Note: Descriptions are shown in the official language in which they were submitted.
CA 02613732 2013-01-17
1
THERMOREVERSIBLE IMMUNO-ADJUVANT EMULSION
The present invention relates to the field of vaccines; more particularly, the
invention
relates to the field of vaccines comprising an adjuvant emulsion.
Many vaccines which contain one or more adjuvants exist in the prior art. US
patent
6 299 884 discloses in particular an adjuvant formulation comprising an oil-in-
water
emulsion, in which the size of the oil droplets is between 100 and 1000 nm.
This
emulsion is obtained by means of a high pressure homogenizer (microfluidizer),
in the
course of a manufacturing process using high mechanical energies in order to
obtain
shear forces that are sufficiently great to reduce the size of the oil drops.
According to
this teaching, while the minimum value of the size range of the drops obtained
is
100 nm, the mean value is much higher and is, at best, in the region of 170
nm, more
generally in the region of 500 nm.
It is desirable to have available a formulation that is an alternative to that
proposed in
that patent, that can be obtained by means of a simpler process (not requiring
any
specific shear technology) that is a low-energy process while at the same time
being
reproducible and completely reliable; in addition, the adjuvant formulation
must make
it possible to effectively adjuvant vaccines, by making it possible in
particular to
increase the immune response obtained or to decrease the dose of antigen
present,
while at the same time exhibiting no sign of toxicity that would be
detrimental to its
completely safe administration.
To achieve this aim, the present invention as broadly disclosed is an oil-in-
water
adjuvant emulsion characterized in that it comprises at least:
= squalene,
- an aqueous solvent,
- a hydrophilic nonionic surfactant which is a polyoxyethylene alkyl ether,
- a hydrophobic nonionic surfactant,
CA 02613732 2013-07-24
la
in that it is therrnoreversible and in that 90% of the population by volume of
the oil
drop has a size less than 200 nm.
The invention as claimed is however more specifically, an oil-in-water
adjuvant
emulsion characterized in that it comprises at least:
- squalene,
- an aqueous solvent,
- a nonionic surfactant which is a polyoxyethylene alkyl ether having a
hydrophilic/lipophilic balance (HLB) greater than or equal to 10, and
- a hydrophobic nonionic surfactant having a hydrophilic/lipophilic balance
(HLB)
less than 9, said hydrophobic nonionic surfactant being a sorbitan ester or a
mannide ester,
wherein said emulsion is thermoreversible and in that 90% of the population by
volume of the oil drops has a size less than 200 nm.
According to the invention, such an emulsion may be obtained by means of a
phase
inversion temperature process, which provides a very large advantage from an
industrial point of view. Such a process provides all the guarantees of safety
and of
profitability necessary for the pharmaceutical industry. In addition, by
virtue of this
CA 02613732 2007-12-28
W02007/006939
PCT/FR2006/001635
- 2 -
process, it is possible to obtain a monodisperse emulsion, the droplet size of
which is
very small, which renders the emulsion thus obtained particularly stable and
readily
filterable by means of sterilizing filters, the cutoff threshold of which is
200 nm.
According to a particular characteristic, 90% of the population by volume (or
d90) of
the oil drops has a size less than 160 nm, and even less than 150 nm.
According to a particular embodiment of the invention, the emulsion according
to the
invention also comprises an alditol; this makes it possible to obtain a phase
inversion
at a temperature below that which would be necessary for the same composition
not
containing any alditol, which makes it possible to reduce the production costs
and also
the risks of thermal denaturation of the constituents of the emulsion.
According to a particularly advantageous embodiment, the hydrophobic nonionic
surfactant of the invention is a sorbitan ester or a mannide ester. Such
surfactants have
the advantage of being able to be used entirely safely in injectable
solutions.
According to a particular embodiment of the invention, the emulsion also
comprises
an alkylpolyglycoside and a cryoprotective agent such as a sugar, in
particular
dodecylmaltoside and/or sucrose.
Thus, it is possible to obtain a lyophilizable emulsion which, after
lyophilization and
reconstitution, recovers its properties, in particular particle size
properties, i.e. the
lyophilized and then reconstituted emulsion is still monodisperse and consists
of oil
droplets, 90% of the population by volume of which has a size of less than 200
nm.
This is particularly important for the field of vaccines which must sometimes,
for
reasons of stability (either of certain antigens, or of certain adjuvants), be
conserved
in lyophilized form.
A subject of the invention is also a process for preparing an immunogenic
composition according to which at least one vaccine antigen is mixed with an
oil-in-
water emulsion, characterized in that the oil-in-water emulsion is obtained by
means
of a phase inversion temperature process.
According to one embodiment, the process according to the invention comprises
at
least one step of preparing the oil-in-water emulsion by cooling a water-in-
oil inverse
emulsion, which comprises at least:
- squalene,
- an aqueous solvent,
- a hydrophilic nonionic surfactant which is a polyoxyethylene alkyl ether,
,
CA 02613732 2007-12-28
. W02007/006939
PCT/FR2006/001635
-3 -
- a hydrophobic nonionic surfactant.
By virtue of such a process, which is very advantageous from an industrial
point of
view, a stable immunogenic composition is obtained which is very effective
even at a
very low dose of antigens.
In addition, by virtue of the preparation process used, the droplets of oil of
the
emulsion are all calibrated over the same very small size; in fact, when the
particle
size properties (size and size distribution) are measured, it is noted that
the emulsion
is a monodisperse emulsion, with a Gaussian-type distribution curve, which is
very
narrow and centered around a low value, generally around 80-90 nm.
According to a specific embodiment of the process according to the invention,
the
water-in-oil inverse emulsion is obtained by mixing squalene, an aqueous
solvent, a
hydrophilic nonionic surfactant which is polyoxyethylene alkyl ether, and a
hydrophobic nonionic surfactant so as to obtain, first of all, an oil-in-water
coarse
emulsion, and this emulsion is then heated to at least the phase-inversion
temperature
so as to obtain the inverse emulsion. Carrying out the process in such a way
has the
advantage of limiting the time during which the various constituents of the
emulsion
are subjected to a high temperature.
According to another embodiment, the process according to the invention
comprises
the following steps:
- firstly, the aqueous phase comprising the aqueous solvent and the polyoxy-
ethylene alkyl ether and, secondly, the oily phase comprising the squalene and
the hydrophobic surfactant are heated, separately, to a temperature at least
equal to the phase-inversion temperature, and then
- the 2 phases are mixed so as to obtain a water-in-oil inverse emulsion.
According to a specific embodiment of the invention, each of the aqueous and
oily
phases is heated separately, before mixing, to a temperature below the phase-
inversion temperature. The 2 phases are then mixed so as to obtain a water-in-
oil
emulsion; the whole is then heated to a temperature at least equal to the
phase-
inversion temperature so as to obtain the water-in-oil inverse emulsion.
CA 02613732 2007-12-28
WO 2007/006939
PCT/FR2006/001635
- 4 -
According to a particular embodiment, the preparation process according to the
invention also comprises a lyophilization step. Thus, the process according to
the
invention can be used for the preparation of immunogenic compositions
comprising
antigens which must be conserved in lyophilized form for reasons of stability.
Many other advantages of the present invention will become apparent in the
course of
the following description.
For the purposes of description of the invention, the parameters d50 and d90
mentioned in the present patent application are values relating to volume; the
d50
value signifies the value of 50% of the drop population by volume.
For the purpose of the invention, the term "oil-in-water emulsion" is intended
to mean
a dispersion of an oily phase in an aqueous phase which may consist either of
water or
of a saline solution, that is optionally buffered. According to a particular
embodiment
of the invention, the aqueous phase of the emulsion consists of a buffer, such
as
Dulbecco's phosphate buffered solutions (D-PBS, without calcium or magnesium).
The term "adjuvant emulsion" is intended to mean an immunoadjuvant emulsion,
i.e.
an emulsion capable of modifying the response of the immune system induced
during
the administration of an antigen, compared with the response obtained in the
absence
of the emulsion; this immune system response may be reflected by antibody
production or by activation of certain cells, in particular antigen-presenting
cells (for
example, dendritic cells), T lymphocytes and B lymphocytes. This cellular
activation
can be demonstrated by the presence of activation markers at the surface of
the cells
or by the release of cytokines. The modification of the immune response
induced by
the adjuvant emulsion may be quantitative in nature, i.e. an increase in the
induced
response is obtained, or qualitative in nature, i.e. a response that is
different in nature
or has a different orientation is obtained, or else an additional response is
obtained.
The term "adjuvant emulsion" is also intended to mean an emulsion that makes
it
possible to reduce the amount of antigens administered for the same induced
response.
For the purpose of the present invention, the term "immunogenic composition"
is
intended to mean a composition comprising at least one antigen and that can be
CA 02613732 2007-12-28
W020071006939
PCT/FR2006/001635
-5 -
administered to humans or to animals in order to induce an immune system
response.
This response may be a humoral response (antibody production) or a cellular
response
(proliferation and/or activation of immune cells). The immunogenic composition
may
be a composition for prophylactic purposes or for therapeutic purposes, or
else both.
The immunogenic composition obtained according to the invention can be
administered via any of the routes normally used or recommended for vaccines:
parenterally or mucosally, and can be in various forms, in particular a liquid
or
lyophilized form. It can be administered by means of a syringe or by means of
a
needle-free injector for intramuscular, subcutaneous or intradermal injection,
or by
means of a nasal spray.
For the purpose of the present invention, the term "antigen" is intended to
mean any
antigen that can be used in a vaccine, whether it involves a whole
microorganism or a
subunit antigen, regardless of its nature; the antigen may in fact be a
peptide, a
protein, a glycoprotein, a polysaccharide, a glycolipid, a lipopeptide, etc.
The adjuvant emulsion according to the invention is particularly suitable for
viral
antigens; particularly good results have in fact been obtained with antigens
of the
human cytomegalovirus, of the human immunodeficiency virus, and of the flu
virus.
As regards the flu virus antigens, it is possible to use antigens that come
from a single
viral strain, or from a mixture of various strains. It is possible to use
antigens derived
from viruses cultured conventionally on eggs, or on cells. By means of the
invention,
it has been noted that whether for a single strain or for a mixture of
strains, it is
possible to obtain a satisfactory response of the immune system while at the
same
time very substantially reducing the amount of antigens present in the vaccine
dose.
This may be of particularly great value in the case of the preparation of a
vaccine
against a flu pandemic, where it must be possible to produce, in a very short
period of
time, very large amounts of vaccine doses.
According to the invention, the oil-in-water emulsion comprises squalene,
which is an
oil initially originating from shark liver; it is an oil whose empirical
chemical formula
is C30H50, comprising 6 double bonds; this oil is metabolizable and has
qualities that
allow it to be used in an injectable pharmaceutical product. Squalene of plant
origin,
extracted from olive oil, also exists. Good results have in particular been
obtained
using the squalene provided by the company Fluka, which is of animal origin.
The
CA 02613732 2007-12-28
WO 2007/006939
PCT/FR2006/001635
- 6 -
amounts of squalene used for the preparation of a concentrated emulsion are
advantageously between 5 and 45%; this concentrated emulsion is subsequently
diluted during the preparation of immunogenic compositions so as to prepare
immunizing doses in which the amount of squalene is between 0.5 and 5%. This
dilution can be carried out by simply mixing the adjuvant emulsion according
to the
invention and the suspension comprising the antigen.
According to the invention, the emulsion comprises a nonionic hydrophilic
surfactant,
the value of the hydrophilic/lipophilic balance or HLB of which is greater
than or
equal to 10 and which belongs to the chemical group of polyoxyethylene alkyl
ethers
(PAEs), also known as polyoxyethylenated fatty alcohol ethers, or n-alkyl
polyoxyethylene glycol ethers. These nonionic surfactants are obtained by
chemical
condensation between a fatty alcohol and ethylene oxide. They have a general
chemical formula of the type CH3(CH2)x-(0-CH2-CH2)n-OH in which n denotes the
number of ethylene oxide units and is usually between 10 and 60, and x + 1 is
the
carbon number, depending on the fatty alcohols used. In general, these
products are
mixtures of polymers with hydrocarbon-based chains of similar length.
The emulsion according to the invention usually comprises a single hydrophilic
PAE.
A mixture of several PAEs is also suitable insofar as the overall HLB value is
10.
The polyoxyethylenated fatty alcohol ethers suitable for the subject of the
invention
can be in a liquid or solid form at ambient temperature. Among the solid
compounds,
preference is given to those which dissolve directly in the aqueous phase or
which do
not require substantial heating.
Insofar as the number of ethylene oxide units is sufficient, the
polyoxyethylenated
ethers of lauryl, myristyl, cetyl, oleyl and/or stearyl alcohols are
particularly suitable
for the subject of the invention. They can in particular be found in the range
of
products known under the trade names Brij for the products sold by the
company
ICI America's Inc., Eumulgine for the products sold by the company Cognis, or
Simulsol for the products sold by the company SEPPIC.
A particularly preferred emulsion according to the invention contains, as
nonionic
hydrophilic surfactant, a polyoxyethylene alkyl ether chosen from the group
CA 02613732 2007-12-28
WO 2007/006939
PCT/FR2006/001635
- 7 -
consisting of ceteareth-12 (sold under the name Eumulgin B1), ceteareth-20
(Eumulgin B2), steareth-21 (Eumulgin S21), ceteth-20 (Simulsol 58 or
Brij 58), ceteth-10 (Brij 56), steareth-10 (Brij 76), steareth-20 (Brij
78),
oleth-10 (Brij 96 or Brij 97) and oleth-20 (Brij 98 or Brij 99). The
number
assigned to each chemical name corresponds to the number of ethylene oxide
units in
the chemical formula.
Good results have been obtained with the product Brij 56. A compound that is
particularly suitable and preferred due to its semisynthetic origin is the
polyoxy-
ethylene (12) cetostearyl ether supplied by the company Cognis under the name
EumulginTmB1 . This compound is a mixture of CH3(CH2)15-(0-CH2-CH2)12-0H and
of CH3(CH2)17-(0-CH2-CH2)12-0H.
According to the invention, the adjuvant emulsion also comprises a hydrophobic
nonionic surfactant; it must be a surfactant that can be used in the
pharmaceutical
industry; among surfactants that are suitable in this regard, mention may be
made of
sorbitan esters and mannide esters; the sorbitan esters are obtained by
reaction of a
fatty acid and of a mixture of partial esters of sorbitol and its mono- and
dianhydrides;
this may involve a mono-, a di- or a triester, or even a mixture; they are
hydrophobic
surfactants for which the overall hydrophilic-lipophilic balance (HLB). is
less than 9,
and preferably less than 6. They can be found in particular in the range of
surfactants
sold by the company ICI Americas Inc. under the name Span , or by the company
Cognis under the name DehymulsTm, or by the company ICI under the name
ArlacelTM; as examples of surfactants that are particularly suitable, mention
may be
made of the sorbitan monooleate sold under the name Dehymuls SMOTm or Span 80.
Among the surfactants consisting of mannide esters, mention may be made of the
mannide monooleate sold by the company Sigma, or by the company Seppic under
the name Montanide 80TM
By virtue of the selection of these specific surfactants among all the
surfactants
proposed in the prior art for preparing emulsions, it has now been found that
it is
possible, very advantageously, to produce an oil-in-water adjuvant emulsion
using a
phase-inversion process.
CA 02613732 2007-12-28
WO 2007/006939
PCT/FR2006/001635
- 8 -
For this, the amounts of squalene and of each of the surfactants used are
advantageously chosen so as to obtain a mixture, the phase diagram of which
comprises a zero mean curvature phase (microemulsion or lamellar phase type)
for
which the interfacial tensions are extremely low.
In the case of the use of squalene, it has been noted that the emulsions
obtained are
stable and monodisperse, with the oil droplets being very small in size (d90
less than
200 nm), when the overall HLB value of the various surfactants used is between
8.5
and 10, and more particularly between 8.6 and 9.6. To determine the respective
concentrations of hydrophilic and hydrophobic surfactants in the composition
of the
emulsion, the following formula can be used:
HLBm = (HLBe x M) + HLBpae (1-M) in which,
HLBm corresponds to the HLB of the mixture, which is preferably between 8.5
and
10, and more particularly between 8.6 and 9.6,
HLBe corresponds to the HLB of the hydrophobic surfactant,
M corresponds to the percentage by mass of the hydrophobic surfactant in the
mixture
consisting of the hydrophobic surfactant and the polyoxyethylene alkyl ether
(PAE),
HLBpa, corresponds to the HLB of the PAE.
It has been noted that, by using a concentration of squalene of between 5 and
45%, an
emulsion whose phase-inversion temperature is less than 95 C is very
advantageously
obtained.
For such an emulsion, it is possible to use a polyoxyethylene alkyl ether at a
concentration of between 0.9 and 9% and a hydrophobic nonionic surfactant at a
concentration of between 0.7 and 7%; the remainder of the emulsion consisting
of an
aqueous solvent.
According to a particular embodiment of the invention, the immunogenic
composition
also comprises an alditol such as, in particular, glycerol, erythritol,
xylitol, sorbitol or
mannitol. Good results have in particular been obtained with the mannitol sold
by the
CA 02613732 2007-12-28
* W02007/006939
PCT/FR2006/001635
,
- 9 -
company Roquette Freres. The amounts of alditol used in the preparation
process can
range between 1 and 10%, and more particularly between 2 and 7.
According to a particular embodiment of the invention, the adjuvant emulsion
also
comprises a cryoprotective agent which makes it possible to lyophilize the
emulsion
obtained; among the cryoprotective agents, sugars are particularly preferred,
and
especially sucrose. In addition, the emulsion according to the invention can
comprise
an alkylpolyglycoside, which is a surfactant with a sugar head; it may in
particular be
sodium decyl-D-galactoside uronate, or, according to a preferred embodiment,
dodecy1-13-ma1toside available from the company Roche.
By virtue of the process for preparing the emulsion according to the invention
by
phase inversion obtained by varying the temperature, an oil-in-water emulsion,
the
size of the oil droplets of which is very homogeneous, is very readily and
very
reproducibly obtained: the d90 value (by volume) is less than 200 nm,
preferably less
than 150 nm, and even close to 100 nm while the d50 value is less than 100 nm,
or
even 90 nm. Most of the emulsions prepared according to the process of the
invention
have made it possible to attain d50 values of around 80 nm, with d90 values of
around 100 nm (measurements carried out with a Coulter LS230). It is thus
possible
to perform a sterilizing filtration of the emulsion obtained, on the condition
that the
latter is sufficiently diluted.
Such emulsions in which the size of the drops is homogeneous and very small
are
stable over time. It has thus been possible to note that an emulsion prepared
according
to the invention and stored at 4 C, conserved, after 2 years, a monodisperse
profile
with a d50 value of 90 nm and a d90 value of 116 nm, which proves that the
emulsion
is very highly stable.
The size of the drops can be measured by various means, and in particular by
LASER
diffraction particle sizes, such as the Beckman Coulter devices of the LS
range (in
particular the LS230) or Malvern devices of the Mastersizer range (the
Mastersizer
2000 in particular). The principle of measurement of these devices is based on
analyzing the intensity of the light scattered by the particles as a function
of the angle
(large, medium and small angle detectors) when the sample is illuminated by a
CA 02613732 2007-12-28
WO 2007/006939
PCT/FR2006/001635
- 10 -
LASER beam. This analysis is carried out by means of mathematical models
chosen
according to the size and the nature of the material used. In the case of the
measurement of the size of submicronic particles, it is necessary to apply a
specific
optical model (Mie theory) taking into account the refractive indices of the
sample
(here, 1.495 for squalene) and of its medium (here, 1.332 for water); it is
also
necessary to be capable of detecting the weak intensities emitted by the very
fine
particles, which requires an optimization of the analysis:
- an additional detection cell for the large-angle polarized intensity
differential
scattering measurement (PIDS system from Coulter, which allows measurement
from
40 nm),
- a detection system combining 2 wavelengths, blue and red light, from
Malvern. The
source of blue light of shorter wavelength, associated with wide-angle
scattering and
backscattering detectors, reinforces the performance levels of the analysis in
the
submicronic range.
According to the devices used, the measurements may vary slightly as a
function of
the components of the device and of the data processing software used. Thus,
the
same emulsion according to the invention was analyzed with the 2 devices and
gave
the following results:
- using the LS230, with the following parameters: IR particle = 1.495; IR
medium = 1.332; absorption value = 0; d50 = 80-90 nm and d90 = 120-
130 nm;
- using the Mastersizer 2000, with the following parameters: IR particle =
1.495; IR medium = 1.332; absorption value = 0; obscuring = 4-7%; "general
purpose" optical model; d50 = 90-100 nm and d90 = 140-150 nm.
The process according to the invention may be carried out in the following
way: a
concentrated crude oil-in-water emulsion is prepared by incorporation of the
aqueous
phase (buffer solution, to which alditol is optionally added, comprising the
polyoxyethylene alkyl ether) into the oily phase (squalene and hydrophobic
nonionic
surfactant); or, conversely, by incorporation of the oily phase into the
aqueous phase.
A noncalibrated oil-in-water emulsion is then obtained, which rapidly
manifests its
instability. This emulsion is stirred and heated until a phase inversion is
obtained, i.e.
a water-in-oil emulsion is obtained. The phase inversion or transition can be
CA 02613732 2007-12-28
WO 2007/006939
PCT/FR2006/001635
- 11 -
monitored by conductimetry. In fact, during the rise in temperature, the
conductivity
increases until phase inversion occurs; at this time, a relatively abrupt drop
in
conductivity is observed. The temperature at which the change in curvature of
the
curve for following the conductivity occurs reflects the passage from one type
of
emulsion to another; it is the phase inversion temperature. In reality, this
temperature
is rather a temperature range than a very precise point value; in fact, it may
be
considered that this temperature is a temperature determined to within 1 or 2
degrees
so that the entire emulsion undergoes the phase inversion phenomenon. Once
this
phase inversion temperature has been reached, and therefore once in the
presence of a
water-in-oil emulsion, the heating is stopped and the mixture is cooled. The
cooling
can be carried out passively, by simply allowing the emulsion to return
spontaneously
to ambient temperature, or more actively, by immersing the emulsion, for
example, in
an ice bath. When the temperature passes through the phase inversion
temperature, the
water-in-oil emulsion will invert again so as to once more give an oil-in-
water
emulsion, in which the size of the oil droplets is this time very homogeneous
and
small; the emulsion obtained is then very stable. It can be stored as it is,
while
awaiting dilution with a solution comprising the vaccine antigen.
This emulsion is thermoreversible, which means that, if it is again brought to
a
temperature above the phase inversion temperature, it will once again become a
water-in-oil emulsion. It is noted that the curves for following the
conductivity are
superimposable for the same emulsion, irrespective of the number of
thermoinversions undergone, and that the emulsions obtained always have the
same
particle size profile.
Advantageously according to the invention, the formulation of the emulsion is
chosen
so as to have a phase inversion temperature that is less than 95 C, and more
particularly between 45 and 80 C, and more particularly still between 50 and
65 C.
This temperature range is advantageous since there is no risk of the emulsion
changing state if it is stored at a relatively high temperature (= 37 C).
Furthermore, as
in the process for preparing the thermoreversible emulsion, the heating of the
components is not too severe, this contributes to maintaining the structural
integrity of
the components. When the phase-inversion temperature of the emulsion is high,
in
particular when it is greater than or in the region of 80 C, it may be useful
to reduce it
by adding to the composition of the emulsion an alditol, which is normally
chosen
CA 02613732 2007-12-28
W02007/006939
PCT/FR2006/001635
- 12 -
from sorbitol, mannitol, glycerol, xylitol or erythritol. When the alditol is
used in a
concentration range of from 1 to 10% (w/w), and in particular in a
concentration
range of from 2 to 7% (w/w), the phase-inversion temperature of the emulsion
is
reduced by approximately 10 C. It is also possible to reduce the phase-
inversion
temperature of the emulsion by replacing the aqueous phase consisting only of
water
with a buffered saline aqueous phase. A Tris buffer, or a phosphate buffer
such as
PBS, or the Dulbecco PBS buffer without Ca2+ and without Mg2+, is normally
used.
Alternatives to the process that has just been described exist. Specifically,
it is
possible, as has just been described, to mix the 2 aqueous and oily phases in
order to
obtain the crude emulsion which will then be heated and then cooled.
Alternatively, the 2 phases that have been prepared can be heated separately
to a
temperature slightly above the phase inversion temperature, before being mixed
so as
to give a water-in-oil inverse emulsion, which will be cooled until the oil-in-
water
submicronic emulsion is obtained.
It is also possible to slightly heat each of the phases before carrying out
the mixing,
which will result in an oil-in-water emulsion, and then to heat this emulsion
to phase-
inversion before carrying out the cooling.
All these operations can be carried out in separate containers for a batch
preparation
but it is also possible to use an on-line process.
The process for preparing the emulsion on-line may notably consist of a
mixing,
under hot conditions, of the two aqueous and oily phases prepared separately
beforehand, through a thermostatted static mixer, followed by on-line cooling
through
a refrigerated heat exchanger connected at the outlet of the static mixer, and
then final
recovery of the emulsion according to the invention in an appropriate
container (flask
or reactor). A static mixer consisting of a succession of mixing elements made
up of
cross blades inclined relative to the axis of the tube into which they are
introduced
was successfully used. The energy required for the mixing is provided by the
pumps
that transport the fluids and the mixing is carried out without any mobile
part, through
the mixing elements by virtue of the separating, displacing and successive
combining
of the constituents of the mixture.
CA 02613732 2007-12-28
W02007/006939
PCT/FR2006/001635
- 13 -
The on-line production process is carried out in the following way: the
aqueous phase
(buffered solution comprising the polyoxyethylene alkyl ether) and the oily
phase
(squalene and hydrophobic nonionic surfactant) are prepared separately in two
flasks
or reactors. The two phases are heated with stirring to a temperature slightly
above the
phase inversion temperature. The two phases are then introduced into a
thermostatted
static mixer by means of 2 pumps, the flow rates of which are regulated so as
to
obtain the composition of the emulsion according to the invention. The water-
in-oil
inverse emulsion is obtained during the passage of the two phases in the
static mixer.
The inverse emulsion is subsequently cooled by passing it on-line through a
refrigerated heat exchanger connected at the outlet of the static mixer. The
water-in-
oil emulsion will then invert through the refrigerated heat exchanger to give
an oil-in-
water emulsion, which will be collected in a flask or reactor, and the
characteristics of
which are identical to those of the emulsion obtained by a batch process.
The adjuvant emulsion according to the invention is then used for the
preparation of
an immunogenic composition. A simple embodiment consists in mixing a solution
comprising at least one vaccine antigen with an emulsion obtained according to
one of
the embodiments which have just been described. The immunogenic composition
obtained is in the form of an oil-in-water emulsion or in the form of a
thermoreversible oil-in-water emulsion when the amount of squalene represents
at
least 5% by mass of the total mass of the immunogenic composition.
Alternatively, it
is possible to mix the antigen with the aqueous phase or with the oily phase
before
preparing the emulsion. Carrying out the process in such a manner implies, of
course,
that said antigens are antigens which are compatible with the thermoinversion
process.
The solutions of the antigen may also contain mineral salts or one or more
buffers,
and also any other compound normally used in vaccines, such as stabilizers,
preserving agents or, optionally, also other adjuvants.
For the preparation of a lyophilizable emulsion, a concentrated liquid
emulsion is first
of all prepared, as has just been described, but preferably choosing water
rather than a
buffered solution as aqueous phase, and then this emulsion is diluted with a
solution
CA 02613732 2007-12-28
WO 2007/006939
PCT/FR2006/001635
- 14 -
comprising an alditol, a sugar and an alkylpolyglycoside, for example with a
solution
comprising mannitol, sucrose and dodecylmaltoside.
The emulsion obtained is then divided up into samples (for example, 0.5 ml)
and
subjected to a lyophilization cycle, which can be carried out in the following
way:
- loading of the samples at +4 C,
- approximately 2 hours of freezing at a set temperature of -45 C,
- 14 to 19 hours of primary desiccation at a set temperature of 0 C,
- 3 hour 30 min of secondary desiccation at a set temperature of + 25 C.
The emulsion obtained can then be conserved until it is used for the
preparation of an
immunogenic composition, i.e. until it is combined with a composition
comprising
vaccine antigens. This step for preparing the immunogenic composition can be
carried
out by taking up the lyophilized emulsion with an aqueous solution comprising
the
antigens. The immunogenic composition thus obtained can subsequently be
conversed
in the liquid state, or can be subjected to a further lyophilization cycle in
order to be
conserved in the form of a lyophilisate, if the nature of the antigens allows
this.
Alternatively, it is possible to directly dilute the concentrated emulsion
with an
aqueous solution comprising both the vaccine antigens and also the alditol,
the sugar
and the alkylpolyglycoside, and to subsequently subject the composition
obtained to
the lyophilization. Such a manner of carrying out the process implies, of
course, that
the antigens are antigens that are compatible with a lyophilization process.
The following examples illustrate various embodiments of the invention.
Example 1: Preparation of an adjuvant emulsion according to the invention
3.71 g of EumulginTm B1 and 33.9 g of a 10% solution of mannitol in PBS buffer
were
mixed in a beaker, and the mixture was homogenized with stirring at
approximately
C.
30 In another container, 2.89 g of DehymulsTm SMO and 19.5 g of squalene
were stirred
magnetically.
CA 02613732 2007-12-28
WO 2007/006939
PCT/FR2006/001 635
- 15 -
When homogeneous phases were obtained in each of the containers, the aqueous
phase was incorporated into the oily phase, which was maintained at 30 C with
stirring.
When the incorporation was complete, the crude emulsion obtained was heated
until
the temperature reached 58-60 C, while at the same time maintaining the
stirring.
The heating was then stopped but the stirring was maintained until the
temperature
reached ambient temperature.
An oil-in-water emulsion was then obtained, the size of the oil droplets of
which was
centered around 80 nm (measurement carried out using an LS230), and the
composition by mass of which was as follows:
- 32.5% of squalene,
- 6.18% of polyoxyethylene (12) cetostearyl ether,
- 4.82% of sorbitan monooleate,
- 6% of mannitol.
Example 2: Vaccine composition against AIDS.
Vaccine compositions comprising a detoxified TAT III B protein as antigen were
prepared. The TAT protein was detoxified by means of an alkylation reaction in
an
alkaline medium using iodoacetamide under the following conditions: number of
micromoles of iodoacetamide = 200 X number of micromoles of TAT + number of
micromoles of DTT. This detoxified protein and the process for preparing it
are
described in detail in application W099/33346, where it is identified under
the term
carboxymethylated TAT. This recombinant TAT antigen is conserved in solution,
in
the presence of 50 mM Tris buffer, pH 7.5, at ¨70 C.
The vaccine compositions to be administered were prepared from concentrated
solutions, in order to obtain immunization doses of 200 pi having the
following
quantitative compositions:
- for the composition having only the antigen: 20 pig of TAT in 50 mM Tris
buffer, 100 mM NaC1, at pH 7.5;
- for the composition according to the invention:
= 20 g of TAT,
,
CA 02613732 2007-12-28
µ
WO 2007/006939
PCT/FR2006/001635
- 16 -
= 5 mg of squalene
= 0.75 mg of Dehymuls SMO,
= 0.94 mg of Eumulgin Bl,
= 0.91 mg of mannitol.
Two groups of six 8-week-old female BALB/c mice were provided, and were
injected
subcutaneously with one of the compositions prepared, in a proportion of one
dose of
200 ul per mouse; the injections were given on DO and on D21.
Blood samples were taken from the retroorbital sinus on D14 in order to assess
the
primary response and on D34 for the secondary response. The specific IgG1 and
IgG2a titers were determined by means of standardized ELISA assays.
The mice were sacrificed on D37; their spleen was removed and the splenocytes
were
isolated.
The results obtained regarding the humoral responses are summarized in the
table
below, in which the IgG titers are expressed in arbitrary ELISA units (log10).
For each group of mice, the value indicated in the table is the mean geometric
titer of
the values obtained for each of the mice.
CA 02613732 2007-12-28
WO 2007/006939
PCT/FR2006/001635
- 17 -
Vaccine IgG1 IgG2a IgG1 IgG2a IgGl/IgG2a ratio
composition at D14 at D14 at D34 at D34 at D34
Tat 2.6 1.2 4.4 3.0 25
Tat+ PIT 3.5 2.5 5.7 5.0 5
The results obtained show that the emulsion according to the invention makes
it
possible to increase, overall, the humoral response and also tends to promote
the type
1 T-helper response since the IgG2a response is increased more than the IgG1
response.
As regards the cellular response, it was possible to demonstrate, by ELISPOT
assay
after restimulation of the splenocytes removed with the recombinant TAT
protein, a
clear increase in the number of cells producing y interferon when the
splenocytes
came from mice immunized with a preparation according to the invention (486
spots
per 106 cells versus 39 per 106 for the preparation containing the antigen
alone).
Similarly, the assaying of the cytokines in the culture supernatants showed
the greater
secretion of both y interferon (5028 pg/ml versus 1940 pg/ml) and Interleukin
5
(5365 pg/ml versus 2394 pg/ml).
Example 3: Preparation of a vaccine composition against human cytomegalovirus
infections.
Vaccine compositions comprising, as vaccine antigen, a recombinant protein
derived
from an envelope glycoprotein of the Cytomegalovirus (CMV) Towne strain,
called
gB, the nucleotide and protein sequences of which are described in patent USP
5,834,307, were prepared. This recombinant protein is produced by a
recombinant
CHO line transfected with a plasmid called pPRgB27c1v4 which contains a
modified
gB gene. Specifically, in order to facilitate the production of this
recombinant protein
by the CHO line, the gB gene was modified beforehand by deleting the part of
the
gene that encodes the transmembrane region of the gB protein corresponding to
the
amino acid sequence between Valine 677 and Arginine 752 and by introducing 3
point mutations such that the cleavage site that exists in the native gB was
eliminated.
In fact, the recombinant protein produced by the recombinant CHO line
corresponds
CA 02613732 2007-12-28
,.
WO 2007/006939
PCT/FR2006/001635
- 18 -
to a truncated gB protein devoid of cleavage site and of transmembrane region,
called
gBdTM.
The construction of the plasmid pPRgB27c1v4 and the production of the
truncated gB
protein (gBdTM) by the recombinant CHO line are described in US 6,100,064. The
purification of the truncated gB protein is carried out on an immunoaffinity
chromatographic column using the monoclonal antibody 15D8 described by
Rasmussen L. et al. ( J. Virol. (1985) 55: 274-280).
From a stock composition at 0.975 mg/ml of gB antigen thus obtained and
maintained
in phosphate buffer, the emulsion according to the invention concentrated as
described in example 1, and an emulsion of the prior art obtained by
microfluidization, 50 1 doses of immunizing compositions were prepared,
having the
following compositions:
- 2 lig of gB in citrate buffer at pH 6 (group called gB
alone),
- 2 g of gB; 1.075 mg of squalene; 0.133 mg of MontaneTm VG 85 and
0.125 mg of TweenTm80 in citrate buffer at pH 6 (group called with emulsion
of the prior art ),
- 2 ,g of gB; 1.25 mg of squalene; 0.185 mg of Dehymuls SMO;
0.235 mg of
EumulginTM B1 and 0.230 mg of mannitol in PBS buffer at pH 7.4 (group
called with emulsion of the invention ).
Three groups of ten 8-week-old female Outbred OF1 mice were provided and were
immunized twice, on DO and on D21, subcutaneously, with one of the
compositions
indicated above (each group of mice is given the same composition both times).
Blood samples were taken from the retroorbital sinus on D20 and D34 and were
used
to determine the concentrations of IgGl- and IgG2a-type antibodies specific
for the
gB antigen.
The assays were carried out by means of ELISA assays; the results obtained are
given
in the table below, and are expressed as logio of the ELISA titers. The values
indicated are the mean values obtained for each group of mice.
,
CA 02613732 2007-12-28
' WO 2007/006939
PCT/FR2006/001635
- 19 -
D20 D34
IgGl/IgG2a
Nature of the groups IgG1 IgG2a IgG1 IgG2a ratio at
D34
gB alone 2.474 2.094 3.801 2.941
137
gB + emulsion of the prior art 4.063 2.980 5.493 4.185
143
gB + emulsion according to the 4.615 3.914 5.615 4.854
14
invention
These results show the effectiveness of the emulsion according to the
invention,
which allowed a greater induction of antibodies, both those of IgG1 type and
of IgG2a
type, with, in addition, compared with the emulsion according to the prior
art, the
obtaining of the desired result in the context of a vaccine against human
cytomegalovirus, which is that of orienting the immune response toward a TH1
response (an indicator of which is the IgG2a titer), while at the same time
maintaining
the TH2-type response (an indicator of which is the IgG1 titer) at a
sufficient level.
Example 4: Vaccine composition against the flu.
Flu virus antigens were available, obtained according to the process described
in the
examples of application WO 96/05294, with the exception of the fact that the
viral
strain used was the A/New Caledonia H1N1 strain.
Using this preparation of antigens, the concentrated emulsion according to the
invention obtained in example 1, and a suspension of aluminum provided by
REHEIS
under the name AlOOH Rehydra, 50 pl immunization doses were prepared, which
doses had the composition indicated hereinafter, in which the amounts of flu
antigens
are expressed by weight of hernagglutinin HA:
- either 1 ps of HA in PBS buffer,
- or 5 pg of HA in PBS buffer,
- or 1 pg of HA and 60 lig of aluminum hydroxide,
- or 1 lig of HA; 1.25 mg of squalene; 0.185 mg of DehymulsTm SMO; 0.235 mg
of EumulginTm B; 0.21 mg of mannitol; the entire mixture in PBS buffer.
CA 02613732 2007-12-28
WO 2007/006939
PCT/FR2006/001635
- 20 -
Eight groups of five 8-week-old female BALB/c mice were provided and were
administered, on DO, with the compositions prepared according to the following
distribution:
- one group received the composition having 1 pg of HA,
subcutaneously,
- one group received the same composition having 1 lig of HA,
intradermally,
- one group received the composition having 5 g of HA, subcutaneously,
- one group received the same composition having 5 pg of HA, intradermally,
- one group received the composition having 1 g of HA and 60 lig of
aluminum, subcutaneously,
- one group received the same composition having 1 pg of HA and 60 pg of
aluminum, intradermally,
- one group received the composition having 1 pg of HA and the emulsion
according to the invention, subcutaneously,
- one group received the composition having 1 g of HA and the emulsion
according to the invention, intradermally.
Blood samples were taken from each of the mice on D14, D28, D41, D56 and D105.
The sera from the immunized mice were assayed, firstly, by the ELISA technique
in
order to evaluate their content of total antibodies induced against the flu
strain
A/H1N1 of the trivalent vaccine (the antibody titers are expressed as the
log10 value
of arbitrary ELISA units, with a detection threshold of 1.3 log10) and,
secondly, by
the HAI (hemaglutination inhibition) technique in order to determine their
content of
functional antibodies against the flu strain A/H1N1. The antibody titers are
expressed
as the inverse of the dilution in arithmetic value, with a detection threshold
at 5.
The results obtained are reiterated in the tables hereinafter, in which the
values
indicated represent the mean of the titers of the mice of each group.
,
CA 02613732 2007-12-28
W02007/006939 PCT/FR2006/001635
-21 -
ELISA titers:
Group of Immunization D14 D28 D41 D56
D105
mice route
HA 1 ps subcutaneous 2.838 3.288 3.556 3.581
3.535
HA 5 Kg subcutaneous 3.203 3.642 3.836 3.732
3.844
HA 1 ilg + subcutaneous 2.860 3.363 3.843 3.951
3.982
AlOOH
HA 1 g + subcutaneous 4.043 4.511 4.753 4.723
4.681
emulsion
HA 1 jig intradermal 2.174 2.814 3.143 3.075
2.735
HA 5 jig intradermal 2.839 3.297 3.496 3.549
3.479
HA 1 jig + intradermal 2.654 3.004 3.137 3.020
2.724
AlOOH
HA 1 Kg + intradermal 4.134 4.692 4.895 4.911
4.823
emulsion
HAI titers:
Group of Immunization D14 D28 D41 D56 D105
mice route
HA 1 g subcutaneous 5 6 5 40 23
HA 5 jig subcutaneous 5 9 20
HA 1 tig + subcutaneous 5 15 23 121 160
AlOOH
HA 1 ttg + subcutaneous 6 160 368 422 485
emulsion
HA 1 ps intradermal 5 5 5 23 8
HA 5 pig intradermal 5 8 10
HA 1 jig + intradermal 5 5 5 26 7
AlOOH
HA 1 lig + intradermal 7 557 1640 1557 1844
emulsion
,
CA 02613732 2007-12-28
W02007/006939
PCT/FR2006/001635
- 22 -
These results show the advantage of the present invention; specifically,
aluminum
hydroxide, which is a well known adjuvant widely used in the prior art, did
not make
it possible to increase the immunogenicity of the flu antigens with the same
rapidity
and the same strength as the formulation according to the invention; it is
also noted
that the composition according to the invention was particularly effective,
whether
given subcutaneously or intradermally.
Example 5: Vaccine composition against the flu.
The intention was to evaluate, in mice, the advantage of the present invention
for
decreasing the amount of vaccine antigen when the vaccine involved is a
vaccine
against the flu which contains, as antigens, 3 flu virus strains and which
would be
administered intradermally.
To this end, the concentrated emulsion of example 1 was diluted with PBS
buffer in
order to obtain a 5% squalene emulsion, which was further diluted by half with
a
composition comprising the antigens.
A composition comprising flu virus originating from 3 different viral strains
obtained
in the manner described in patent application WO 96/05294 was in fact
available, the
3 strains being in this case the A/New Caledonia (H1N1) strain, the A/Wyoming
(H3N2) strain and the B/Jiangsu strain. Such a trivalent vaccine composition
is
conventional for a flu vaccine and corresponds to the vaccine sold in the
northern
hemisphere during the 2004 flu campaign. The amounts of antigens of each of
the
viral strains are assessed through their amount of hemagglutinins HA.
The immunization doses prepared, having a volume of 50 pl, had the
compositions
indicated below:
- 0.33 pg of HA of each of the viral strains in PBS buffer at pH 7.4;
- 1.31 pg of HA of each of the viral strains in PBS buffer at pH 7.4;
- 5.25 pg of HA of each of the viral strains in PBS buffer at pH 7.4;
- 10.5 pg of HA of each of the viral strains in PBS buffer at pH 7.4;
- 21 lig of HA of each of the viral strains in PBS buffer at pH 7.4;
- 0.33 pg of HA of each of the viral strains; 1.25 mg of squalene; 0.185 mg
of
DehymulsTM SMO; 0.235 mg of EumulginTm B1 and 0.230 mg of mannitol in
PBS buffer at pH 7.4;
CA 02613732 2007-12-28
WO 2007/006939
PCT/FR2006/001635
- 23 -
- 1.31 pg of HA of each of the viral strains; 1.25 mg of squalene;
0.185 mg of
DehymulsTM SMO; 0.235 mg of EumulginTm B1 and 0.230 mg of mannitol in
PBS buffer at pH 7.4;
- 5.25 jig of HA of each of the viral strains; 1.25 mg of squalene;
0.185 mg of
DehymulsTM SMO; 0.235 mg of EumulginTm B1 and 0.230 mg of mannitol in
PBS buffer at pH 7.4.
Eight groups of ten 6- to 8-week-old female BALB/c mice were provided and were
administered intradermally (internal face of the ear) with one of the
compositions
prepared, at a rate of one composition per group.
Three weeks after immunization, blood samples were taken and, for each of the
groups, the IgGs induced against each of the viral strains were assayed by
ELISA; a
hemagglutinin inhibition assay against each viral strain was also carried out
for each
of the groups.
The results obtained are represented in the table below in the form of means
for each
of the groups. The ELISA results are expressed as logio of arbitrary units,
and the
HAI results are the mean arithmetic titers of the inverses of the dilutions.
Nature H1N1 H3N2
Composition ELISA HAI ELISA HAI ELISA HAI
0.33 ptg HA ' 3.51 35 4.38 243 4.28 25
1.31 lig HA 3.96 65 4.74 640 4.35 32
5.25 pg HA 4.16 92 5.05 970 4.62 53
10.5 pg HA 4.38 197 5.18 1810 4.71 130
21 gg HA 4.63 260 5.37 2389 4.98 184
0.33 pg HA 4.27 226 5.20 2389 4.91 149
+ emulsion
1.31 pg HA 4.46 279 5.37 3880 4.95 171
+ emulsion
5.25 lig HA 4.68 394 5.58 5487 5.12 22
+ emulsion
CA 02613732 2007-12-28
. W02007/006939
PCT/FR2006/001635
- 24 -
These results demonstrate the particular advantage of the invention for
reducing the
amount of antigens; specifically, by virtue of the emulsion according to the
invention,
it was possible, for the same immune response induced, to very substantially
decrease
the amount of antigens present in the immunization dose.
Example 6: Trivalent vaccine composition against the flu in a non-naive
population
The intention was to test the effectiveness of the emulsion of the invention
in the case
of a flu vaccine that would be administered to individuals whose body has
already
been in contact with flu virus antigens, as is frequently the case, either
because the
individuals have already been in contact with the flu virus, or because they
have
already been previously immunized with a flu vaccine.
According to the information published by C.W. Potter in Vaccine, 2003, 21:940-
5, it
is possible to use, as animal model for carrying out this test, BALB/c mice
pre-
immunized intramuscularly with a trivalent vaccine.
50 ill immunization doses comprising either PBS buffer only, or trivalent
vaccine
from the 2004 campaign, i.e. a vaccine comprising the A/New Caledonia (H1N1)
strain, the A/Wyoming (H3N2) strain and the B/Jiangsu strain, in a proportion
of 5 g
of HA of each of the strains, in PBS buffer at pH 7.4, were therefore
prepared.
Six groups of seven BALB/c mice were provided; 3 groups were immunized with
the
doses comprising only buffer, and 3 others with the trivalent vaccine,
intramuscularly.
I immunization doses were also prepared from the concentrated emulsion of
example 1 and a vaccine composition comprising the 3 viral strains of the 2004
campaign mentioned above, these immunization doses having the following
25 compositions:
- PBS buffer alone,
- 0.3 jig of HA of each of the viral strains in PBS buffer,
- 0.3 jig of HA of each of the viral strains; 0.75 mg of squalene; 0.11 mg
of
DehymulsTM SMO; 0.143 mg of EumulginTm B1 and 0.138 mg of mannitol in
30 PBS buffer at pH 7.4.
,
CA 02613732 2007-12-28
=
WO 2007/006939 PCT/FR2006/001635
-25 -
Each of the compositions thus prepared was used to immunize, on D34
intradermally
(in the internal face of the ear), both a group of mice having previously
received only
PBS buffer, and a group of mice having received a dose of trivalent vaccine.
On D56, a blood sample was taken from each of the mice and the antibodies
produced
against the H1N1 strain (A/New Caledonia) were titered by means of a
hemagglutinin
inhibition assay.
The results obtained have been summarized in the table below and represent the
mean
values obtained for each group of mice having followed the same immunization
protocol.
Mouse group Nature of the dose Nature of the dose for HAI titer with respect
identification for the im priming the id boost to the H1N1
strain
A PBS PBS 5
Trivalent vaccine
B PBS containing 0.3 ug 59
HA/strain
Trivalent vaccine
C PBS containing 0.3 ug 320
HA/strain + emulsion
D Trivalent vaccine
containing 5 tig PBS 145
HA/strain
E Trivalent vaccine Trivalent vaccine
containing 5 lig containing 0.3 ug 476
HA/strain HA/strain
F Trivalent vaccine Trivalent vaccine
containing 5 ug containing 0.3 lig 861
HA/strain HA/strain + emulsion
These results show how advantageous the invention is, even in individuals who
are
non-naïve with respect to the antigen administered. In fact, contrary to the
CA 02613732 2007-12-28
WO 2007/006939
PCT/FR2006/001635
- 26 -
observations by C.W. Potter when using this model, who, himself, has only been
able
to show a weak adjuvant effect of Iscoms when they were used to immunize mice
pre-
infected or pre-immunized with flu antigens, here it is seen that the emulsion
according to the invention made it possible to significantly increase the
response
induced, whether this was with naïve mice or with mice having already been
immunized with flu vaccine.
Example 7: Trivalent vaccine composition against the flu comprising low doses
of
antigens
30 ill immunization doses were prepared from the concentrated emulsion of
example
1 and a vaccine composition comprising the 3 viral strains of the 2004
campaign (the
A/New Caledonia (H1N1) strain, the A/Wyoming (H3N2) strain and the B/Jiangsu
strain), these immunization doses having the following compositions:
- 0.1 tg of HA of each of the viral strains in PBS buffer,
- 0.4 pg of HA of each of the viral strains in PBS buffer,
- 1.6 it g of HA of each of the viral strains in PBS buffer,
- 6.3 vtg of HA of each of the viral strains in PBS buffer,
- 0.1 lig of HA; 0.75 mg of squalene; 0.11 mg of DehymulsTm SMO;
0.143 mg
of EumulginTm B1 and 0.138 mg of mannitol in PBS buffer at pH 7.4,
- 0.4 lig of HA; 0.75 mg of squalene; 0.11 mg of DehymulsTm SMO;
0.143 mg
of EumulginTm B1 and 0.138 mg of mannitol in PBS buffer at pH 7.4.
Six groups of eight 8-week-old female BALB/c mice were provided and were
administered, on DO, intradermally (internal face of the ear) with a dose of
30 ill of
one of the compositions indicated below (1 composition per group).
In each group, a 2nd dose having The same nature as the 1st dose administered
was
again administered, intradermally, to half the mice on D29.
Blood samples were taken on D22 and on D43 in order to determine the amounts
of
antibodies induced.
The antibody titers were assayed by ELISA for the antibodies induced at D22
and at
D43, with respect to all the strains administered: H1N1, H3N2 and B, and by
HAI
with respect to the H1N1 strain only, both at D22 and at D43. The results
obtained
have been summarized in the table below, in which the titers expressed are the
means
CA 02613732 2007-12-28
W02007/006939 PCT/FR2006/001635
- 27 -
obtained for each group of mice. As regards the results at D43, the means were
determined separately within the same group, for the mice having received 2
doses of
vaccine and those having received only one.
Vaccine H1N1 H3N2 B ELISA H1N1 HAI H1N1 HAI titer at
composition ELISA titer ELISA titer at D22 titer at
D22 D43
at D22 titer at D22 Boosted
Non-
mice
boosted
mice
0.1 lig HA 3.467 4.131 4.063 57 95 80
0.4 tig HA 3.816 4.527 4.313 73 226 80
1.6 lig HA 4.069 4.831 4.750 147 1280 135
6.3 ttg HA 4.534 5.298 4.947 320 1522 320
0.1 jig HA
+ emulsion 4.200 5.015 4.807 207 2153 538
0.4 lig HA
+ emulsion 4.612 5.482 5.001 453 2560 640
Vaccine H1N1 ELISA titer at H3N2
ELISA titer at B ELISA titer at D43
composition D43 D43
Boosted Non- Boosted
Non- Boosted Non-
mice boosted mice boosted mice boosted
mice mice mice
0.1 j_tg HA 3.833 3.425 4.782 4.174 4.479 3.911
0.4 pg HA 4.385 3.599 5.250 4.354 5.053 4.079
1.6 pg HA 4.906 3.876 5.526 4.779 5.458 4.487
6.3 pg HA 5.287 4.176 6.073 5.033 5.678 4.773
0.1 pg HA 5.210 4.523 5.990 5.264 5.723 4.943
+ emulsion
0.4 pig HA 5.394 4.790 6.171 5.640 5.962 5.144
+ emulsion
These results show that, by virtue of the emulsion according to the invention,
even
with low doses of antigens, very substantial humoral responses were obtained.
Thus,
CA 02613732 2007-12-28
WO 2007/006939
PCT/FR2006/001635
- 28 -
it can be noted that the best results are those obtained with a dose of 0.4 g
of HA of
each of the viral strains and an emulsion according to the invention; these
results are,
very surprisingly, much better than those obtained by using a dose of 6.3 g
of HA
alone. In addition, it is noted that, even in the individuals not given a
booster dose, the
immune system continues to induce antibodies, whereas this is not the case for
the
individuals given the non-adjuvanted vaccine antigens.
Example 8: Trivalent vaccine composition against the flu
30 I immunization doses were prepared from the concentrated emulsion of
example
1 and a vaccine composition comprising the 3 viral strains of the 2004
campaign (the
A/New Caledonia (H1N1) strain, the A/Wyoming (H3N2) strain and the B/Jiangsu
strain), these immunization doses having the following compositions:
- 0.1 ps of HA of each of the viral strains in PBS buffer,
- 0.4 fig of HA of each of the viral strains in PBS buffer,
- 1.6 g of HA of each of the viral strains in PBS buffer,
- 6.3 g of HA of each of the viral strains in PBS buffer,
- 0.1 pg of HA; 0.75 mg of squalene; 0.11 mg of DehymulsTM SMO; 0.143 mg
of EumulginTm B1 and 0.138 mg of mannitol in PBS buffer at pH 7.4,
- 0.4 pg of HA; 0.75 mg of squalene; 0.11 mg of DehymulsTM SMO; 0.143 mg
of EumulginTm B1 and 0.138 mg of mannitol in PBS buffer at pH 7.4.
Six groups of eight 8-week-old female C57BL/6J mice were provided and were
administered, on DO intradermally (internal face of the ear), with a dose of
30 1 of
one of the compositions indicated above (1 composition per group).
Blood samples were taken on D23 in order to determine the amounts of
antibodies
induced.
The antibody titers were assayed by ELISA and by HAI for the antibodies
induced
with respect to all the strains administered: H1N1, H3N2 and B.
The results obtained have been summarized in the table below, in which the
titers
expressed are the means obtained for each group of mice.
CA 02613732 2007-12-28
WO 2007/006939
PCT/FR2006/001635
- 29 -
Composition ELISA ELISA ELISA HAI HAI HAI
H1N1 H3N2 B H1N1 H3N2
B
0.1 jig HA 2.924 3.506 3.920 26 174 95
0.4 [Is HA 3.673 4.227 4.431 135 269 147
1.6 pz HA 3.948 4.593 4.786 160 381 190
6.3 g HA 4.446 5.106 5.190 587 1174 453
0.1 jig HA
+ emulsion 4.456 5.192 5.064 349 1974 320
0.4 jig HA
+ emulsion 4.659 5.337 5.174 761 2348 494
Again, the results obtained show the great advantage of the emulsion according
to the
invention, by virtue of which it is possible to very substantially reduce the
amounts of
antigens present. Specifically, it can be considered, overall, that with only
0.1 jig of
HA adjuvanted with the emulsion according to the invention, results are
obtained that
are as good as with an amount of 6.3 jig of HA.
Example 9: Trivalent vaccine composition against the flu comprising an
emulsion
according to the invention or according to the prior art
30 I immunization doses were prepared from the concentrated emulsion of
example
1 and a vaccine composition comprising the 3 viral strains of the 2004
campaign (the
A/New Caledonia (H1N1) strain, the A/Wyoming (H3N2) strain and the B/Jiangsu
strain), these immunization doses having the following compositions:
- 0.3 g of HA of each of the viral strains in PBS buffer,
- 6.3 g of HA of each of the viral strains in PBS buffer,
- 0.3 g of HA; 0.21 mg of squalene; 0.031 mg of DehymulsTM SMO; 0.040 mg
of EumulginTM B1 and 0.039 mg of marmitol in PBS buffer at pH 7.4
(emulsion at 0.7%),
- 0.3 g of HA; 0.75 mg of squalene; 0.11 mg of DehymulsTM SMO; 0.143 mg
of EumulginTM B1 and 0.138 mg of mannitol in PBS buffer at pH 7.4
(emulsion at 2.5%),
CA 02613732 2007-12-28
WO 2007/006939
PCT/FR2006/001635
- 30 -
- 0.3
jtg of HA; 0.645 mg of squalene; 0.075 mg of TweenTm 80; 0.075 mg of
SpanTM 85 (emulsion according to the prior art obtained by microfluidization).
Five groups of eight 8-week-old female BALB/c mice were provided and were
administered, on DO intradermally (internal face of the ear), with a dose of
30 pi of
one of the compositions indicated above (1 composition per group).
To evaluate the amount of antibodies induced, blood samples were taken on D21
and
the activity against the A/H1N1 strain, the A/H3N2 strain and the B strain was
determined on said blood samples by HAI (hemagglutination inhibition).
The results obtained for each group of mice are represented in the table
below.
Composition tested HAI against HAI against HAI
against
H1N1 H3N2
0.3 lig HA 26 174 8
6.3 pg HA 247 905 73
0.3 ps HA + invention emulsion 95 640 37
at 0.7%
0.3 fig HA + invention emulsion 269 1974 73
at 2.5%
0.3 lig HA + prior art emulsion 135 987 57
These results show that, with an emulsion obtained according to the invention
by
virtue of a very simple preparation process consisting of phase inversion by
means of
a change in temperature, an adjuvant was obtained which is as good as, and
even
slightly better than, the emulsion of the prior art obtained using very high
shear rates.
Example 10: Trivalent vaccine composition against the flu comprising an
emulsion
according to the invention at various concentrations
1 immunization doses were prepared from the concentrated emulsion of example
1 and a vaccine composition comprising the 3 viral strains of the 2004
campaign (the
CA 02613732 2007-12-28
WO 2007/006939
PCT/FR2006/001635
-31 -
A/New Caledonia (H1N1) strain, the A/Wyoming (H3N2) strain and the B/Jiangsu
strain), these immunization doses having the following compositions:
- 0.3 pig of HA of each of the viral strains in PBS buffer,
- 6.3 pig of HA of each of the viral strains in PBS buffer,
- 0.3 pig of HA of each of the viral strains; 0.12 mg of squalene; 0.018 mg
of
DehymulsTM SMO; 0.023 mg of EumulginTM B1 and 0.022 mg of mannitol in
PBS buffer at pH 7.4 (emulsion at 0.4%),
- 0.3 pig of HA of each of the viral strains; 0.299 mg of squalene; 0.044
mg of
DehymulsTM SMO; 0.057 mg of EumulginTM B1 and 0.055 mg of mannitol in
PBS buffer at pH 7.4 (emulsion at 1%),
- 0.3 pig of HA of each of the viral strains; 0.75 mg of squalene; 0.11 mg
of
DehymulsTM SMO; 0.143 mg of EumulginTM B1 and 0.138 mg of mannitol in
PBS buffer at pH 7.4 (emulsion at 2.5%).
Five groups of eight 8-week-old female BALB/c mice were provided and were
administered, on DO intradermally (internal face of the ear), with a dose of
30 jil of
one of the compositions indicated above (1 composition per group).
In order to evaluate the amount of antibodies induced, blood samples were
taken at
D21 and the anti-H1N1 antibodies, the anti-H3N2 antibodies and the anti-B
antibodies were determined on these blood samples by ELISA, and the activity
against the AJH1N1 strain, the A/H3N2 strain and the B strain was determined
by
HAI (hemagglutination inhibition).
The results obtained are represented in the table below in the form of means
for each
of the groups; the ELISA results are expressed in logio of arbitrary ELISA
units and
the HAI results are the mean arithmetic titers of the inverses of dilutions.
CA 02613732 2007-12-28
WO 2007/006939
PCT/FR2006/001635
- 32 -
Anti-H1N1 Anti-H3N2 Anti-B
Composition tested ELISA HAI ELISA HAI ELISA HAI
0.3 jig HA 3.864 67 4.594 320 4.406 31
6.3 jig HA 4.478 269 5.041 1660 5.053 135
0.3 jig HA + invention 4.053 108 4.827 525 4.545 54
emulsion at 0.4%
0.3 lig HA + invention 4.312 269 5.074 1174 4.733 123
emulsion at 1%
0.3 lig HA + 4.425 293 5.200 1974 4.840 123
invention emulsion at 2.5%
These results confirm once again that, whatever the strain evaluated, the
emulsion
according to the invention made it possible, with a very low dose of antigens,
to
obtain a very substantial immune system response.
Example 11: Preparation of a lyophilizable composition
The process was carried out as in example 1, but using water instead of the
buffer; the
emulsion obtained was subsequently diluted with an aqueous solution comprising
mannitol, sucrose and dodecylmaltoside, in order to obtain an emulsion whose
final
composition was as follows:
- 5% of squalene,
- 0.95% of polyoxyethylene cetostearyl ether,
- 0.75% of sorbitan monooleate
- 3% of mannitol,
- 2% of dodecylmaltoside,
- 6% of sucrose.
This emulsion was lyophilized and conserved at 4 C for 3 months; then, after
reconstitution, it was noted that its properties were conserved, in particular
its
monodisperse emulsion qualities, with d50 and d90 values close to those
measured
before lyophilization.
This emulsion was able to be diluted 50/50 with a solution comprising vaccine
antigens in order to obtain a vaccine composition.
CA 02613732 2007-12-28
W02007/006939
PCT/FR2006/001635
- 33 -
Example 12: Comparison of the at_ljuvant effect of the emulsion according to
the
invention and of a surfactant present in the emulsion
The intention was to evaluate the adjuvant activity of the emulsion according
to the
invention, compared with that of the surfactant EumulginTM B1 which is present
in the
emulsion.
For this, a test was carried out on mice using flu antigens.
To this end, flu virus antigens were available, obtained according to the
process
described in the examples of application WO 96/05294, with the exception of
the fact
that the viral strain used was the A/New Caledonia H1N1 strain. A vaccine
composition comprising the 3 viral strains of the 2004 campaign (the A/New
Caledonia (H1N1) strain, the A/Wyoming (H3N2) strain and the B/Jiangsu strain)
was also available.
100 gl immunization doses were prepared from the concentrated emulsion
obtained
according to the invention and described in example 1, from EumulginTM Bl, and
from the flu virus antigen compositions, these immunization doses having the
following compositions:
- 1 jig of HA of the H1N1 strain in PBS buffer at pH 7.4;
- 5 jig of HA of the H1N1 strain in PBS buffer at pH 7.4;
- 1 pg of HA of the H1N1 strain; 2.5 mg of squalene; 0.37 mg of DehymulsTm
SMO; 0.48 mg of EumulginTm B1 and 0.46 mg of mannitol in PBS buffer at
pH 7.4;
- 1 pg of HA of the H1N1 strain and 0.48 mg of EumulginTm B1 in PBS buffer
at pH 7.4 ;
- 0.33 pg of HA of each of the viral stains in PBS buffer at pH 7.4;
- 1.66 pg of HA of each of the viral stains in PBS buffer at pH 7.4;
- 0.33 gg of HA of each of the viral stains; 2.5mg of squalene; 0.37
mg of
DehymulsTM SMO; 0.48 mg of EumulginTm B1 and 0.46 mg of mannitol in
PBS buffer at pH 7.4;
- 0.33 jig of HA of each of the viral stains and 0.48 mg of EumulginTm B1
in
PBS buffer at pH 7.4.
CA 02613732 2007-12-28
WO 2007/006939
PCT/FR2006/001635
- 34 -
Eight groups of 8 female BALB/c mice were provided and were immunized by means
of a single intramuscular injection on DO. Blood samples were taken on D21 and
D35
in order to evaluate by ELISA assay their content of total antibodies induced
against
the A/H1N1 flu strain or against each of the strains of the trivalent vaccine.
The
antibody titers, indicated in the table below, are expressed as the log10
value of
arbitrary ELISA units, with a detection threshold of 1.3 log10.
Titers at D21 Titers at D35
Immunization dose compositions H1N1 H3N2 B H1N1 H3N2 B
H1N1 at 1 jig 2.745 2.848
H1N1 at 5 ps 3.057 3.139
H1N1 at 1 jig + invention emulsion 4.002 4.128
H1N1 at 1 + EumulginTm B1 3.019 3.119
Trivalent vaccine at 1 jig of total HA 3.111 3.718 3.706 3.218 4.147 3.935
Trivalent vaccine at 5 jig of total HA 3.692 4.386 3.975 3.776 4.632 4.225
Trivalent vaccine at 1 jig of total HA 4.252 5.002 4.596 4.186 5.214 4.897
+ invention emulsion
Trivalent vaccine at 1 ps of total HA 3.146 3.712 3.950 3.241 4.177 4.242
+ EumulginTM B1
The results obtained in this test confirm those already obtained in previous
tests,
namely that the emulsion according to the invention makes it possible to
greatly
reduce the dose of antigens for the same immune system response; a better
response
was in fact obtained using the emulsion according to the invention and only 1
pig of
HA rather than using a dose of 5 jig of HA without adjuvant.
It is also observed that the surfactant used does not really exhibit any
adjuvant effect
when it is used alone, whereas the emulsion according to the invention itself
produces
a highly adjuvant effect with respect to all the strains tested.
