy DNA traces.
Finally, the anion exchange chromatography step
allows the possible nucleic acid traces, as well as DNA
and RNA still remaining and proteins to be adsorbed.
Other advantages and characteristics of the
invention will be clear from reading the following
description, of a non limitative example.
1. PREPARATION OF T~E CELLULAR CULTURE
ZS
A CHO cellular colony clone transfected by a
recombining plasmid (patent application FR-A 84 03564
published under the number 2 560 890) carrying the gene
of the hepatitis B virus encoding for the HBS antigen
(pre S and S regions), is multiplicated from a cellular
bank vial, in fermentation units of increasing volumes.
The~ last multiplication is carried out in a 300
fermenter, the cells propagating on microballs in
suspension in the culture medium. For these
mul~iplying steps, the growth medium contains 4 to 10%
o CS (calf serum), preferably 5~.
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2. CULTURES, HARVESTING AND CONCENTRATION
The first harvest is made after 3 to 4 days of
culture in -the 300 l fermenter: after decantation of the
balls supporting the cells, 250 l of supernatant are
drawn off and replaced by an equivalent volume of new
medium containing 0.2 to 4~ of CS, preferably 1 to 2%.
The cellular culture is con~inued in the fermenter for 3
to 4 days at the end of which a new harvest of 250 l is
made. This operation may thus be carried out up to 10
successive harvests, which are kept at +4C for
purification thereof. As a general rule, the first
harvest and harvest N7 and the following have a low HBs
antigen content, so that they are not retained for the
purification steps.
In the present example, harvest numbers 2 to 6
were kept to be purified and contained 1~ of CS.
After mixing the harvest, the preparation,
having a volume of 1250 l, is filtered on a 0.2
micrometer membrane, then concentrated by ultrafiltration
about 50 times, on a membrane with a cut off point of 100
000 daltons, which gives the concentrated harvest R.
3. FRACTIONATED PRECIPITATIONS
The concentrated harvest R is subjected to
fractionation by selective precipitations in a first
stage of the high molecular weight components,
particularly heavy classes of DNA and retroviral
particles, the HBs antigen remaining in suspension and in
a second stag0, the HBs antigen. The precipitating agent
is chosen so that the retroviral par~icles maintain their
original size and density on the one hand and, on the
other, so that the antigenicity of the HBs particles is
not adversely affected. The preferred precipitating agent
is polyethylene glycol, preferably of 6000 MW.
A concentration of 5% of PEG 6000 is used for
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the first precipitation and the precipitate obtained is
elimina-ted by centrifugation. PEG 6000 is added to the
supernatant S for a final concentration of 9.5%; after
centrifugation, the precipitate is totally dissolved in a
volume equal to about a 625th of the treated volume of
harvest, namely about 2 l, of EC antigenic preparation.
At this stage, the performances in eliminating
th~ cellular DNA and non HBsAg protein are given in the
following table:
,
Volume DNA Non HBsAg proteins
l in l ~g/ml ~g total % ~g/ml ~g total %
15 mix- _ _ _ . _ _
ture 1250 520x10-3650000 100 740 925X106 100
20 vest 25 15 41x10 31031 0 6 14860 374x10 ~.4
med 27.84 1.3xlO 3 36~25 0.0056 8666 241~10~ 2.6
EC 2.03 7.67xlO 9 15.6 0.00~ 11850 24.06xlO 2.6
~5
Whereas the recovery rate of the H~s antigens
equals 80~, elimination factors of 42000 for DNA and 38.S
for the proteins were noted.
~he reverse transcriptase activity reflecting
the retrovirus content is not detec~able in the harvests
and is sometimes slightly positive in the harvest
concentrated 50 times, and , in all cases, it is negative
in the supernatant after treatment with 5~ PEG.
The power in eliminating a retrovirus by this
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treatment step with 5~ PEG was measured by supplementing a
concentrated harvest with MuLV (Murine LeukPml~ Virus): an
elimination factor of 7 was obtained.
4. RATE ZONAL CENTRIFUGATION
A sucrose gradient (O - AO%) is formed in a
KZ~Electronucleonics rotor (Ref~ 2): when th rotation
speed reaches 35000 rpm, the rotor is fed at a continuous
flow rate of 2 l/h first of all with 500 ml of EC sample to
be purified (concentrate), then for the next 30 minutes
with a phosphate buffer 65 mM PH 6.8. After stopping the
rotor, the contents of the rotor is fractiona-ted and the
fractions titrated for their HBS antigen content. The HBsAg
rich fractions are found again in the less dense layers of
U~ the gradient, containing less than 5% of s crose; they are
mixed, under identical conditions, with those coming from
the treatment of the other 500 ml fractions of concentrate.
The mixture of these fractions is dialyzed so as
to eliminate the sucrose and then concen~rated by ultra-
filtration to a volume of 0.361 1. A preparation Z10 is
ob-tained. The HBsAg yield is 80~. The performances in
eliminating ADN and non HBsAg proteins were:
25Volume DNA Non HBsAg proteins
_ in 1 ~g/ml ~g total ~ ~g/ml ~g t~tal
30EC 2.03 7.67xlG 3 15.6~ 0.0024 11850~24.06xlO ~2.6
Z10 0.36 16.2x10-3l5 825l0.00o93 173301 6.24x10~10.67
Although giving appreciable results in elimin-
ating DNA and non H~s~g proteins, this ra~e centrifugationis particularly interesting for its power of discrim-
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inating between the antigen and the retroviral particles.In fact, when the concentrate is deliberately
contaminated wi~h a murine retrovirus (Murine Leukemia
Virus), then subjected to centrifugation under the condi-
tions mentioned, the retroviral particles titrated inreverse transcriptase are found again in the dense layers
of the gradient with an enzymatic activity peak at about
33-35% of sucrose.
The retroviral particles have joined their iso-
dense zone of the gradient by travelling through thegreatest part of the gradien~, whereas at the same time
the HBsAg particles, of lower sedimentation constant
(40s) have only travelled over a small path in the grad-
ient. Separation thereof is provided by a short centrifu-
gation time.
The reverse transcriptase activity measured inthe mixture of the HBsAg rich fractions represents 0.013%
of that found in the retroviral peak, namely an elimin-
ation fac-tor of 77.
5. ISOPYCNIC FLOTATION TYPE CENTRIFUGATION
The rotor is a zonal rotor of type TI15 - Beck-
man sold by the firm Beckman, Palo Alto, California, USA,
of a total capacity of about 1700 ml (reference 3). A
density gradient is formed by introducing at the peri-
phery of the rotor successive volumes of potassium
bromide solution (KBr) so as to form a gradient of dens-
ities between 1.17 and 1.35. One of ~he layars introduced
is formed by the sample to which K~r has been added so
that the density of the layer is equal to 1.25, so that
at the beginning of centrifugation all the impurities and
the antigen are found in the dense part of the gradient.
After centrifugation at 31000 rpm for 20 h, and
after fractionation of the contents of the rotor, the
HBsAg particles are found in their isodense zone (d 1.22
-
~0~9
g
1.23) whereas practically the whole of the other proteins
and the DNA are found in the dense layers of the grad-
ient. The mixture of the H~sAg rich fractions is dialyzed
against phosphate buffer 65mM pH 6.8 so as to elimin-
ate the Ksr and balance the sample in the buffer whichwill be used for the chromatography. The preparation Z20
is obtained.
Whereas the HBsAg yield in this centrifugation
is close to 100~, very high DNA and non HBsAg protein
elimination factors were obtained.
_ Volume DNA Non HBsAg proteins
in l ~g/ml ~g total % ~g/ml ~g total %
Z10 0.360 0 0162 r 82 9.3xl0-4 l7330 6.24x10`6 -0.67
z20 0.4sa 36xlO-61o~o166 2.7x10 234 107x103 0.011
They are respectively 350 for DNA and 58 for
the non HBs~g proteins.
A retrovirus elimination factor could not be
calculated in this step, for the reverse transcriptase
activity is totally des~royed in a KBr medium. This inac-
tivation is probably due to the chaotropic effect of the
salt on the virus. It is interesting to note that this
chaotropic effect ought to release the viral genome (RNA)
in the medium which would be present after centrifugation
in the dense l~yers of the gradient like the cellular
~NA, and thus separatPd from the anti~en peak.
6. ANION EXCHANGE CHROMATOGRAPHY
The antigenic preparation of Z20 dialysed after
isopycnic centrifugation is passed over an anion
exchanger, preferably on DE 52-cellulose Whatman
prevlously balanced ~n a phosphate buffer 65 mM pH 6.8.
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Under these condi~ions, the HBs antigen is not adsorbed
on the exchan~er, whereas the residual dna traces and the
contaminating proteins are fixed on the exchanger:
5_ Vol. DNA nonHBsAg proteins
in l ~g/ml ~g total ~ ~g ml ~g total %
.
10a ter 0.458 36~10-6 0.0166 2.7xlO 234 107x103 0.011
chrom 0 . 600 IZ 5~1U 5 <0 . 0075<1.2xlO 7 4200 4.2
A high elimination factor, of 25 times, is
obtained for the non HBsAg proteins whereas the DNA is no
longer detectable by the dot-blot technique after
hybridization with a probe marked with P32 (sensitivity
limit = 12.5 pg/ml).
To measure the elimination factor for DNA, to a
Z20 preparation was added a known amount of DNA and
chromatographed on the anion exchanger. The DNA rate
found in the effluent of the column was 1000 ~o 10000
times less than the initial rate.
The antigenic preparation thus obtained at
after purification has th following characteristics:
.HBsAg protein........... 1 ~g
.non HbsAg protein...... ~50 ng
~DNA~ ~10~S
Pg
After electrophoresis on polycrylamide gel in a
dissociating medium, the polypeptides coloured by the
silver nitrate spread out over three ma;or bands:
.P22 of an apparent molecular weight of
22.000 daltons
.P26 of an apparent molecular weight of
26.000 daltons
.P34 of an apparent molecular weight of
.
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34000 daltons which are all three recognized by immuno
impression with an anti-S monoclonal antibody, where only
the P34 polypeptide is recognized with an anti-pre S 2
monoclonal antibody.
7. PREPARATION OF VACCINES AND IMMUNOGENICITY
The chromatographed preparation is filtered on
a sterilizing membrane~ then it is heated to +60C for 60
minutes and, after cooling, formaldehyde (100 ~g/ml) is
added and it incubated for 48h at +30C. This antigenic
preparation is diluted in a physiological buf~er and an
additive is added thereto such as aluminium hydroxide for
obtaining vaccines.
Different doses of HBsAg antigens were injected
subcutaneously in guinea pigs and the antibody responses
(anti-S and an-ti-pre S 2) were measured and compared with
those obtained in animals injected with equal doses of
antigen derived from human plasma (Hevac B vaccine -
Pasteur Vaccins MARNES LA COQUETTE ~ FRANCE).
.
RESPONDEX ANIMALS/INJECTED ANIMALS
.
Anti-S response Anti-pre S2 response
HEVAC B CHO HEVAC B CHO
lO~g 5/10 8/10 7/lO 10/10
:~g 4/9 4/10 2/10 10/10
30 2.5~g 1/10 0/10 2/10 5/10
: power 1.0 1.2 _ 3.0
The anti-S ant~ho~ies, generally called
anti-HBsAg, were measured by the Ausab-Abbot~ technique
(Abbot~ Laboratories, USA) and the anti-pre S2 antibodies
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were measured by the immunoenzymatic method using a
synthetic peptide of 31 amino acids on ~he solid phase,
including the pre S2 epitope, so as to capture the
antibodies which were recognized in a second stage by an
S anti-IgG antibody conjugated with peroxydase; after
addition of the substra~e (H202) and o a chromogene
(orthophenylene diam;ne) the coloration developed was
measured at 492 nm, whose intensity was proportional to
the amount of antibodies captured.
As the results in the table show, an equivalent
anti-S response is obtained with the two types of
hepatitis B vaccin~, whersas a higher anti-pre S2
response is observed with the recombined vaccine (CHO).
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.
References.
1. M. Mi~hel (1984) Proc. Natl. Acad. Sci. USA
81,7708-7712
2. Progress in Separation And Purification, edition Perry
and Van Oss, Copyright 1971 by John Wiley & Sons Inc.
USA.
3. A. Fritsch, Les Centrifugations Preparatives en
Gradient de densite, 2nd edition.
