Note: Descriptions are shown in the official language in which they were submitted.
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Vaccine composition comprising iron phosphate as a
pharmaceutical aid to said vaccine
The present invention relates to the vaccines field,
and in particular to the field of vaccine compositions
comprising an adjuvant. More particularly, the present
invention relates to a vaccine in which the adjuvant
comprises iron phosphate.
Vaccine compositions comprising adjuvants intended to
increase or to qualitatively modify the immune response
induced when an antigen is administered are known in
the prior art. In fact, the developments realized in
the vaccine industry result in the use of antigens
obtained from viruses or from bacteria, but with
increasingly developed purification methods, and also
of antigens derived from biotechnology. These antigens
have the advantage of generally being purer than some
of the antigens used in the past, but, in return, they
are often less immunogenic and therefore require the
use of an adjuvant.
Many adjuvants have already been described: saponins,
emulsions, cationic lipids, etc.
However, to date, the only adjuvants commonly used in
the marketed products are aluminum-based adjuvants.
Now, it would be desirable to have other adjuvants.
Among the prior art relating to adjuvants, mention may
in particular be made of US patent 5,895,653, which
describes, as adjuvant, iron hydroxide compounds in the
form of a colloidal solution, the adsorbent capacity of
which is thought to be improved compared to the iron
hydroxides previously described in the form of a gel.
However, such adjuvants, while they present an
alternative to the aluminum-based adjuvants currently
used, cannot always effectively replace them, and it is
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therefore desirable to have other compounds, the
characteristics of which are such that at the same time
they exhibit good tolerance with respect to the
organisms to which they are administered, they can be
produced under conditions compatible with the
constraints of the pharmaceutical industry and,
especially, they make it possible to increase or to
modify the immune response to vaccine antigens in a
manner which is at least as effective as the aluminum-
based compounds currently used.
With this aim, the subject of the present invention is
a vaccine composition comprising at least one vaccine
antigen and at least one adjuvant, characterized in
that the adjuvant comprises iron phosphate.
According to a particular characteristic, the iron
phosphate is present in the form of a suspension of
particles, the size of which is between 0.01 pm and
300 pm, and more particularly between 1 and 40 pm, with
in particular a large proportion of particles, the size
of which is approximately 7 pm.
According to a particular embodiment of the present
invention, the iron phosphate is prepared from a
solution of iron salt and of phosphate salt.
Such a preparation makes it possible to obtain a
whitish non-crystalline product providing all the
safety conditions required for administration to
humans, generally in good health.
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Many advantages of the present invention will emerge in the course of the
description which will follow with reference to the figures which illustrate
results
obtained in the tests described in example 3.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 represents the IgG1 results after a 1st immunization.
Figure 2 represents the IgG1 results obtained after the 2nd immunization.
Figure 3 represents the IgG2a results obtained after the 1 st immunization.
Figure 4 represents the IgG2a results obtained after the 2nd immunization.
For the purpose of the present invention, the term
"vaccine composition" is intended to mean a composition
which can be administered to humans or to animals in
order to induce an immune system response; this immune
system response can result in a production of
antibodies or simply in the activation of certain
cells, in particular antigen-presenting cells,
T lymphocytes and B lymphocytes. The vaccine
composition can be a composition for prophylactic
purposes or for therapeutic purposes, or both.
For the purpose of the present invention, the term
"antigen" is intended to mean any antigen which can be
used in a vaccine, whether it involves a whole micro-
organism or a subunit, and whatever its nature:
peptide, protein, glycoprotein, polysaccharide, glyco-
lipid, lipopeptide, etc. They may be viral antigens,
bacterial antigens, or the like; the term "antigen"
also comprises the polynucleotides, the sequences of
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which are chosen so as to encode the antigens whose
expression by the individuals to which the
polynucleotides are administered is desired, in the
case of the immunization technique referred to as DNA
immunization. They may also be a set of antigens, in
particular in the case of a multivalent vaccine
composition which comprises antigens capable of
protecting against several diseases, and which is then
generally referred to as a vaccine combination, or in
the case of a composition which comprises several
different antigens in order to protect against a single
disease, as is the case for certain vaccines against
whooping cough or the flu, for example.
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Particularly good results have in particular been
obtained with a vaccine composition comprising antigens
against diphtheria, tetanus and poliomyelitis.
For the purposes of the present invention, the iron
phosphate is an inert mineral compound based on
amorphous, crystalline or pseudocrystalline iron
phosphate. Very satisfactory tests have been achieved
with an amorphous iron phosphate.
Such a compound is prepared from a solution of iron
salt and of phosphate salt.
As iron salt, mention may in particular be made of iron
citrate or, particularly advantageously, iron chloride.
As phosphate salt, use may in particular be made of
sodium phosphate.
The iron phosphate can be prepared by simultaneously or
successively mixing the two starting components. The
order of the mixing can be either the addition of iron
chloride to sodium phosphate or, conversely, the
addition of sodium phosphate to iron chloride. The rate
of addition can be very rapid or, on the contrary,
dropwise.
To obtain an iron phosphate suitable for the needs of
the invention, it has been noted that a ratio of
2 times more iron salt than phosphate salt is
satisfactory.
Alternatively, the iron phosphate can be prepared using
a nebulizer. In this case, the sodium phosphate can be
nebulized in a solution of iron chloride or,
conversely, the iron chloride in the sodium phosphate.
During the preparation of the vaccine compositions
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according to the invention, the iron phosphate can be
introduced into the vaccine composition at the very
beginning of the formulation. The iron phosphate is
then diluted to a certain concentration, and the
5 antigens of interest are then added thereto.
Alternatively, it is possible to add the iron phosphate
to a lyophilisate already comprising the portion of the
vaccine composition which contains the antigens of
interest.
According to the invention, the iron phosphate is
present in the vaccine composition in a sufficient
amount to exert an adjuvant action.
When it is the only adjuvant used, the dose of vaccine
composition administered can comprise between 0.2 and
1.4 mg of iron phosphate.
When it is combined with another adjuvant, the amounts
present can be reduced, as a function of the desired
adjuvant effect and of the potency of the other
adjuvants present.
Local and systemic toxicity tests on rabbits have shown
that iron phosphate is as well tolerated as aluminum
hydroxide.
The following examples illustrate particular
embodiments of the present invention.
Example 1: Preparation of iron phosphate and of iron
hydroxide
The iron phosphate is prepared by mixing FeC13.6H20 at
0.5 M (supplied by SIGMA under the batch reference
55H 1251) in a solution of Na2HPO4.2H2O at 0.25 M
(supplied by Merck with the batch reference FA004911).
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The 2 products are mixed in the following proportions:
1 1 of ultrafiltered water comprising 44.5 g of
Na2HPO4.2H20 is combined with a solution of 800 ml of
ultrafiltered H2O comprising 108.12 g of FeC13.6H20. The
mixing is performed at ambient temperature.
Stirring is performed for 15 minutes by means of a rod-
stirrer and then successive centrifugations are carried
out with the precipitate being washed with water. After
each wash, the presence of chloride ions is detected by
assaying the chloride ions with silver nitrate. When
there are no longer any chloride ions, i.e. after
10 washes, the precipitate is taken up in water and
autoclaved for 1 h at 118 C in order to ensure its
sterility.
The solution obtained in this manner is an iron
phosphate solution containing 16 g Fe/1.
The iron hydroxide is prepared by mixing 0.5 M iron
citrate and 2 M NaOH, at ambient temperature. NaOH is
added dropwise to 100 ml of iron citrate until a pH of
8 is obtained. In the same way as for the preparation
of the iron phosphate, stirring is performed for
15 minutes and then successive centrifugations are
carried out with the precipitate being washed with
water, and the chloride ions being assayed with silver
nitrate. When chloride ions are no longer detected, the
precipitate is taken up in water and autoclaved for 1 h
at 118 C in order to be sterile. The solution then
obtained is an iron hydroxide solution containing 6.3 g
Fe/l.
Example 2: Preparation of the vaccine compositions
Using the solutions prepared as indicated above, an
aqueous suspension of aluminum hydroxide (supplied by
Reheis) comprising 10.8 g of Al/l, and also a
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preparation of PTP (purified tetanus protein) at a
concentration of 330 flocculation or Lf units/ml (i.e.
a protein concentration of 0.9125 g/1), the following
compositions are prepared:
= PTP alone: 840 pl H2O + 120 pl 9% NaC1 +
240 PTP at 100 Lf/ml,
= PTP and A1OOH : 618 pl H2O + 222 pl A100H at
10.8 g of Al/l + 120 pl 9% NaC1 + 240 PTP at
100 Lf/ml,
= PTP and FePO4: 690 pl H2O + 150 pl. FePO4 at
16 g/l + 120 p1 of a 10 times-concentrated PBS
buffer solution (i.e. 95 mM Na2HPO4.2H2O) +
240 PTP at 100 Lf/ml,
= PTP and FeOOH: 458 pl H2O + 382 pl FeOOH at
6.3 g of Fe/1 + 120 p1 of a 10 times-
concentrated PBS buffer solution + 240 PTP at
100 Lf/ml.
Example 3: Assaying of the anti-tetanus antibodies
4 groups of six 6-week-old female OF1 mice are used.
One of the formulations prepared in example 2 is
administered to each group of mice. The administration
is performed intramuscularly in the quadriceps under
anesthesia, at a rate of 2 injections of 50 pl on
days Dl and D22. The amount administered is therefore
2 flocculation or Lf units of PTP and 0.2 mg of mineral
material (either aluminum or iron) in the case of the
formulations comprising an adjuvant.
On D15, a blood sample of approximately 300 pl is taken
from the retro-orbital sinus in each mouse.
On D36, the mice are bled out under anesthesia, by
sectioning of the carotid.
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The PTP-specific IgGls and IgG2as are assayed, by the
ELISA method, on each of the sera corresponding to the
bloods collected.
The results obtained are given in figures 1 to 4 and
show that the iron phosphate is a good adjuvant; as
regards the IgGis, the results obtained are clearly
superior to those obtained when the antigen is
administered alone, even though they are not quite as
good as those obtained with the aluminum hydroxide; as
regards the IgG2as, the titers obtained are as high as
those obtained with the aluminum hydroxide.
Example 4: Verification of the anti-tetanus activity
The anti-tetanus activity of the compositions according
to the invention is verified by means of a test
consisting in evaluating, in mice, the activity of
these compounds with respect to a lethal dose of toxin
administered subcutaneously.
The activity of the compositions tested is determined
by comparing the dose required to protect 50% of the
animals (ED50) against the effects of the toxin, with
that of a reference vaccine calibrated in IU/ml.
The formulations tested are obtained by mixing the
adjuvant (0.6 mg/dose) with water, 30 Lf of PDT
(diphtheria toxoid) and 10 Lf of PTT (tetanus toxoid),
and then adding 20 pg/dose of merthiolate and a saline
solution (solution containing 90 g/l of NaC1 and 5 mM
Na2HP04.2H20) .
The vaccine compositions tested comprise either
aluminum hydroxide or iron phosphate or iron hydroxide.
The results obtained with each of the adjuvants tested
are reported in the table below, and show that iron
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phosphate is a good adjuvant, clearly better than iron
hydroxide under the same conditions:
Adjuvants Tetanus activity (IU/dose)
A100H 103 (76-139)
FePO4 32 (24-43)
FeOOH 8.2 (5.4-12.5)
