FR2850872A1 - Composition containing an antigen of human immune deficiency virus, for use as a vaccine, comprises multilamellar vesicles with an internal liquid crystal structure - Google Patents

Abstract

A composition (A) comprises a dispersion or suspension of multilamellar vesicles (B) having an internal liquid crystal structure, where (B) are formed by stacking concentric bilayers, based on an amphiphilic agent (I), alternating with layers of water, aqueous solution, polar liquid or a solution of polar liquid, and they contain an antigenic protein (II) of human immunodeficiency virus (HIV). A pharmaceutical composition (A) comprises a dispersion or suspension, in a vehicle, of multilamellar vesicles (B) having an internal liquid crystal structure, where (B) are formed by stacking concentric bilayers, based on an amphiphilic agent (I), alternating with layers of water, aqueous solution, polar liquid or a solution of polar liquid, and they contain an antigenic protein (II) of human immunodeficiency virus (HIV). An independent claim is also included for a method for preparing (A). ACTIVITY : Anti-HIV. MECHANISM OF ACTION : Vaccine, particularly induction of a Th1-type response. Mice were vaccinated by administration to the nasal mucosa of 3 Microg HIV p24, formulated in vesicles made of macrogol oleate and soya lecithin, twice at an interval of 21 days. Three weeks after the second injection, serum antibodies were determined by enzyme-linked immunosorbent assay. Titers were over 1000 for immunoglobulin (Ig) G and about 60 for IgA; compare about 130 and 1, respectively, when the same amount of p24 was administered as nasal solution.

Description

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  The present invention relates to a vaccine pharmaceutical composition for inducing an immune response against the AIDS virus.

  More specifically, the invention relates to a vaccine composition for administering an antigen of the human immunodeficiency virus (HIV) and for amplifying and orienting the immune response induced against this antigen by both injection and mucosal administration.

  First of all, here are some notions and definitions.

  The immune system provides protection for the body against pathogens and exogenous agents. It establishes a humoral immunity composed of serum antibodies divided into different subclasses or isotypes and cellular immunity. The cellular component is characterized by the activation of cytotoxic TCD4 + and TCD8 + lymphocytes (CTL) and NK cells. It is evidenced by lymphoproliferation, secretion of interleukins and / or lysis of infected or tumor cells. Interleukins (IL) are soluble mediators that act autocrine or paracrine on different cell types of the immune system. CD4 + T cells can be classified into two types according to the profile of interleukins they secrete. Thus, type 1 or Th1 lymphocytes secrete IL-2 and interferon gamma while type 2 or Th2 lymphocytes secrete IL-4, IL-5 and IL-10. The mode of presentation of the antigen to antigen-presenting cells (dendritic cells and macrophages) will also condition early the type of response induced. Thus, the dose, frequency, mode of presentation and route of administration affect the immune response.

  The antigens can be administered by parenteral, systemic or mucosal immunization.

  Mucosal is a non-invasive administration of the antigen to a mucosal site, for example - nasopharyngeal area - oral area bronchial tree - intestine - urogenital tract - conjunctive internal ear - mammary, salivary and lacrymal glands. Among these various constituents of the mucosal-associated lymphoid tissue (MALT), some routes of introduction are of easier access and acceptability: nasal, oral, gastrointestinal, rectal, and vaginal.

  The mucosal response is the mucosal response of the immune system characterized by IgA (isotype at high mucosal concentration) and IgG production and / or cell response within the mucosal site and draining lymph nodes. .

  The systemic response is the response of the generalized immune system resulting in the presence of circulating antibodies (IgG divided into different subclasses and also IgA) and / or by a cellular response (Thelper or CTL) in the secondary lymphoid organs (spleen , 25 lymph nodes).

  An adjuvant is a substance added to the antigen so as to amplify and guide the specific immune response directed against that antigen.

  Vectorization by a synthetic vector consists of incorporating the antigen into and / or on the surface of a particle or vesicle, so as to protect the antigen and / or to facilitate its capture by the competent cells of the immune system and / or or to facilitate the processing of the antigen by said cells, and thus to amplify the immune response. The addition of an empty vector to the free antigen generally provides little or no amplification.

  Human immunodeficiency virus (HIV) antigens are polyproteins or proteins encoded by the HIV genome. Nine genes encode polyproteins or structural proteins, proteins with enzymatic activity: proteases or proteins involved in integration, reverse transcription and replication, and regulatory proteins for this activity.

  These proteins can be glycosylated (glycoproteins).

  For the purposes of the present invention, the term "HIV protein antigen" means all the antigens of the HIV virus in the form of proteins or polyproteins, glycosylated or otherwise.

  AIDS is a scourge that affects more than 30 million people and for which there is no vaccine, prophylactic or therapeutic. However, given the very high prevalence of this disease in the lowest income countries, the vaccine appears to be the only effective means of control, antiviral therapies being prohibitively expensive.

  The difficulties of developing a vaccine against the human immunodeficiency virus are mainly related to the rapidity of mutation of the virus. The various studies on people who are resistant to HIV infection or who are infected with this virus, but who do not develop the disease, have suggested that the simultaneous presence of neutralizing antibodies (systemic IgG antibodies and mucosal IgA antibodies) and induction cytotoxic T lymphocyte responses are the major effectors of anti-viral immunity. The induction of a type 1 TCD4 + response is also a favorable factor in the selection or expansion of cytotoxic cell populations. The orientation of the immune response towards these components is therefore a serious lead for the development of an HIV vaccine. Several antigens are currently used for these developments. The best known are the GP160 type envelope proteins or its GP120 or GP41 derivatives (see, for example, WO 92/06113 by Smithkline Beecham Biologicals), the capsid protein encoded by the GAG motif, and its derivatives such as P24. Or P17 or fusion proteins therebetween (see WO 90/03735 of Pharmacia Genetics Engineering Inc), as well as Nef and Tat proteins modified to lose their regulatory character and their derivatives or fusion proteins (cf. WO 01/00232 to Smithkline Beecham Biologicals).

  One of the technical difficulties in developing these vaccines is obtaining a vehicle capable of protecting the antigenic protein, allowing its administration, and stimulating the immune system to induce an effective immune response. This vehicle must therefore have sufficient adjuvant properties, and be able to direct the response to the most favorable profile.

  Today, the only adjuvants accepted in human medicine are mainly aluminum hydroxide or phosphate. These salts are in the form of a suspension of grains of aluminum salt, on the surface of which the antigen is adsorbed. They have several disadvantages: they induce inflammatory local reactions and IgE production, they are not effective for all antigens and they are incapable of generating CTL type cell-mediated reactions. In addition, recent fears have arisen about the possibility of local toxicity of aluminum-based adjuvants, which has led to restrictions on their use in the development of new vaccines.

  Finally they are not usable in a nasal administration. A substitute for aluminum salts is calcium phosphates, which have recently regained interest, given concerns about the use of aluminum.

  There are numerous documents outlining strategies for developing AIDS vaccines, using the antigens mentioned above, and various adjuvant systems. In addition to the aluminum salts used in MicroGeneSys Inc. applications EP 0.327.180 and WO 92/22654, the main adjuvants are detoxified toxins, for example the heat-sensitive endotoxin of E. coli (cf. WO 98/32461 to The Administrators of the Tulane Educational Fund), or modifications of lipid A, for example 3D Mpl (monophosphoryl 3 deacylated lipid A) as described in WO 92/06113. Many other adjuvants have been developed, such as saponin derivatives, muramyl dipeptide, detoxified tetanus toxins or cholera toxins, but few documents describe their use in vaccines directed against AIDS.

  More recently, new adjuvants have appeared, such as DCChol (obtained by reaction of choloroformate on N, N20 dimethylethylenediamine according to WO 96/40067) in admixture with one of the proteins mentioned above (see WO 02 / 28427 of Aventis Pasteur). Another recent approach is the use of oligonucleotides having a CpG sequence, known for its ability to amplify and guide the immune response. They are described for an application against the AIDS virus in WO 01/00232. Their mode of action is different from more conventional adjuvants since they can be administered before the immunization itself.

  Finally, it can be noted that in certain patent applications, several adjuvants are used simultaneously in order to benefit from a synergistic effect. This is the case of WO 01/00232 which couples an adjuvant based on aluminum and a CpG oligonucleotide. In other applications, the adjuvant and / or the antigen are formulated in the form of an emulsion, generally of oil-in-water type (see WO 92/06113 and WO 02/28427).

  There is little literature teaching the use of liposomes or other lipid-based vesicles or amphiphiles coupled to HIV proteins in an AIDS vaccine strategy. The use of liposomes is cited in WO 02/28427 but the reading of Example 8 teaches that only DC-Chol adjuvant is incorporated into the liposomes. WO 99/00142 teaches the use of lyophilized liposomes for orally administering a viral protein, particularly HIV.

  WO 96/40243 from the US Department of the Army teaches the use of liposomes comprising GP120 protein and lipid A to induce an immune response. The bibliographic part of this document demonstrates, with the aid of numerous citations, that the use of liposomes to amplify an immune response against a given antigen is not obvious, and that no predictive tool exists to anticipate the result. .

  In an infection such as HIV, the route of administration of the vaccine certainly plays an important role. The pathogen having a mucosal entryway, the generation of IgA antibodies, specific for mucosal tissue, and moreover, at the level of the urogenital mucosa is a characteristic sought in a prophylactic vaccine against AIDS.

  Most of the work presented in the cited applications uses the injectable route, although other routes of administration are described in the text of the applications. WO 99/00142 describes and teaches only the oral route, while WO 02/28427 proposes in addition to the injectable route a mucosal (nasal) route. It is known that systems based on aluminum or calcium salts can not be used in mucosal administration because of their local intolerance to nasal mucosa and in particular to ciliate function.

  None of the antigen delivery or adjuvant technologies has yet resulted in an AIDS vaccine on the market. The methods of presenting the antigen must therefore be further improved and the development of new adjuvants or antigen vectors remains an urgent need in the development of an HIV vaccine.

  Of the vesicles, spherical objects formed from molecular arrangements of amphiphilic molecules, multilamellar vesicles with onion structure have been the subject of extensive research and have given rise to several patents (WO 93/19735; WO 95/18601; WO 97/00623, WO 98/02144, WO 99/16468). They are distinguished from liposomes by: their mode of preparation which starts from a lamellar phase to the thermodynamic equilibrium their internal crystal-liquid structure formed of a regular stack of concentric bilayers of amphiphiles alternating with layers of water, polar liquid, aqueous solution or solution of a polar liquid (glycerol, for example)> the varied nature of the amphiphilic molecules that can be used to constitute them, alone or as a mixture.

  The international application WO 99/16468 describes vesicles with onion structure incorporating within them an antigen and for amplifying the immune response of this antigen. This international application, which for the first time describes the incorporation (also referred to herein as encapsulation) of antigens into a multilamellar vesicle with an onion structure, makes it clear in its examples that such an encapsulation allows very clearly to amplify the immune response during parenteral administration of an antigen encapsulated in a multilamellar vesicle with onion structure.

  International patent application WO 01/19335 has also described a new mode of mucosal administration of an antigen incorporated into the same type of multilamellar vesicle and demonstrated that this type of mucosal administration makes it possible to generate an immune response not only within the different mucosal sites but also in the systemic compartment.

  The inventors of the present invention have now discovered that the subcutaneous administration of a composition comprising an HIV protein antigen incorporated into vesicles of the type described in WO 99/16468, elicits an immune response. directed against this antigen and that in addition, this response is oriented, with a significant modification of the distribution of antibody subclasses in favor of IgG2a antibodies. In addition, the administration of the same composition by the mucous route induces the induction of both IgG antibodies, but also of HIV-specific IgA in the systemic compartment and in different mucosal sites. In the following description and examples, it has been found that compositions in which the HIV antigen is incorporated into an onion-structured vesicle as defined above, when administered mucosally to generate a delocalized mucosal IgA immune response (pulmonary, intestinal and vaginal) and thus to stimulate the common mucosal lymphoid tissue. Such capabilities are of particular importance when addressing vaccinating potential antigens against invasive pathogens with mucosal tropism such as HIV, whose gateway is precisely mucosal.

  The dual possibility of inducing both a mucosal response and a systemic response is of great interest in vaccination because it simplifies administration while offering immunity at several levels: mucosal to defend the access routes of the virus and systemic 30 to fight more disseminated or generalized infection.

  This is a particularly remarkable advantage of the present invention.

  On the other hand, the finding that the compositions of the invention modify isotype to an IgG2a response suggests orientation of the cell response to a type 1 profile (polarization to IgG2a being interferon dependent secretion dependent). , y). This orientation is a particular advantage of the compositions of the invention since this profile is particularly sought after when it comes to fight against viruses and in particular against HIV.

  On the other hand, the multilamellar vesicles used according to the invention can be prepared from biocompatible constituents known for their safety.

  In addition, the preparation method is simple to implement and only uses current devices of chemistry. The fact that the process uses an initial lamellar phase at thermodynamic equilibrium gives it an excellent reproducibility and allows a high stability of the vesicles obtained.

  Thus, according to one of its essential features, the invention relates to a pharmaceutical composition containing a dispersion or a suspension in a pharmaceutically acceptable vehicle of multilamellar vesicles with a liquid-crystal internal structure, formed of a stack of concentric bilayers based on amphiphilic agents alternating with layers of water, aqueous solution, polar liquid or solution of a polar liquid and in which at least one protein antigen of the HIV virus is incorporated.

  By the term "incorporated" whose use seems to us preferable to that of the term "encapsulated" is meant that the antigen or antigens are an integral part of the entity constituted by the vesicle. Indeed, one can find antigen molecules (s) in any layer 30 between the center and the periphery of said vesicle.

  As is apparent from the following detailed description as well as examples, the pharmaceutical compositions according to the invention can be used as a vaccine intended to induce a systemic or mucosal response.

  As is clear from the examples, the onion-structured vesicles as defined previously incorporating an HIV antigen induce, in humans or animals, the production of specific antibodies directed against HIV and this, whether by injection or by mucous administration, in particular nasal.

  According to another of its essential characteristics, the invention relates to a method for manufacturing the compositions of the invention, comprising the preparation of vesicles with a liquid-crystal structure by transformation of a liquid-crystal phase containing at least one amphiphilic agent and at least one minus one HIV protein antigen and dispersing said vesicles in a pharmaceutically acceptable carrier.

  The vesicles used according to the invention generally have diameters of between 0.1 and 25 μm, preferably between 0.2 and 15 μm.

  More specifically, the vesicles contained in the compositions of the invention consist of several layers of amphiphilic agents alternating with layers of aqueous or polar phase. The thickness of each of these layers is a molecular thickness, typically of the order of 5 to 10 nanometers. For a stack of ten to a few hundred layers, a diameter of between 0.1 μm and a few tens of micrometers is thus obtained. This is observed experimentally, the vesicles being observable by optical microscopy (in polarized light in order to have a better contrast related to their birefringence), or as unresolved points for the smaller ones of them. as birefringent spheres for larger ones. The size profile can be studied using a laser granulometer (using the static scattering of a laser beam, it is analyzed from several angles). In general, a Gaussian profile centered on a value varying between 0.1 and 25 μm is obtained, showing a low heterogeneity of size for a given formulation, under given preparation operating conditions.

  The vesicles in which the antigen is incorporated have, as previously stated, a multilamellar structure in onion and consist, from their center to their periphery, of a succession of lamellar layers separated by a liquid medium. These vesicles may be obtained by a process comprising the preparation of a liquid-crystal lamellar phase and its shearing transformation. Such a process is in particular described in the patent WO 93/19735 issued from the French patent FR-2,689,418 or WO 95/18601.

  According to the French patent FR-2,689,418, this transformation can be done during a homogeneous shear step of the crystalline phase, which leads to vesicles also called microcapsules of controlled size. However, by acting on the formulation of the liquid-crystalline lamellar phase, in particular on the nature of the surfactants used in its composition, the transformation of this crystalline phase into vesicles can be obtained by simple mechanical stressing, in particular when mixing the components.

  Such vesicles have, among others, the advantage of being able to be prepared by a particularly simple preparation process making it possible to use a wide variety of surfactants.

  Another advantage, also essentially related to the process used to prepare the onion-structure vesicles used according to the invention, is that the active ingredients and additives are incorporated prior to the formation of the vesicles, which allows excellent encapsulation efficiency, o better efficiency and a very important saving for extremely expensive molecules.

  Such structures are advantageously obtained by incorporating at least one HIV protein antigen in a crystalline lamellar phase comprising at least one surfactant then transformation of this lamellar liquid crystal phase into a dense phase of small multilamellar vesicles.

  Thus, the vesicles used according to the invention can be obtained according to a process according to which a lamellar crystalline phase incorporating at least one antigen is prepared and the rearrangement of said liquid-crystal phase in multilamellar vesicles 10 is obtained by application of shear.

  This shear may be a homogeneous shear, which has the advantage of leading to vesicles of perfectly uniform size. However, simple mechanical agitation may be sufficient to lead to the formation of the multilamellar vesicles of the invention.

  The antigen may be any protein antigen of the HIV virus.

  It may be both an antigen in the form of a protein or a polyprotein, glycosylated or not.

  It may be in particular a protein or a glycoprotein called structural protein, derived from the envelope of the virus or its nucleocapsid or a regulatory or enzymatic protein or any protein derived from this type of protein. .

  The antigen may in particular be an HIV virus envelope protein, in particular a GP 160 protein or a GP 120 or GP 41 derived protein encoded by the env gene.

  The antigen may also be an enzymatic protein involved in the integration, transcription, replication of the virus and its genetic material.

  It may also be a regulatory protein for transcription or replication.

  It may also be a protein of the HIV virus capsid encoded by the gag gene or a protein derived from such a protein such as P 24 or P 17 protein or a fusion protein.

  An example of such a preferred protein according to the invention is the P24 protein.

  The multilamellar vesicles with onion structure are prepared according to the methods described above, in particular in the international application WO 99/16468. The amphiphilic molecules used for their preparation will be selected, without this being an obligation, among the molecules described in the pharmacopoeia, or already used in mucosal or parenterally injected drugs.

  According to an advantageous variant, the bilayers of the vesicles contained in the compositions of the invention contain at least one surfactant chosen from the group consisting of: hydrogenated or non-hydrogenated phospholipids, saturated C 6 to C 30 fatty acid esters and mono- or polyunsaturated, linear or branched, and macrogol (polyethylene glycol), ethoxylated or non-ethoxylated esters, linear or branched, saturated or mono- or polyunsaturated C6 to C30 fatty acids, and> sucrose sorbitan, mannitol, glycerol or polyglycerol, glycol, linear or branched C6 to C30 fatty alcohol ethers, saturated or mono- or polyunsaturated, and macrogol (polyethylene glycol) ethers, ethoxylated or non-ethoxylated, linear or branched, saturated or mono- or polyunsaturated C 6 to C 30 fatty alcohols, ethoxylated or not, and> sucrose,> sorbitan,> mannitol,> glycerol or polyglycerol , At these surfactants, which can be used alone or as a mixture, co-surfactants may optionally be added in order to improve the rigidity and / or the tightness of the bilayer forming the vesicle. Among these molecules, mention may be made of: cholesterol and its derivatives, in particular charged or neutral cholesterol esters such as cholesteryl sulphate; other derivatives with a sterol backbone, in particular those of plant origin commonly called phytosterols (sitosterol, sigmasterol ...) - ceramides.

  The formulation advantageously involves a mixture of surfactant molecules. It is generally used at least two different surfactants having different hydrophilic-lipophilic balance, which allows to continuously adjust the properties of the bilayers and thus to control the appearance of instability that governs the formation of multilamellar vesicles.

  Thus, the surfactants above will advantageously be chosen from two surfactants having relatively different properties, in particular a different hydrophilic-lipophilic balance (HLB). The first surfactant will advantageously have a hydrophilelipophilic balance of between 1 and 6, preferably between 1 and 4, while the second surfactant will have a hydrophilelipophilic balance greater than 3, preferably greater than 4.

  According to a particularly advantageous variant of the invention, the amphiphilic agents used for the preparation of the vesicles according to the invention will consist of a mixture of a phospholipid and a nonionic surfactant.

  The nonionic surfactant will advantageously be a fatty acid ester, preferably a fatty acid ester of polyethylene glycol or sorbitan or a polysorbate; the phospholipid will, for its part, advantageously a phosphatidylcholine.

  According to a particularly preferred variant of the invention, the constituent amphiphiles of the vesicles will consist of a mixture of phosphatidylcholine and macrogol oleate (polyethylene glycol oleate) or a mixture of phosphatidylcholine and polysorbate 80.

  The preparation obtained after transformation of the lamellar liquid-crystal phase into multilamellar vesicles may then be diluted, in particular with an aqueous solvent such as, for example, a buffer solution, saline solution or physiological solution, thereby obtaining a suspension aqueous vesicles.

  The encapsulation technique used according to the present invention makes it possible to easily reach very high encapsulation yields, even close to 100%. However, such yields are not always essential depending on the applications concerned.

  Thus, the encapsulation efficiency of the antigen (s) in the compositions of the invention is advantageously greater than%, preferably greater than 80%. It seems that the structure of the vesicles is responsible for the results

  The multilamellar vesicles of this invention are particularly advantageous in that they allow the antigen to arrive intact to the antigen presenting cells (APCs) and promote its capture by these cells or their activation. It therefore seems that the function of the vesicles of the invention is to vectorize, protect and improve the capture of the antigen by the immune system.

  Another advantage of the technology is that one can add to this formulation natural or artificial polymers such as polysaccharides (alginates, chitosan, etc.) in order to reinforce the solidity of the vesicle, and allow it to stay longer on the site of administration or in the body, thereby delivering the antigen over a longer time. These polymers can be incorporated into the vesicle as well as deposited around in the form of a coating. In this case the vesicle or particle formed vesicles embedded in the polymer matrix has a diameter greater than that of the vesicles alone. These polymers may optionally be crosslinked to further enhance their strength.

  Moreover, and this is one of the other advantages of the technology, the formulation can be supplemented by the addition of immunomodulatory molecules (chitosan, interleukins, etc.) which, by their intrinsic properties, will enhance amplification and / or orientation of the immune response.

  The vesicles incorporating the antigens are advantageously prepared in a process consisting of preparing, initially, the lamellar phase. This is achieved by simple mixing of the ingredients in an order determined by the experimenter based on the miscibility of each of the components. It may be necessary to heat some pasty or solid constituents to facilitate their incorporation. In this case, the antigen is preferably added at the end of the mixture, after cooling to prevent it from being subjected to too high a temperature. A mixture of all the components except the antigen or its aqueous solution can also be prepared in the form of a "stock" mixture which will be used as needed to prepare the lamellar phase. The aqueous solution may contain various constituents intended to ensure its biological compatibility and in particular buffer mixtures but also various antigens. The lamellar phase thus prepared is then subjected to moderate shear (from 0 to 1000 s -1) for a limited time (from 0 to 60 minutes).

  In most cases, this shear is obtained directly by the action of the device used for mixing. For very small quantities, it can be obtained by hand by mixing the preparation for example with a microspatule in a 1.5 ml plastic conical tube.

  The sheared lamellar phase is then dispersed in a final medium, generally water or a buffer, identical or different from that used in the preparation of the lamellar phase. This dispersion is advantageously at room temperature (20-250C) by slow addition of the medium to the lamellar phase with constant stirring.

  A preservative and optionally other additives to supplement the dosage formulation may be added to the product.

  The pharmaceutical compositions described above constitute vaccine compositions formulated for both parenteral and mucosal administration, in particular nasally.

  All the compositions described above comprising at least one HIV protein antigen incorporated into the vesicles with onion lamellar structure have the advantage that they can be used for mucosal administration, and especially nasally, and to induce a response. mucosa and / or systemic.

  The invention also relates to a vaccination method for immunizing a human being against the AIDS virus by parenteral or mucosal administration, in particular by the nasal route of a pharmaceutical composition as defined above, with the advantages exposed. previously, particularly with the possibility of inducing a THi-type immune response.

  According to a last aspect, the invention relates to the use of multilamellar vesicles incorporating at least one protein antigen of the HIV virus, as described above, for the manufacture of a vaccine composition intended for immunization against the virus responsible for AIDS. .

  In such a use, the administration can be carried out both parenterally and mucosally, in particular nasally, with all the advantages described above.

  The examples below illustrate in a nonlimiting manner the invention.

  More specifically, Example I given below clearly demonstrates such an effect by nasal administration of the HIV P24 protein. Example II illustrates a subcutaneous immunization method using HIV P24 protein.

  Figures 1 to 4 given with reference to Example I show the titers obtained after nasal administration of the free protein and the protein incorporated in the vesicles of the invention as well as the standard deviations (denoted SD):> FIG. serum circulating P24-specific IgG titres, after nasal administration> Figure 2, serum circulating P24-specific IgA titres, following nasal administration> Figure 3, P24-specific IgG antibody titers , present in bronchoalveolar lavages (BAL), in vaginal washings (LV) and in intestinal washes (LI), after nasal immunization> FIG. 4, the P24-specific IgA antibody titers present in the broncho washings alveolar (BAL), in the vaginal wash (LV) and in the intestinal wash (LI), after nasal administration FIGS. 5 and 6 given with reference to Example II show the titers obtained after subcutaneous immunization with the free P24 protein and with the protein incorporated in the vesicles of the invention: FIG. 5, the P24 specific IgG1 antibody titers, as well as the standard deviations in the sera after subcutaneous immunizations.

  Figure 6, P24 specific IgG2a antibody titers, as well as standard deviations in sera after subcutaneous immunizations.

EXAMPLES

  EXAMPLE 1 PRODUCTION OF SPECIFIC ANTIBODIES OF HIV-1 P24 FOLLOWING MUCENT ADMINISTRATION I-Preparation of vesicles containing HIV-i antigen P24 Formulation (in percentage by mass) O Macrogol oleate (Simulsol 2599, SEPPIC): 8.0% * Soya lecithin Phospholipon 90G (NATTERMANN): 57.0% 20 * HIV-1 P24 (TEBU) at 1 mg / ml in sodium phosphate buffer pH8: 35.0% Procedure The components are sterilized by UV irradiation for min. The containers and accessories (spatulas, stirrers, etc.) are flame sterilized just before use.

  Constituents O and O are introduced into a plastic tube of 1.5 ml, in any order, mixed with the spatula and then heated at 370C for 60 min in the oven. One component is introduced at room temperature in one step. The whole is homogenized using a spatula sterilized and left to rest overnight at 40C.

  The preparation is then dispersed at 28.46% in sodium phosphate buffer at pH 8.

  II - Nasal immunization protocol In order to test the effect of the vesicles according to the invention during mucosal administration, 7-week-old female BALB / c mice (n = 7) were twice nasally administered (at 30 and 21), various preparations described below. Nasal administration was carried out after anesthesia of the animals with a solution, a mixture of Ketamine, Valium and Atropine, injected intraperitoneally. Three weeks after the last immunization, the animals are sacrificed, the sera are collected and the mucosal secretions are taken, except for the vaginal washings that are taken from the living mouse during the four days preceding the end of the experiment. .

  Immunization Groups The mice were divided into 2 groups marked I and II, subjected to an immunization protocol as defined above by means of the following products: encapsulated HIV-1 group P24: 15 μl per dispersion nostril vesicles according to the invention HIV-1 P24 which corresponds to 3 μg of HIV-1 P24 per mouse at each immunization> group II: HIV-1 P24: 15 μl per nostril of a corresponding HIV-1 P24 solution at 3 μg per mouse at each immunization Samples secretions All samples are made with cold solutions (40C) and are stored on ice as and when to limit degradation by proteolytic enzymes.

  Broncho-alveolar lavage: The trachea of the mice is cannulated with a probe and 750 μl of PBS are injected slowly so as not to create haemorrhage, the lungs are washed 3 times with the same solution, is then centrifuged to remove the lung cells and separated into 10 aliquots stored at -201C until the assay.

  Vaginal washings These samples are made on the vigilant mouse, by injecting 50 μl of PBS into the opening of the vagina, the vagina is washed 3 times with the same solution. This type of sampling is repeated for 4 consecutive days in order to cover the variations due to the hormonal cycle of the mouse. Secretions are grouped and stored at -200C.

  Intestinal lavage The intestine is removed, released from the mesentery and rinsed in water to remove external blood. It is then cut longitudinally and incubated on ice in 1 mil of a protease inhibitor-enriched wash solution. The whole is centrifuged and the supernatant is recovered and frozen at -200C.

  Antibody assay The HIV-1 P24 specific antibodies present in the sera and secretions are assayed by ELISA technique in which the IgA and IgG specific for HIV-1 P24 (biotinylated anti-IgA (Jackson laboratories) / Streptavidin peroxidase are determined. anti-IgG peroxidase (Pasteur Diagnosis)). The results are expressed as a geometric mean of the antibody titers with respect to a reference pool (sampling of 30-nave mice). The antibody titre corresponds to the inverse of the dilution greater than or equal to the reference threshold (average values of naves + 2SD mice).

  III-Results The results of the determination of the specific antibodies are presented in the 4 figures (FIGS. 1 to 4) respectively illustrating> FIG. 1, the titres of P24-specific IgG antibodies, circulating in the serum, after nasal administration> FIG. titers of P24-specific IgA antibodies, circulating in the serum, after nasal administration> FIG. 3, the P24-specific IgG antibody titers, present in the bronchoalveolar lavages (BAL), in the vaginal washings (LV) and in the intestinal washes (LI), after nasal administration> FIG. 4, the P24-specific IgA antibody titers, present in the bronchoalveolar lavages (BAL), in the vaginal washings (LV) and in the intestinal washes (LI), after nasal administration Example I demonstrates the ability of the invention to induce antibodies specific for the HIV virus. The incorporation of the viral antigen and its nasal administration make it possible to amplify the serum IgG and IgA responses relative to the free antigen (amplification of a factor of 100 and 500, respectively for IgG and IgA).

  Only the P24 antigen does not induce mucosal responses. Incorporation into the vesicles of the invention allows the induction of local immunity (IgA and IgG) in both the lungs and the vagina.

  EXAMPLE II PRODUCTION OF SPECIFIC ANTIBODIES OF HIV-1 P24 following subcutaneous administration

ORIENTATION OF THE ANTIBODY RESPONSE

  I - Preparation of vesicles containing HIV-1 antigen P24 Formulation (in percentage by weight) O Macrogol oleate (Simulsol 2599, SEPPIC): 8.0% O Soy lecithin Phospholipon 90G (NATTERMANN): 57.0% * HIV-1 P24 (TEBU) at 1 mg / ml in sodium phosphate buffer at pH 8: 35.0% Procedure The components are sterilized by UV irradiation for min. Containers and accessories (spatulas, stirrers ...) are flame sterilized just before use.

  Constituents O and O are introduced into a 1.5 ml plastic tube, in any order mixed with the spatula and then heated at 370C for 60 min in the oven. The component O is introduced at ambient temperature in one step. The whole is homogenized using a sterilized spatula and then left standing overnight at 41 ° C.

  The preparation is then dispersed at 5.7% in sodium phosphate buffer at pH 8.

  II-Immunization Protocol Subcutaneous In order to test the effect of the vesicles according to the invention during parenteral administration, female BALB / c mice (n = 7) of 7 weeks were subcutaneously administered at the base of the tail, twice (on D0 and D21), various preparations described below. Three weeks after the last immunization, the sera are collected.

  Immunization groups The mice were divided into 2 groups marked I and II, subjected to an immunization protocol as defined above by means of the following products:> group I: HIV-1 P24 encapsulated: 150 μl of dispersion of vesicles according to the invention HIV-1 P24 which corresponds to 3 μg of HIV-1 P24 by injection, 10> group II: HIV-1 P24: 150 μl of a solution of HIV-1 P24 corresponding to 3 μg per injection Preparation of the sera The blood samples are taken on anesthetized animals. The sera are collected after blood exudate and centrifugation to eliminate the blood clot. They are stored at -200C until analysis.

  Antibody assay The HIV-1 P24 specific antibodies present in the sera were assayed by ELISA using peroxidase labeled conjugates (anti-IgG1 and IgG2a, SouthernBiotechnology Associates). The antibody titres obtained are determined according to a protocol identical to that described in Example 1.

  III-Results The results of the assay of the specific antibodies are shown in FIGS. 5 and 6 and Table I below illustrates FIG. 5, the P24 specific IgG1 antibody titers, as well as the standard deviations (SD) in FIGS. sera after subcutaneous immunizations.

  Figure 6, P24 specific Ig2a antibody titers, as well as standard deviations (SD) in sera after subcutaneous immunizations.

  Table I: the ratios of IgG1 subclass on IgG2a to P24 specific antibodies, after subcutaneous injection of P24 antigen, into vesicles according to the invention or free; (n = 7). 10

TABLE I

  IgG1 subclass titers on IgG2a P24 specific antibodies, after subcutaneous injection of P24 antigen, into vesicles according to the invention or free; (n = 7) P24 in free IgG1 / IgG2a P24 vesicles 3,3 The incorporation into the vesicles of the invention makes it possible to induce very high titres of serum antibodies compared to the free antigen. The study of the antibody subclasses reveals a change in the IgG1 and IgG2a ratio following the incorporation of the antigen into the vesicles of the invention.

  The production of IgG2a is particularly increased by the administration of vesicles containing P24 antigen.

  Modification of the IgG1 / IgG2a ratio in favor of IgG2a by the compositions of the invention suggests that encapsulation in the vesicles of the invention alters the presentation, processing or activation of the antigen presenting cells. and / or promotes the secretion of a type 1 interleukin profile.

Claims (24)

  1. A pharmaceutical composition containing a dispersion or a suspension in a pharmaceutically acceptable vehicle of multilamellar vesicles with a liquid-crystal internal structure, formed of a stack of concentric bilayers based on amphiphilic agents alternating with layers of water, of solution aqueous, polar liquid or solution of a polar liquid, and within which is incorporated at least one protein antigen of the HIV virus.   2. Pharmaceutical composition according to claim 1, characterized in that said protein antigen is a protein or an envelope glycoprotein of said HIV virus, in particular a GP 160 protein or a GP 120 or GP 41 derived protein.   3. Pharmaceutical composition according to claim 1 characterized in that said antigen is an enzymatic protein involved in the integration, transcription, replication of the virus and its genetic material.   4. Pharmaceutical composition according to claim 1, characterized in that said antigen is a regulatory protein for transcription or replication.   5. Pharmaceutical composition according to claim 1 characterized in that said antigen is a capsid protein of said HIV virus encoded by the gag gene or a protein derived from such a protein such as the protein P 24 or P 17 or a protein of fusion.   6. Pharmaceutical composition according to claim 5, characterized in that said protein antigen is the P 24 protein.   7. Pharmaceutical composition according to one of claims 1 to 6 characterized in that said vesicles have diameters between 0.1 and 25 pm, preferably between 0.2 and 15 pm.   8.Composition according to one of claims 1 to 7 characterized in that the bilayers of said vesicles contain at least one surfactant selected from the group consisting of: hydrogenated or non-hydrogenated phospholipids, esters of C6 to C30 fatty acids saturated or mono- or polyunsaturated, linear or branched, and macrogol (polyethylene glycol), - esters, ethoxylated or not, linear or branched, saturated or mono- or polyunsaturated C 6 to C 30 fatty acids, and> sucrose,> sorbitan,> mannitol,> glycerol or polyglycerol,> glycol, ethers of C6 to C30 fatty alcohols, saturated or mono- or polyunsaturated, linear or branched, and macrogol (polyethylene glycol) ethers, ethoxylated or non-ethoxylated, linear or branched, saturated or mono- or polyunsaturated fatty alcohols, and> sucrose,> sorbitan,> mannitol,> glycerol or polyglycerol,> glycol .   9. Composition according to one of claims 1 to 8 characterized in that said bilayers contain, in addition, at least one agent 25 for improving the rigidity and / or the tightness of said bilayers.   10. Composition according to claim 9 characterized in that said agent is selected from the group consisting of cholesterol and its derivatives, in particular charged or neutral cholesterol esters such as cholesterol sulfate, other sterol backbone derivatives, particularly those of plant origin commonly called phytosterols such as sitosterol or sigmasterol, ceramides.   11 Composition according to one of claims 1 to 10 characterized in that said bilayers contain as amphiphilic agents a phospholipid in combination with a nonionic surfactant.   12.Composition according to claim 11 characterized in that said nonionic surfactant is a fatty acid ester.   13. Composition according to claim 12, characterized in that said fatty acid ester is a polyethylene glycol or sorbitan ester or a polysorbate.   14.Composition according to one of claims 11 to 13 characterized in that said phospholipid is a phosphatidylcholine.   15.Composition according to one of claims 11 to 14 characterized in that the amphiphilic agents consist of a mixture of phosphatidylcholine and polyethylene glycol oleate or polysorbate 80.   16. Composition according to one of claims 1 to 15 characterized in that said vesicles further comprise a natural or artificial polymer to enhance their strength.   17.Composition according to one of claims 1 to 16 characterized in that it further contains immunomodulatory molecules enhancing the amplification and / or the orientation of the immune response.   18. Pharmaceutical composition according to one of claims 1 to 17, characterized in that it is a vaccine composition formulated for parenteral administration.   19. Pharmaceutical composition according to one of claims 1 to 17, characterized in that it is a vaccine composition formulated for mucosal administration, in particular via the nasal route.   20.Procédé for manufacturing a composition according to one of claims 1 to 19 characterized in that it comprises the preparation of vesicles with a liquid-crystal structure by transformation of a lamellar crystalline phase containing at least one amphiphilic agent and at minus one HIV protein antigen and dispersing said vesicles in a pharmaceutically acceptable carrier.   21.Utilization of multilamellar vesicles incorporating at least one protein antigen of the HIV virus, as defined in one of claims 1 to 17 for the manufacture of a vaccine composition for immunization against the virus responsible for AIDS.   22.Use according to claim 21, characterized in that said vaccine composition is intended for parenteral administration.   23.Use according to claim 21, characterized in that said vaccine composition is intended for administration by the mucous route, in particular via the nasal route.   24. Use according to one of claims 21 to 23 characterized in that said vaccine composition is intended to induce a TH1 type immune response. 20