Link:  Pharm/Biotech Resources

Title:  Vaccine composition comprising iron phosphate as vaccine adjuvant

United States Patent:  6,927,235

Issued:  August 9, 2005

Inventors:  Sauzeat; Elisabeth (Lentilly, FR)

Assignee:  Sanofi Pasteur S.A. (Lyons Cedex, FR)

Appl. No.:  845883

Filed:  May 14, 2004

The present invention relates to vaccine compositions comprising iron phosphate as adjuvant. This iron phosphate is in the form of particles, the size of which is between 0.01 μm and 300 μm; it can be obtained by mixing a solution of iron salt and of phosphate salt.

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 U.S. Pat. No. 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 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, wherein 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 μm and 300 μm, and more particularly between 1 and 40 μm, with in particular a large proportion of particles, the size of which is approximately 7 μm.

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.

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 microorganism or a subunit, and whatever its nature: peptide, protein, glycoprotein, polysaccharide, glycolipid, lipopeptide, etc. They may be viral antigens, bacterial antigens, or the like; the term "antigen" also comprises the polynucleotides, the sequences of 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.

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 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 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.

EXAMPLE 1

Preparation of Iron Phosphate and of Iron Hydroxide

The iron phosphate is prepared by mixing FeCl₃.6H₂O 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) . The 2 products are mixed in the following proportions: 1 l of ultrafiltered water comprising 44.5 g of Na₂HPO₄.2H₂O is combined with a solution of 800 ml of ultrafiltered H₂O comprising 108.12 g of FeCl₃.6H₂O. 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/l.

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 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/l), the following compositions are prepared:

PTP and FeOOH: 458 μl H₂O+382 μl FeOOH at 6.3 g of Fe/l+120 μl of a 10 times-concentrated PBS buffer solution+240 PTP at 100 Lf/ml.
 

Claim 1 of 8 Claims

1. A composition comprising at least one vaccine antigen and at least one adjuvant, wherein the adjuvant comprises iron phosphate.

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