FR2808194A1 - Improving the immunomodulating activity of a substance, comprises admixture of a material that modulates immune response with a hydrophilic solid optionally coated with an amphiphilic compound - Google Patents

Abstract

Improving the immunomodulating activity of a substance, other than an antigen, capable of modulating an immune response, comprises mixing the substance with a carrier having a non-liquid hydrophilic core, preferably with an external layer of amphiphilic material bonded to the core through hydrophobic interactions and/or ionic bonds. Independent claims are also included for the following: (1) the use of a mixture as described to prepare medicaments for treatment of cancers and viral diseases; and (2) vaccines comprising antigens and specific carriers.

Description

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 USE OF HYDROPHILIC PARTICLES WITH OR WITHOUT IONIC LIGANDS FOR IMPROVING THE IMMUNOMODULATORY PROPERTIES OF A SUBSTANCE OTHER THAN AN ANTIGEN, AND THE PHARMACEUTICAL COMPOSITIONS THUS OBTAINED.

 The present invention relates to a method for improving the immunomodulatory properties of a substance, other than an antigen, capable of modulating the immunological response of mixing said substance with hydrophilic particles bearing or without ionic ligands and optionally coated with a layer of amphiphilic compounds. The invention relates to the treatment and / or prevention of diseases comprising an immunogenic character such as cancers or infections. The invention is particularly interested in a particular therapeutic vaccine composition and their preparation process.

 The Applicant has developed its expertise in the preparation of synthetic particulate vectors, hereinafter also referred to as "BVSMTM".

 A first type of BVSM, and its method of preparation, have been described in European Patent No. 344 040. These are particles comprising from the inside to the outside: a non-liquid hydrophilic central core consisting of a matrix of naturally or chemically cross-linked polysaccharides or oligosaccharides, which can be modified by various ionic groups; a layer of fatty acids grafted by covalent bonds to the nucleus; one or more lipid layers consisting in particular of phospholipids.

 The developments of this first generation of BVSM have led the Applicant to design and prepare new BVSM, with improved properties including the active ingredients transported.

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 The patent applications published under the numbers WO 94/20078, WO 96/06638 and EP 782 851 describe these BVSM, their manufacture, their combination with various active ingredients and their use for the preparation of pharmaceutical compositions.

 The know-how currently available to the Claimant on the preparation of BVSM allows it to have a wide range of BVSM type. These BVSM are formed of a crosslinked hydrophilic polymer, preferably polysaccharides, in the form of nanoparticulate gel, these BVSM are said to be of the PSC (or NPS in French) type.

 This polymer may optionally carry positive ionic ligands, BVSM said cationic, or negative, so-called anionic BVSM. This charged or unloaded polymer is optionally covered with a layer of amphiphilic compounds, preferably phospholipids, BVSM, referred to as Light type, described in patent application PCT WO94 / 20078.

 The size of the BVSM is between 20 and 200 nm, preferably between 50 and 100 nm and more preferably between 60 and 80 nm.

 The BVSM can be sterilized by filtration and their combination with the active ingredients is done on completely manufactured BVSM (Major et al., Biochem. & Biophys.

Acta (1997), 1327, 32-40). This makes it possible to work the biological molecules, particularly fragile, in optimal conditions.

 BVSMs protect biological molecules from degradation (Prieur R. et al., Vaccines (1996) Vol 14, N 6 pp. 511-520 Combination of recombinant hCMV IE1 protein with 80 nm cationic biovectors: protection from proteolysis and potentiation of presentation CD4).

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 They have also been shown to increase the biological activity of peptides and proteins such as enzymes, antigens, etc.

 BVSMs are known to be used as transporters of active substances, for example peptides, antigens or oligonucleotides. In this use, there is formation of a real complex between the BVSM and the active substance, such as ionic interactions between the cationic core of the BVSM and the anionic oligonucleotide. There is thus formation of a stable ionic conjugate in a biological medium. In this type of preparation, the oligonucleotide / BVSM ratio is 5 to 10% and the measured association yield is greater than 90%.

 The PCT patent application published under the number WO 96/06638 reports the increase of the immunogenicity of an antigen, obtained by a simple antigen / BVSM mixture.

The antigen is also associated with the particulate vector by ionic and / or hydrophobic bonds. To obtain this result, for example, 1 mg of GST-e4 protein is incubated per 10 mg of BVSM, which makes it possible to obtain average association levels of 90% regardless of the type of BVSM used (Prieur R. et Vaccines (1996) Vol 14, No. 6 pp 511-520).

 The Applicant has now discovered that the BVSM make it possible to improve the immunomodulatory properties of a substance other than an antigen capable of modulating the immunological response. These substances are more particularly: adjuvants capable of amplifying, regulating or modifying the immune response, cytokines and chemokines whose intrinsic property is to modify the activity of the cells of the immune system,

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 immunosuppressants due to their use in the treatment of allergies, and / or graft rejection, substances capable of modifying the Th1 / Th2 balance.

 It may be recalled that the immune response is relayed by lymphocyte cells, in particular B cells and T lymphocytes. The latter can be classified into two subtypes on the basis of their expression of CD4 and CD8 surface antigens. CD4 + cells are usually involved in helper functions. In particular, they secrete cytokines that induce the proliferation and maturation of other lymphocyte cells. Thus, two profiles of helper T lymphocytes can be defined: on the one hand, a Th1 profile, which characterizes a cell-mediated response, with the induction of cytokines, in particular IL2, IL7 and interferon gamma, stimulation of CTL and induction of IgG2a antibodies, and secondly, a Th2 profile, which characterizes a humoral mediated response, with the induction of cytokines including IL4, IL5 and IL10, and the presence of IgG1 and IgE antibodies.

 Protein antigens, whose presentation by antigen-presenting cells (APCs) is predominantly made by MHC class II, induce a Th2-directed response. Conversely, DNA that allows after transfection to present the antigens

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 directly on MHC class I, directs the immune response to Th1.

 Some adjuvants are known to guide the response predominantly to Th1 or Th2. For example, aluminum salts that induce a serum IgG1 response are considered Th2. In contrast to adjuvants such as lipid A (MPL) derivatives or QuilA derivatives, which induce an IgG2a and CTL response, are considered to be Thl. It would therefore be useful to have means for acting on the Thl / Th2 balance, which is precisely what the present invention proposes to achieve.

 The research work carried out by the Applicant in the context of the present invention show that the BVSM does not act as an adjuvant since it alone does not induce activation of the immune system, but as a carrier allowing better penetration and / or a better presentation of the antigen to the presenting cells. Thus, the results obtained showed the contribution of BVSM on the adjuvant power of CTB, MPL and ODN. Surprisingly, the adjuvant power is visualized in a different way: - on the serum IgG response, for which a strong synergy, in the case of the CTB, or an addition of the responses, in the case of the CpG ODNs, was obtained - on the IgA response, where a synergy is clearly demonstrated for MPL, - on the Thl / Th2 balance, where even in the case of a quantitatively equivalent response (IL2), a qualitative difference in the response is observed in particular

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 in the Igl / IgG2a balance that reflects a differential in cellular response.

 The invention therefore firstly relates to a method for improving the immunomodulatory properties of a substance, other than an antigen, capable of modulating the immune response, characterized in that it comprises the mixing of said substance with hydrophilic particles with or without ionic ligands and optionally coated with a layer of amphiphilic compounds.

 The hydrophilic particles bearing or not ionic ligands and optionally coated with a layer of amphiphilic compounds, or BVSM, are advantageously vectors of the type comprising: a non-liquid hydrophilic nucleus, consisting of a matrix of polysaccharides or oligosaccharides naturally or chemically crosslinked, on which are grafted ionic ligands, - an outer layer consisting at least in part of amphiphilic compounds, associated with the nucleus by hydrophobic interactions and / or ionic bonds.

 Preferably, the ionic ligands are positively charged, such as quaternary ammoniums, and the outer layer is dipalmitoyl phosphatidyl choline (DDPC) and cholesterol.

 In the process of the invention, the substance / particle weight ratio is between about 1% and 20%, preferably between about 5% and 10%.

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 A first class of non-antigen substances capable of modulating the immune response includes proteins of therapeutic interest that play a role in the functioning of the immune system. It is more particularly a cytokine or a chemokine, preferably chosen from the group comprising interleukins, interferons, TNFs, TGFs, GCSF, GM-CSF, MIF, RANTES, or a mixture of these.

 A second class of substances, other than an antigen, capable of modulating the immune response, partially covering the previous one, comprises the adjuvants. By adjuvant is meant a molecule for amplifying, regulating or directing the immune response specific for an antigen. Thus, in order to evaluate the properties of the BVSM on the immunomodulatory power of substances other than antigens, various molecules belonging to the main categories of adjuvants have been tested in the context of the present invention, such as bacterial products (endotoxins, wall), cytokines, oligonucleotides (CpG). More particularly, mention may be made of: bacterial enterotoxins, such as CT, CTB, LT, liposaccharide derivatives, such as MPL and lipid A derivatives, saponin derivatives of the QS21 type, ammonium salts, and their derivatives, like alum,

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proteins of the type DT, TT, or a mixture thereof
The substance, other than an antigen, capable of modulating the immune response is preferably chosen from: adjuvants capable of amplifying, regulating or modifying the immune response, cytokines and chemokines, the intrinsic property of which is to modify the activity of the cells of the immune system, - immunosuppressants because of their use in the treatment of allergies and / or transplant rejection, substances capable of modifying the Thl / Th2 balance.

 Among these, the invention more particularly envisages, as a substance whose immunomodulatory properties are to be improved, the cytokines which stimulate the activity of the immune cells, and among these, IL2, IL11 and GM-1. CSF. Indeed, cytokines stimulate or inhibit the activity of immune cells.

 The invention also relates to the use of hydrophilic particles bearing or not ionic ligands and optionally coated with a layer of amphiphilic compounds for the preparation of a medicament for the treatment of cancers and / or particularly viral infections, said particles being associated in the

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 drug with at least one substance, other than an antigen, capable of modulating the immune response. In this use, the substance, other than an antigen, capable of modulating the immune response is a protein of therapeutic interest or an adjuvant that plays a role in the functioning of the immune system. As indicated above, it is in this use of the invention, a protein of therapeutic interest which is advantageously a cytokine or a chemokine, preferably selected from the group comprising interleukins, interferons, TNF, TGF, G-CSF, GM-CSF, MIF, RANTES, or a mixture thereof. It may also be an adjuvant, advantageously chosen from the group comprising bacterial enterotoxins, liposaccharide derivatives, saponin derivatives, ammonium salts and their derivatives, DT or TT type proteins, or mixture of these. In this use, the invention is concerned with the treatment of cancers of the non-immunogenic or weakly immunogenic type, and / or with the treatment of particularly viral infections such as AIDS or chronic hepatitis where immunomodulators have been proposed in addition to therapeutic treatments. to activate the immune response. A composition for the implementation of this use comprises hydrophilic particles bearing or not ionic ligands and optionally coated with a layer of amphiphilic compounds in which are incorporated proteins of therapeutic interest or adjuvants as defined above. Such a composition does not include antigen.

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 The weight ratio of substances (proteins of interest or adjuvant) to particles is between about 1% and 20%, preferably between about 5% and 10%.

 The invention therefore also relates to a therapeutic composition characterized in that it comprises hydrophilic particles bearing or not ionic ligands and optionally coated with a layer of amphiphilic compounds, and at least one protein of therapeutic interest and / or an adjuvant, the protein of therapeutic interest and the adjuvant being defined as above.

 The invention more particularly relates to the use of hydrophilic particles bearing or not ionic ligands and optionally coated with a layer of amphiphilic compounds for the preparation of a therapeutic or prophylactic vaccine composition, said particles being mixed in the composition with least one antigen and at least one substance capable of modulating the immunological response to said antigen. In the case of a therapeutic use, the invention is particularly interested in the treatment and / or prevention of viral diseases, including AIDS and chronic hepatitis, but also cancers having an immunogenic nature.

 The invention therefore relates especially to a vaccine composition characterized in that it comprises an antigen or a mixture of antigens, hydrophilic particles bearing or not ionic ligands and optionally coated with a layer of compounds

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 amphiphilic, and at least one substance capable of modulating the immunological response to said antigen.

 The substance capable of modulating the immunological response to the antigen present in the composition of the invention is preferably an adjuvant. Among these, the invention contemplates bacterial products (endotoxins, wall components), cytokines, oligonucleotides (CpG). More particularly, mention may be made of: bacterial enterotoxins, such as CT, CTB, LT, liposaccharide derivatives, such as MPL and lipid A derivatives, saponin derivatives of the QS21 type, ammonium salts, and their derivatives, such as alum, DT, TT type proteins, or a mixture thereof.

 As indicated above, the substance / particle weight ratio is between about 1% and 20%, preferably between about 5% and 10%.

 The invention is particularly suitable for the preparation of a composition, more particularly for mucosal administration, in particular nasal administration, but any other mode of administration, for example parenteral, may be envisaged.

 Other features and advantages of the invention will become apparent from the following examples

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 concerning (i) the effect in mice of the BVSM on the immunogenicity of a split trivalent Influenza to that of different adjuvants, and (ii) the effect in mice of the co-administration of the formulation and different adjuvants on the immunogenicity of a split trivalent Influenza.

The various formulas (adjuvant BVSM Ag) are administered at 0 and 21 according to the following scheme:
PBS control in

 C: Naive control i. not.

 AS: Ag s. c.

 AN: Ag i. not.

 FA: Ag + BVSM i. not.

 Ax: Ag + Adj X i. not.

 FX: Ag + BVSM + Adj X i. not.

 The immunogenicity is evaluated after the booster, on day 35: - at the serum level, ELISA detection of the specific IgGs of each monovalent split - at the mucosal level, detection by ELISA in the nasal and / or vaginal secretions of the specific IgAs of each monovalent split .

Reference will be made hereinafter to the appended drawings in which:
Figure 1 shows the Biacore analysis (surface plasmon resonance) of the association of CTB with the BVSM as a function of the concentration of CTB.

 Figure 2 shows the Biacore analysis of the association of MPL with BVSMTM as a function of MPL concentration.

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 Figure 3 shows the inhibition of the split B Harbin association on BVSM in the presence of an increasing amount of CTB.

 Figure 4 shows the inhibition of split B Harbin association on BVSM in the presence of an increasing amount of MPL.

 Figure 5 shows the sensorgrams obtained by comparing the two modes of preparation of CTB and influenza antigens on BVSM immobilized on HPA sensorship. First, the CTB and the antigen are deposited successively on the BVSM (curve A) and vice versa (curve B).

 FIG. 6 represents the sensorgrams obtained by comparing the two modes of preparation of the MPL and the antigens of the flu on the immobilized BVSMs on the HPA sensorship. At first, the MPL and then the antigen are deposited successively on the BVSM (curve A) and vice versa (curve B).

 FIG. 7 represents the specific B / Harbin split titration of sera pools (IgG) and mouse nasal secretions (IgA) administered by the trivalent formulations and the antigen controls, which may or may not be associated with BTC.

 FIG. 8 shows the specific B / Harbin split titration of mouse pools (IgG) and mouse nasal secretions (IgA) administered by trivalent formulations and antigen controls with or without MPL.

 FIG. 9 represents the specific B / Harbin split titration of serum pools (IgG) and

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 mouse nasal secretions (IgA) administered by the trivalent formulations and the antigenic controls with or without oligonucleotides.

 I - Materials and methods.

 1) Material.

 The animals used are female BALB / cJ / Rj mice (10 weeks old at the beginning of the study) from the January breeding center (Dry Oaks Road - BP5 - Le Genest-Saint-Isle). acclimated for 7 days, at a rate of 6 mice per cage before the start of the study.

 2) Products. a) BVSMTM.

 The BVSMTM used is of type KY: QAE = 2 mEq DPPC / Cholesterol. It corresponds to a polysaccharide nucleus grafted with glycidyl trimethylammonium and surrounded by a layer of DPPC / Cholesterol. This type of vector is described in EP 687,173. (b) Adjuvants.

 The following 4 adjuvants were used: - Subunit B cholera toxin (CTB) (ref.

C9903, Sigma, St Quentin Fallavier, France).

 Monophosphoryl, Lipid A (MPL) (Ref R-350, Ribi Immunochem Research, Inc., Hamilton, MO).

 Recombinant IL-2, Proleukin, activity 16.3.

106 U / mg (Chiron France, Suresnes, France).

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 Oligonucleotides ODN: 5TCCATGACGTTCCTGAC '(Eurogentec, Brussels, Belgium). c) Split trivalent Influenza.

 A 250 μg solution of HA / ml (83.3 μg / strain) of trivalent split influenza is prepared with 3 split monovalent eggs from Biochem Pharma (Canada).

 - the monovalent split #uf produced from strain B / Harbin 7/94.

 - the monovalent split #uf produced from the A / Johannesburg 82/96 strain.

 - the monovalent split #uf produced from the strain A / Nanchang 933/95.

 These split consist of a mixture of viral proteins, including haemagglutinin (HA) and neuraminidase. d) Formulation.

 - Antigen formulation + BVSMTM.

 A formulation of 250 μg of HA / ml (83.3 μg / strain) of trivalent split influenza is prepared with the 3 split monovalent eggs above to obtain a ratio of HA / BVSM equal to 1/89 250 μg of trivalent HA 22.25 mg BVSM / ml.

 This formulation is obtained by mixing, at equal volume, 3 formulations with 250 μg HA / 22.25 mg BVSMTM / ml, each of the monovalent split.

 - Antigen formulation + BVSMTM + adjuvant.

 From the antigen + BVSMTM formulation, four other adjuvant formulations were prepared.

These formulations are obtained by diluting the trivalent formulation with 250 μg of HA / ml in the presence and

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 by adding adjuvant. The dilution makes it possible to adjust the dose of monovalent HA / mouse / immunization to 1.2 μg, ie 60 μg of monovalent HA / ml and to add the adjuvant in the volume used to dilute. e) Witness.

 Three types of controls were prepared: trivalent split controls, hereinafter AS and AN, prepared by mixing, at equal volume, 3 solutions at 250 μg HA / ml each of the monovalent split, trivalent split controls + adjuvant, - naive control (0.22X PBS).

 Depending on the subcutaneous (AS) or intranasal (AN) route of administration, the trivalent split solution is prepared in Phosphate Buffered Saline or in sterile water, respectively.

 3) Methods. a) Immunization of the mice.

 - Administrations.

The administration of the formulas (BVSM Adj Ag) is carried out without anesthesia either by:. subcutaneous: 100 l in the 1 ml syringe at the dorsal level for controls, nasal: 20 l (10 μl / nostril) at the 10 l micropipette for the tested samples and controls,
For each group the immunization comprises two administrations, OJ and J21 performed according to the same mode.

 - Blood sample.

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 A blood sample of approximately 0.3 ml is made at the retroorbital vein at OJ (control) and J35.

 After formation of a clot and centrifugation the serum is frozen at -20 C until use.

 . Collection of nasal secretions.

 The washing of the nasal cavities is carried out using 500 l of phosphate buffered saline - 1% BSA introduced into the trachea towards the nasal turbines. The operation is thus repeated 3 times with the same PBS-BSA 1%. The nasal sample is stored in an Eppendorf tube at -20 ° C. Nasal secretions are recovered on D35. b) Analysis of samples.

 The analysis of sera and nasal secretions is performed by an ELISA test. Briefly, the microplates (Nunc, Immunoplate maxisorb, polylabo) are coated with the influenza vaccine (100 ng HA / well in carbonate buffer, pH 9.6, 2 hours at 37 ° C). After rinsing (three times in PBS-tween buffer pH 7.6) and then saturation with 250 μl / well of a solution of PBS-BSA 3% (1 h 00 at 37 ° C.), the ranges of sera or nasal secretions are incubated 1 hour at 37 ° C. and rinsed again with a PBS-tween solution, pH 7.6. After rinsing (three times) the antibodies are detected by murine IgG antibodies (ref A4416 Sigma) or murine anti IgA (ref A4789 Sigma), coupled with peroxidase. After a final rinsing step (five times), the OPD substrate is added (ref P8287, Sigma 1 tablet in 5 ml of revealing buffer + 50 l of H2O2, 100 μl / well). After incubation for 30 minutes at 37 ° C., 1N hydrochloric acid (reference 30024-290, Prolabo) is

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 added (50 μl / well) and the absorbance measured at 490 nm. The titles are defined as the inverse of the dilution allowing to obtain an OD of 0.1.

 II - Example 1 Influence of the Mode of Preparation of the BVSM / Adjuvant / Influenza Formulations

 In order to evaluate the best mode of preparation of BVSM / Adjuvant / Influenza formulations, the association of split Influenza on BVSM is studied in the presence of adjuvant (CTB or MPL) using Biacore X surface plasmon resonance (RPS). (Pharmacia Biosensor Inc.). In this study, 20 l of a suspension of BVSM (50 μg / ml in 0.3 X PBS) are introduced on hydrophobic sensors (HPA, Biacore, BR-1000-30) at 5 l / min.

 Figure 1 shows the sensorgram obtained by association of CTB and BVSM immobilized on the sensorchip HPA as a function of the concentration of CTB (1) 2.5ug / ml; 2) 25 μg / ml; 3) 250ug / ml in 45mM PBS (which corresponds to 0.3 X).

 Figure 2 shows the sensorgram obtained by association of MPL and BVSM immobilized on the HPA sensorchip as a function of the concentration of MPL (1) 1, 56ug / ml; 2) 3.125ug / ml; 3) 6.25ug / ml; 4) 12.5ug / ml; 5) 25 μg / ml; 6) 50ug / ml in 45mM PBS).

 In both cases, CTB and MPL, the sensorgrams obtained confirm the association capacity of the BVSM and adjuvants.

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The method of preparation of the BVSM / Adjuvant / split Influenza formulations is then studied. Two methods are used:
In the first, an adjuvant solution is introduced before adding the split Influenza.

 In the second, the split Influenza is introduced before adding the adjuvant.

 Figure 3 summarizes the results obtained in the case of a split B Harbin. Variable amounts of CTB are introduced on the immobilized BVSM (1) No CTB, 2) 2.5Mg / ml, 3) 25ug / ml, 4) 250ug / ml in 45mM PBS). The monovalent B Harbin split at 25 μg / ml is then deposited on the adjuvant BVSM. There is clearly an inhibition of the association of the split on the BVSM when the CTB is added before the split.

 In the same way, figure 4 summarizes the results obtained in the case of the MPL and a split B Harbin. Variable amounts of MPL are introduced on the immobilized BVSM (1) 1, 56ug / ml; 2) 3.12ug / ml; 3) 6.25ug / ml; 4) 12.5ug / ml; 5) 25 μg / ml; 6) 50ug / ml in 45mM PBS). Split at 25 μg / ml is then deposited on adjuvant BVSM. As for BTC and in proportion to the amount of MPL, there is an inhibition of split association on BVSM when adjuvant is added before split.

 Figure 5 shows the sensorgrams obtained by comparing the two modes of preparation in the case of BTC. In A, BTC is added to the immobilized BVSM, then the monovalent split is introduced.

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 In B, the monovalent split is added to the immobilized BVSM, then the CTB is introduced.

 It is found that when the antigen is added before the CTB, there is a decrease of this association of the split on the BVSM. Conversely, when the CTB is added after the split, the BVSM allows the association of an additional amount of material corresponding to the CTB. Thus, to preserve the role of the BVSM as Antigen Carrier, it is important to maintain a mode of preparation in which BTC is added to the BVSM / split formulations.

 In the same way, FIG. 6 represents the sensorgrams obtained by comparing the two modes of preparation in the case of the MPL.

 In A, the MPL is added before the split. In B, it's the opposite.

 When the antigen is added before the MPL, there is no significant change in the split / BVSM combination, but the BVSM allows to associate an additional amount of material corresponding to the MPL.

 It therefore seems possible to define a mode of preparation of the adjuvanted formulations in which: the adjuvant is added to the BVSM / antigen formulations - the role of the BVSM as Antigen Carrier is preserved.

 III - Example 2 Effect of BTC on the Immunological Response of BVSM / Influenza Formulations

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 1) Protocol.

The antigen + BVSMTM formulation is prepared as mentioned in the Materials and Methods section.

 A control is carried out under the same conditions but in the absence of BVSMTM resulting in a trivalent Split at 250ug HA (83ug / strain) / ml.

 A solution of CTB is added to each of these preparations in order to obtain the corresponding adjuvant formulations and controls.

 36 female BALB / cJ / Rj mice (10 weeks old at the start of the study) are divided into 6 groups at a rate of 6 mice per group. For each group the treatment is performed as previously described. Table 1 below summarizes the treatment of each group.

Table 1

<Tb>
<tb> Code <SEP> Group <SEP> (number <SEP> Route <SEP> Formula <SEP> Administered
<tb> of <SEP> mice) <SEP> Quantity / <SEP> dose
<tb> C <SEP> Control <SEP> naive <SEP> i. <SEP> n. <SEP> 20 <SEP>) it <SEP> PBS <SEP> 80mOsm / kg
<tb> FA <SEP> Ag <SEP> + <SEP> BVSM <SEP> in <SEP> 3.6 <SEP> g <SEP> HA <SEP> + <SEP> 320.4 <SEP> g <SEP > BVSM
<tb> AS <SEP> Ag <SEP> sc <SEP> sc <SEP> 3.6 <SEP> g <SEP> HA
<tb> AN <SEP> Ag <SEP> in <SEP> in <SEP> 3.6 <SEP> g <SEP> HA
<tb> FCTB <SEP> Ag <SEP> + <SEP> BVSM <SEP> + <SEP> CTB <SEP> i <SEP>. <SEP> n <SEP>. <SEP> 3 <SEP>, <SEP> 6 <SEP> g <SEP> HA <SEP> + <SEP> 320.4 <SEP> g <SEP> BVSM
<tb> + <SEP> 1 <SEP> g <SEP> CTB
<tb> ACTB <SEP> Ag <SEP> + <SEP> CTB <SEP> i <SEP>. <SEP> n <SEP>. <SEP> 3.6 <SEP> g <SEP> HA <SEP> + <SEP> 1 <SEP> g <SEP> CTB
<Tb>

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For each group, blood samples and nasal secretions are taken and analyzed as indicated in the Materials and Methods section.

 2) Results.

 Figure 7 summarizes the results obtained in split B / Harbin specific titration of pools of sera (IgG) and mouse nasal secretions (IgA). It makes it possible to compare the results of the trivalent formulations containing the CTB (F-CTB) with different controls.

These controls are either antigen-only controls, or BTC-associated antigen controls, or the reference formulation (HA / BVSM ratio: 1/89). These results were confirmed by the analysis of the individual response against split B / Harbin and A / Nanchang.

 Expectedly, the reference formulation (FA) induces a level of antibodies of type G equivalent to the control antigen alone administered subcutaneously (AS) and a higher level of IgG than the control alone administered nasally ( AN) (22.4X).

 The BTC-associated antigen-associated controls (ACTB) administered nasally induced significantly higher IgG than the free antigen administered by the same route (22.4X increase). In parallel, these formulations (Ag + CTB) (ACTB) induce a strong mucosal immunity.

 CTB adjuvant formulation (FCTB) induces a specific IgG response that is significantly greater than the reference formulation (FA) and its reference control

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 (ACTB). For this adjuvant, there is a significant synergistic effect between the CTB and the BVSMTM thus, the serum IgG levels obtained are multiplied by a factor of 3.7 compared to the reference formulation FA. Conversely, and in the presence of a significant mucosal adjuvant activity of CTB, with certainly a saturation effect of the biological response, it is difficult at this stage to define the synergistic potential of the IgA response for this adjuvant type. and the BVSMTM.

 IV - Example 3: Effect of MPL on the immunological response of BVSMTM / Influenza formulations.

 1) Protocol.

 A trivalent formulation and the corresponding control are prepared at 250 μg HA / ml (83.3 μg / strain) / split Influenza.

 A solution of MPL is added to each of these preparations in order to obtain the corresponding adjuvant formulations and controls.

 36 female BALB / cJ / Rj mice (10 weeks old at the start of the study) are divided into 6 groups at a rate of 6 mice per group. For each group the treatment is performed as described in the Material and Methods part. Table 2 below summarizes the treatment of each group.

Table 2

<Tb>
<tb> Code <SEP> Group <SEP> Route <SEP> Formule <SEP> Administered
<tb> (number <SEP> of <SEP> mice) <SEP> Quantity / dose
<Tb>

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<Tb>
<tb> C <SEP> Control <SEP> naive <SEP> (6) <SEP> in <SEP> 20 <SEP> l <SEP> PBS <SEP> 80mOsm / kg
<tb> FA <SEP> Ag <SEP> + <SEP> BVSM <SEP> in <SEP> 3.6 <SEP> g <SEP> HA <SEP> + <SEP> 320.4 <SEP> g <SEP > BVSM
<tb> AS <SEP> Ag <SEP> sc <SEP> sc <SEP> 3.6 <SEP> g <SEP> HA
<tb> AN <SEP> Ag <SEP> in <SEP> in <SEP> 3, <SEP> 6 <SEP> HA
<tb> FMPL <SEP> Ag <SEP> + <SEP> BVSM <SEP> + <SEP> MPL <SEP> i <SEP>. <SEP> n <SEP>. <SEP> 3 <SEP>, <SEP> 6 <SEP> g <SEP> HA <SEP> + <SEP> 320.4 <SEP> g <SEP> BVSM
<tb> + <SEP> 5 <SEP> g <SEP> MPL
<tb> AMPL <SEP> Ag <SEP> + <SEP> MPL <SEP> in <SEP> 3 <SEP> g <SEP> HA <SEP> + <SEP> 5 <SEP> g <SEP> MPL
<Tb>

For each group, blood samples and nasal secretions are taken as described in the methods section.

 The analysis of sera and nasal secretions is performed by ELISA.

 2) Results.

 Figure 8 summarizes the results obtained in split B / Harbin specific titration of pooled sera (IgG) and mouse nasal secretions (IgA) administered with the MPL adjuvanted formulations. These results were confirmed by the analysis of the individual response against split B / Harbin and A / Nanchang.

 Split influenza viruses associated with nasally administered BVSM or MPL induce significantly higher serum IgG responses than free antigen administered by the same route (22.4X and 7.3X increase, respectively). In parallel, these formulations induce a strong mucosal immunity.

 The formulation associated with the MPL adjuvant induces a specific IgG response similar to the reference formulation. Conversely, for this adjuvant there is a significant synergistic effect between the MPL and the BVSMTM.

<Desc / Clms Page number 25>

 Thus, the IgA levels obtained are multiplied by a factor of 2.7 with respect to the ref. (Ag / BVSM) (FA).

Thus, unlike the IgG response, a high synergistic potential between MPL and BVSM can be demonstrated for the mucosal response.

 Example 4: Oligonucleotides on the immunological response of BVSM / Influenza formulations.

 1) Protocol.

A trivalent formulation and the corresponding control are prepared at 250 μg HA / ml (83.3 μg / strain) / split Influenza
An oligonucleotide solution (ODN) is added to each of these preparations to obtain the corresponding adjuvant formulations and controls.

 42 female BALB / cJ / Rj mice (10 weeks old at the beginning of the study) are divided into 7 groups at the rate of 6 mice per group. For each group the treatment is performed as described in the Material and Methods part. Table 3 summarizes the treatment of each group.

Table 3

<Tb>
<tb> Code <SEP> Group <SEP> Route <SEP> Formule <SEP> Administered
<tb> (number <SEP> of <SEP> mice) <SEP> Quantity / dose <SEP>
<tb> C <SEP> Control <SEP> naive <SEP> i. <SEP> n. <SEP> 20 <SEP> l <SEP> PBS <SEP> 80mOsm / kg
<tb> FA <SEP> Ag <SEP> + <SEP> BVSM <SEP> in <SEP> 3.6 <SEP> g <SEP> HA <SEP> + <SEP> 320.4 <SEP> g <SEP > BVSM
<tb> AS <SEP> Ag <SEP> sc <SEP> sc <SEP> 3.6 <SEP> g <SEP> HA
<tb> AN <SEP> Ag <SEP> in <SEP> in <SEP> 3.6 <SEP> g <SEP> HA
<tb> FODN <SEP> Ag <SEP> + <SEP> BVSM <SEP> + <SEP> ODN <SEP> in <SEP> 3.6 <SEP> g <SEP> HA <SEP> + <SEP> 320 , 4 <SEP> g <SEP> BVSM
<tb> + <SEP> 1 <SEP> g <SEP> ODN1
<Tb>

<Desc / Clms Page number 26>

<Tb>
<tb> AODN <SEP> Ag <SEP> + <SEP> ODN <SEP> in <SEP> 3 <SEP>, <SEP> 6 <SEP> g <SEP> HA <SEP> + <SEP> 1 <SEP > g <SEP> ODN1
<Tb>

For each group, blood samples and nasal secretions are taken as indicated in the method section.

 The analysis of sera and nasal secretions is performed by ELISA as described previously.

 2) Results.

 Figure 9 summarizes the results obtained in split B / Harbin specific titration of sera pools (IgG) and mouse nasal secretions (IgA) administered with the oligonucleotide adjuvanted formulations. These results were confirmed by the analysis of the individual response against split B / Harbin and A / Nanchang.

 Split influenza virus associated with nasally administered BVSMTM or ODN induce a significantly higher serum IgG response than free antigen administered by the same route (22.4X and 7.3X increase, respectively). In parallel, these formulations induce a strong mucosal immunity.

 The formulation associated with the ODN adjuvant induces an IgG response higher than the reference formulation and its reference control (Ag + ODN). For this adjuvant, there appears to be an additivity of the responses, the IgG titers obtained for the formulation being equal to the sum of the responses obtained for the antigen + ODN1 (AoDN1) and for the reference formulation (Ag + BVSM) (FA) .

<Desc / Clms Page number 27>

 VI - Example 5: the IgG2a / IgG1 balance by adjuvantation of the BVSM / Influenza formulations.

 1) Protocol.

 A trivalent formulation and the corresponding control are prepared at 250 μg HA / ml (83.3 μg / strain) / split Influenza. From the trivalent formulation, three adjuvanted formulations are obtained: BVSM / Ag / CTB: By adding to the trivalent formulation of a solution of CTB.

BVSMTM / Ag / IL2: By adding to the trivalent formulation of a BVSMTM / Ag / MPL recombinant IL-2 solution: By adding to the trivalent formulation of an MPL solution
54 female BALB / cJ / Rj mice (10 weeks old at the start of the study) are divided into 9 groups at the rate of 6 mice per group. For each group the treatment and the samples are carried out as described in the Material and methods part.

 Table 4 below summarizes these treatments.

Table 4

<Tb>
<tb> Code <SEP> Group <SEP> Route <SEP> Formule <SEP> Administered
<tb> (number <SEP> of <SEP> mice) <SEP> Quantity / dose <SEP>
<tb> C <SEP> Control <SEP> naive <SEP> i. <SEP> n. <SEP> 20 <SEP> {it <SEP> PBS <SEP> 80 <SEP> mOsm / kg
<tb> FA <SEP> Ag <SEP> + <SEP> BVSM <SEP> in <SEP> 3 <SEP>, <SEP> 6 <SEP> fig <SEP> HA <SEP> + <SEP> 320.4 <SEP> g <SEP> BVSM
<tb> AS <SEP> Ag <SEP> sc <SEP> sc <SEP> 3, <SEP> 6 <SEP> g <SEP> HA
<tb> Will <SEP> Ag <SEP> + <SEP> BVSM <SEP> + <SEP> CTB <SEP> in <SEP> 3.6 <SEP> g <SEP> HA <SEP> + <SEP> 320 , 4 <SEP> g <SEP> BVSM
<tb> ~~~~~~~~~~ <SEP> + <SEP> 1 <SEP> g <SEP> CTB
<Tb>

<Desc / Clms Page number 28>

<Tb>
<tb> ACTB <SEP> Ag <SEP> + <SEP> CTB <SEP> i <SEP>. <SEP> n <SEP>. <SEP> 3 <SEP>, <SEP> 6 <SEP> g <SEP> HA <SEP> + <SEP> 1 <SEP> g <SEP> CTB
<tb> FMPL <SEP> Ag <SEP> + <SEP> BVSM <SEP> + <SEP> MPL <SEP> i <SEP>. <SEP> n <SEP>. <SEP> 3 <SEP>, <SEP> 6 <SEP> g <SEP> HA <SEP> + <SEP> 320.4 <SEP> g <SEP> BVSM
<tb> + <SEP> 5 <SEP> g <SEP> MPL
<tb> AMPL <SEP> Ag <SEP> + <SEP> MPL <SEP>(<SEP> 6 <SEP>) <SEP> i <SEP>. <SEP> n <SEP>. <SEP> 3 <SEP> g <SEP> HA <SEP> + <SEP> 5 <SEP> g <SEP> MPL
<tb> FIL <SEP> Ag <SEP> + <SEP> BVSM <SEP> + <SEQ> IL-2 <SEP> in <SEP> 3.6 <SEP> g <SEP> HA <SEP> + <SEP > 320.4 <SEP> g <SEP> BVSM
<tb> + <SEP> 0.613 <SEP> g <SEP> IL-2
<tb> AIL <SEP> Ag <SEP> + <SEQ> IL-2 <SEP> in <SEP> 3 <SEP>, <SEP> 6 <SEP> g <SEP> HA <SEP> + <SEP> 0.613 <SEP> g <SEP> IL-2
<Tb>

The analysis of the sera is carried out by ELISA test as described above, using either a murine IgG2a anti or a murine IgG1 anti.

 2) Results.

 Table 5 below summarizes the results obtained in terms of gamma globulin subtype (IgG2a and IgG1) of the various BVSMTM / adjuvanted influenza formulations and the corresponding controls after nasal administration. Compared with the free antigen administered subcutaneously, the nasally administered BVSM / influenza formulation induces a modest increase in specific IgG2a production.

 By comparison, the addition of CTB to the Ag / BVSMTM formulation induces a clear increase in Ig2a production (multiplication by 5.4 of the Ig2a / IgI index versus Ag S.c). It is important to note that this change in the Thl / Th2 balance is not predictable from the results obtained by the association of BTC with antigens. Indeed, in this control group, there is a lowering of the Ig2a / IgG1 index.

Table 5

<Tb>
<tb> Route <SEP> IgG2A <SEP> IgG1 <SEP> Index <SEP> Ratio <SEP> versus
<Tb>

<Desc / Clms Page number 29>

<Tb>
<tb> IgG2a / IgG1 <SEP> Ag <SEP> sc
<Tb>

Control <SEP> naive <SEP> in <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0,0
<tb> Ag <SEP> sc <SEP> sc <SEP> 157 <SEP> 23355 <SEP> 6.7 <SEP> 1.0
<tb> Ag <SEP> in <SEP> in <SEP> 0 <SEP> 9793 <SEP> 0.0 <SEP> 0.0
<tb> Ag + BVSM <SEP> in <SEP> 572 <SEP> 65699 <SEP> 8.7 <SEP> 1.3
<tb> Ag + CTB <SEP> in <SEP> 87 <SEP> 44771 <SEP> 1.9 <SEP> 0.3
<tb> Ag + BVSM + CTB <SEP> in <SEP> 2506 <SEP> 68877 <SEP> 36.4 <SEP> 5.4
<tb> Ag + MPL <SEP> in <SEP> 143 <SEP> 3003 <SEP> 47.6 <SEP> 7.1
<tb> Ag + BVSM + MPL <SEP> in <SEP> 101 <SEP> 10561 <SEP> 9.6 <SEP> 1.4
<tb> Ag + IL-2 <SEP> in <SEP> 0 <SEP> 3234 <SEP> 0.0 <SEP> 0.0
<tb> Ag + BVSM + IL-2 <SEP> in <SEP> 26 <SEP> 24952 <SEP> 1.0 <SEP> 0.2
<Tb>

Conversely, MPL which, when combined with split influenza, favors IgG2a production (index 47.6 versus 6. 7 for Ags c) only leads to a minor modification of the Thl / Th2 balance after association. with Ag / BVSM formulations.

 Finally, the IL2 association with Ag / BVSM formulations makes it possible to modify the Th1 / Th2 balance as we can see it on the lowering of the IgG2a / Igl index.

 Thus, even in the absence of a quantitative modification of the immunological response, the combination of adjuvant with the Antigen / BVSM formulations makes it possible to induce a qualitative change in the immunological response. This association should allow by a correct choice of the adjuvant used to adapt the balance Thl / Th2 to the proposed vaccine strategy.

Claims (22)

1) Process for improving the immunomodulatory properties of a substance, other than an antigen, capable of modulating the immune response, characterized in that it comprises mixing said substance with hydrophilic particles bearing or not ionic ligands and optionally covered with a layer of amphiphilic compounds.  2) Process according to claim 1, characterized in that the substance / particle weight ratio is between about 1% and 20%, preferably between about 5% and 10%.  3) Method according to one of claims 1 or 2, characterized in that the substance, other than an antigen, capable of modulating the immune response is a protein of therapeutic interest that plays a role in the functioning of the immune system.  4) Method according to claim 3, characterized in that the protein of therapeutic interest is a cytokine or a chemokine, preferably selected from the group comprising interleukins, interferons, TNF, TGF, G-CSF, GM-CSF, MIF, RANTES, or a mixture of these.  5) Method according to one of claims 1 or 2, characterized in that the substance, other than a <Desc / Clms Page number 31>  antigen, able to modulate the immune response is an adjuvant.  6) Process according to claim 5, characterized in that the adjuvant is chosen from the group comprising bacterial enterotoxins, liposaccharide derivatives, oligonucleotides, saponins, ammonium salts and their derivatives, DT type proteins. or TT or a mixture thereof.  7) Method according to one of claims 1 or 2, characterized in that the substance, other than an antigen, capable of modulating the immune response is a substance capable of modifying the balance Thl / Th2.  8) Method according to one of claims 1 or 2, characterized in that the substance, other than an antigen, capable of modulating the immune response is an immunosupressor.  9) Use of hydrophilic particles bearing or not ionic ligands and optionally coated with a layer of amphiphilic compounds for the preparation of a medicament for the treatment of cancers and / or viral diseases, said particles being associated in the medicament with least one substance, other than an antigen, capable of modulating the immune response. <Desc / Clms Page number 32>  10) The use according to claim 9, characterized in that the substance, other than an antigen, capable of modulating the immune response is a protein of therapeutic interest which plays a role in the functioning of the immune system or an adjuvant, or a mixture of these.  11) The use according to claim 10, characterized in that the protein of therapeutic interest is a cytokine or a chemokine, preferably chosen from the group comprising interleukins, interferons, TNFs, TGFs, G-CSF, GM-CSF, MIF, RANTES, or a mixture of these.  12) Use according to claim 10, characterized in that the adjuvant is chosen from the group comprising bacterial enterotoxins, liposaccharide derivatives, oligonucleotides, saponin derivatives, ammonium salts and their derivatives, proteins of type DT or TT, or a mixture thereof.  13) therapeutic composition characterized in that it comprises hydrophilic particles with or without ionic ligands and optionally coated with a layer of amphiphilic compounds, and at least one protein of therapeutic interest and / or an adjuvant, or a mixture of them. <Desc / Clms Page number 33>  14) Therapeutic composition according to claim 13, characterized in that the protein of therapeutic interest is a cytokine or a chemokine, preferably selected from the group comprising interleukins, interferons, TNF, TGF, G-CSF, GM-CSF, MIF, RANTES, or a mixture thereof.  15) Therapeutic composition according to one of claims 13 or 14, characterized in that the adjuvant is selected from the group comprising bacterial enterotoxins, liposaccharide derivatives, oligonucleotides, saponins, ammonium salts and derivatives thereof , DT or TT type proteins or a mixture thereof.  16) Use of hydrophilic particles bearing or not ionic ligands and optionally coated with a layer of amphiphilic compounds for the preparation of a vaccine composition, said particles being mixed in the composition with at least one antigen and at least one substance capable of modulate the immunological response to said antigen.  17) Use according to claim 16, characterized in that substance capable of modulating the immunological response of the antigen is an adjuvant.  18) Use according to claim 17, characterized in that the adjuvant is selected from bacterial enterotoxins, saponin derivatives, <Desc / Clms Page number 34>  ammonium salts and derivatives thereof, DT or TT type proteins, cytokines, oligonucleotides, or a mixture thereof.  19) Use according to any one of claims 16 to 18, for the preparation of a vaccine composition for the treatment and / or prevention of viral diseases, including AIDS and chronic hepatitis, or cancers.  20) Vaccine composition characterized in that it comprises an antigen or a mixture of antigens, hydrophilic particles with or without ionic ligands and optionally coated with a layer of amphiphilic compounds, and at least one substance capable of modulating the response immunological to said antigen.  21) Vaccine composition according to claim 20, characterized in that substance capable of modulating the immunological response of the antigen is an adjuvant.  22) Vaccine composition according to claim 21, characterized in that the adjuvant is selected from bacterial enterotoxins, saponin derivatives, ammonium salts and derivatives thereof, DT or TT type proteins, cytokines, oligonucleotides , or a mixture of these.