AU7428400A - Composition to be administered through mucous membrane - Google Patents

Description

1 COMPOSITION TO BE ADMINISTERED THROUGH MUCOUS MEMBRANE The present invention relates to novel compositions for administration via the mucosa. More particularly, it relates to a pharmaceutical composition, in particular a 5 vaccine composition for administration via the mucosa. It also relates to a method for producing antibodies, in particular IgA. A number of definitions are given below: Administration via the mucosa: non-invasive 10 administration of an antigen to a mucous site, for example: e naso-pharyngeal area; " buccal area; " bronchial tree; 15 e intestine; e uro-genital tract; e inner ear; " conjunctiva; e mammary, salivary and lachrymal glands. 20 Of these different constituents of mucus associated lymphoid tissue (MALT), certain introduction routes are more readily accessible and more readily acceptable: nasal, buccal, gastro-intestinal, rectal and vaginal. Mucous response: The response of the immune system 25 in the mucosa, characterized by production of IgA (isotype in high concentrations in the mucosa) and IgG and/or a cellular response in the mucous site and lymph nodes. Systemic response: The response of the generalized 30 immune system resulting from the presence of circulating antibodies (IgG and IgA) and/or a cellular response (T helper or CTL) in the secondary lymphoid organs (spleen, nodes). Dissemination of response: Installation of a mucous 35 response in compartments other than that used for introduction, because of the circulation and relocalization of lymphocytes induced at the 2 administration site (vaginal IgA after nasal administration, for example). Adjuvant: The substance added to the antigen to amplify and orientate the specific immune response of 5 that antigen. Synthetic vector/vectorization: Incorporation of the antigen into or on the surface of a particle or vesicle, to protect the antigen and/or facilitate its capture by competent cells of the immune system and/or to facilitate 10 presentation of the antigen by said cells, and to amplify the immune response. The addition of an empty vector to the free antigen provides little or no amplification. Until recently, vaccines were produced from dead, 15 attenuated or less virulent micro-organisms. Today's pharmaceutical industry seeks to avoid such an approach, to limit secondary effects, to facilitate production, for safety in administration and for efficacy reasons. Advances in molecular biology have resulted in the 20 industrial production of sub-units of such micro organisms, in particular proteins that are termed recombinant proteins, either membrane, nuclear or cytoplasmic proteins. Unfortunately, such sub-units are not in themselves sufficiently immunogenic to supply a 25 sufficient response to vaccination. They have to be supplemented with adjuvants or vectorized to induce a sufficient response. Today, the only adjuvants that are acceptable for human medicine are aluminum hydroxide or phosphate, also 30 calcium phosphate. These salts are in the form of a suspension of grains of the aluminum or calcium salt, onto the surface of which the antigen is adsorbed. They have a number of disadvantages: they induce local inflammation reactions and the production of IgE, they 35 are not effective for all antigens and they are incapable of causing CTL type cellular mediation reactions. Thus, 3 until now they have not been used for administration via the mucosa. Mucous surfaces are very important, firstly because they are present in all tracts and secondly, because they 5 are the first line of defense against the invasion of pathogenic agents. Mucous surfaces are protected both by innate or non adaptive defense mechanisms (peristalsis, cilia movements and mucous) and by triggering a cellular immune response 10 and an adaptive humoral response specific to the pathogenic agent, which can be generalized to other lymphoid organs. Mucus associated lymphoid tissue (MALT) is responsible for the specific component. In this respect, vaccination via the mucosa 15 represents a considerable advantage. Currently, all vaccines except that for poliomyelitis are injectable, which involves: " medicalization; e low acceptance; 20 e discomfort (fever, injection site pain,...); e risks of contamination (HIV, hepatitis); " high costs in developing countries. Administration via the mucosa would be of advantage for a variety of reasons: 25 e production costs would be lower due to the less stringent preparation conditions compared with those for an injectable vaccine; e its non invasive nature, which eliminates problems linked to injection mentioned above. 30 To this end, it must: e provide immunity at the mucous administration sites, to strengthen the mucous barrier by dint of a specific response, providing an advantage for infections that can be transmitted via the mucosa 35 (respiratory, genital,...); 4 * provide systemic immunity and an effective generalized response following easy administration; e not cause secondary effects (local irritation). 5 A fairly complete review of mucosal immunology can be found in the "Handbook of Mucosal Immunology" edited by Pearay Ogra et al., Academic Press, Inc., San Diego, California, USA. A number of trials and studies have been carried out 10 with a view to developing novel adjuvants and vectors that can induce a mucous and systemic response that is effective as regards protection against the pathogenic agent. Different studies concern the use of live recombinant vectors (attenuated bacteria or viruses), 15 synthetic vectors (immunostimulating complexes also known as iscoms, an abbreviation of the expression "immunostimulating complexes", liposomes, microspheres...) or micro-organism toxins (cholera toxin (CT), or heat labile E. Coli toxin, (LT)). However, a large proportion 20 of these studies are still in the development stage and the majority of formulations have the following disadvantages: e high toxicity (ST and LT), and thus are not possible for human application; 25 e difficulties in preparation (microspheres); e low stability (liposomes); e low efficacy (liposomes). A number of patents concern administration via the mucosa of antigens that are vectorized or supplemented by 30 different systems. The following can be cited: e European patent EP-A-0 440 289 (Duphar), which describes the use of liposomes mixed with an antigen for vaccination against influenza. The lipids act as an adjuvant, the same result being 35 obtained when the antigen is mixed extemporaneously with empty liposomes; 5 e International patent application WO-A-98/10748 (The School of Pharmacy), using cationic liposomes, in the case of injection or of administration via the mucosa; 5 o United States patent US-A-5 679 355 (Proteus), describing non-ionic vesicles, which can be administered parenterally or via the mucosa (especially orally). Further, a great deal of scientific literature 10 describes the use of polymer microparticles as a mucous membrane antigen vector. A good review of that literature can be found in: D. T. O'HAGAN, Adv. Drug. Deliv. Rev., 34 (1198), 305-320. However, none of the above technologies has yet 15 culminated in a commercially available product. Antigen presentation methods must, therefore, be further improved and the development of novel adjuvants or antigen vectors remains an urgent need in the vaccine industry. Vesicles, spherical objects formed by a molecular 20 arrangement of amphiphilic molecules, include mulilamellar vesicles with an onion-like structure which have formed the basis of a large amount of research and have given rise to several patents (WO-A-93/19735; WO-A-95/18601; WO-A-97/00623; WO-A-98/02144; 25 WO-A-99/16468). They are distinguished from liposomes by the following: * their preparation method, which starts from a lamellar phase in thermodynamic equilibrium; e their internal liquid crystal structure formed by 30 a regular stack of concentric bilayers of amphiphilic compounds alternating with layers of water or an aqueous solution or a solution of a polar liquid (for example glycerol); e the varied nature of the amphiphilic molecules 35 that can be used to constitute them, used alone or as a mixture.

6 International patent application WO-A-99/16468 describes vesicles with an onion-like structure incorporating an antigen therein and which can amplify the immune response to that antigen. The examples of 5 that International patent application, which for the first time describes incorporation (also termed encapsulation in that document) of antigens in a multilamellar vesicle with an onion-like structure, clearly show that such encapsulation can substantially 10 amplify the immune response during parenteral administration of an antigen encapsulated in a multilamellar vesicle with an onion-like structure. The present inventors have now discovered that administration via the mucosa, in particular nasal 15 administration, of compositions of the type described in WO-A-99/16468, causes an immune response not only in different mucous sites but also in the systemic compartment. This response is characterized by immunoglobulin A (IgA) induction. Such a result is not 20 at all obvious having regard to WO-A-99/16468 since, under conditions in which injection was carried out in the examples of that application, even in the encapsulated form, the antigen did not induce any detectable IgA response. 25 Immunoglobulins (Ig) or antibodies (Ab) associated with cellular components are essential protagonists in the immune response directed against a pathogen. Igs are proteins that are highly specific as regards the antigen. Different classes of antibodies have been 30 classified, which are distinguished by their mode of induction, their localization and their function (recognition, neutralization of toxic activity or of enzymatic activity). In the blood compartment, the majority of Ig 35 produced is circulating IgG (sub-divided into different sub-classes), IgA and IgE remaining very much in the minority. In contrast, the major isotype in the mucosa 7 is IgA, which is produced by IgA plasmocytes of the mucous lymphoid system and secreted actively by the epithelium of the mucosa. IgA are specialized antibodies and are adapted to 5 defending mucous surfaces that are constantly exposed to pathogens. They are adapted since, because of their biochemical structure (glycosylation, polymerization, secretory piece), they resist the effects of proteases secreted by the micro-organisms and since, in contrast to 10 other isotypes, they limit inflammatory reactions in those compartments in a state of constant activation. They are specialized since they act on a number of levels: secreted, they bind pathogens, limit the effects of toxins and the entry of pathogens by neutralization; 15 they can also act on the epithelium during their transcytosis, or in the lamina propria. Thus, they represent an essential active barrier which, associated with non specific mechanisms, limit invasion by pathogens. 20 Naturally, cytotoxic and specific responses combine with the antibody response to eliminate the pathogen. If the local response is not sufficient, a systemic response is triggered. One highly interesting characteristic of the mucous 25 immune system is the re-circulation of B and T lymphocytes induced at a mucous site and their possible domiciliation in sites other than the induction site, propagating the specific response to other tracts. This phenomenon is very important as regards vaccination. 30 While the mechanisms for induction of a mucous immune response are still in the course of being elucidated, it often remains extremely difficult to induce a mucous response by parenteral administration of an antigen (with or without adjuvant) and even by mucous 35 administration of the antigen. In fact, parenteral administration of an antigen leads to the induction of specific circulating antibodies of the IgG type. This 8 type of injection cannot induce IgA in the mucous (nor the systemic) compartments. It is very clear that parenteral vaccination will not reinforce the natural local defenses required to combat many respiratory or 5 genital infections, for example. For these reasons, it is completely surprising even in the light of the teaching of WO-A-99/16468 that the same antigen incorporated in the same vesicles, administered via the mucosa (nasally) can induce not only 10 a systemic response (IgG in serum) confirming the preceding results by another route, but also a response at mucous sites. Incorporation of the antigen into vesicles with an onion-like structure causes the production of IgA in the compartment linked to the 15 administration site (bronchio-pulmonary) but also in other mucous compartments with a predominance in genital secretions. Further, it should be noted that human serum albumin (HSA) which is used by way of example is a very slightly immunogenic antigen which, when administered 20 nasally alone without incorporation into vesicles, is incapable of inducing any response, whether mucous or systemic. Further, it should be noted that, compared with parenteral administration, administration via the mucosa 25 necessitates loading the antigen into the mucosa and thus penetration of the antigen, which implies that the antigen must be presented in an optimal manner to defeat the non specific defense mechanisms (movement of cilia, mucus) and to resist the enzymes present in the mucosa or 30 on its surface. It appears that these parameters are optimized by the structure of the vesicles during administration via the mucosa. These functions of vesicles in administration via the mucosa are not necessarily present during parenteral administration, 35 where injection directly reaches zones more favorable to capture by cells of the immune system.

9 The result observed by administration via the mucosa is thus not a simple extrapolation of the result obtained by parenteral administration, but a demonstration of a novel characteristic of vesicles with an onion-like 5 structure. The present inventors have now discovered that vesicles identical to those described in International application WO-A-99/16468 incorporating an antigen cause an immune response that is much stronger than 10 administering the free antigen. Further, for certain antigens that induce no response by mucous administration, administering them incorporated into multilamellar vesicles with an onion structure can induce a remarkable response. Finally, the induced response is 15 found not only locally in the mucous compartment (mucous response), but also in the blood circulation (systemic seric response). Furthermore, these multilamellar vesicles are prepared from biocompatible constituents that are known 20 to be innocuous. Still further, the preparation method is simple to carry out and requires only routinely used chemical apparatus. The fact that the process uses a lamellar phase initially in thermodynamic equilibrium endows it with excellent reproducibility, and the 25 vesicles obtained are highly stable. In a first essential characteristic, the invention concerns the use of multilamellar vesicles with an onion like structure having an internal liquid crystal structure formed by a stack of concentric bilayers based 30 on amphiphilic agents alternating with layers of water, an aqueous solution or a solution of a polar liquid and into which at least one antigen is incorporated, for the production of a composition, more particularly a pharmaceutical composition, in particular a vaccine 35 composition, for administration via the mucosa. The term "incorporated", the use of which appears to us to be preferable to that of the term "encapsulated", 10 means that the antigen or antigens form an integral part of the entity constituted by the vesicle. In fact, molecules of antigen(s) can be found in any layer between the center and the periphery of said vesicle. 5 As will become clear from the following detailed description and examples, the pharmaceutical compositions used in the invention allow the preparation of a mucous vaccine intended to induce a mucous response and/or a systemic seric response. 10 As will become clear from the examples, when they are administered via the mucosa, the vesicles with an onion-like structure as defined above incorporating an antigen induce the production of antibodies in man and in animals. 15 As indicated above, one advantage of the invention is to induce a very high production of antibodies characterized by the presence of IgA and IgG. This implies an increase in the frequency of lymphocytes carrying IgA and IgG specific to the antigen. The 20 preparation can thus be used for the purposes of activation and differentiation of antigen-specific B lymphocytes that can then be used in cell fusion to produce monoclonal antibodies. In fact, specific lymphocytes present in large quantities in nodes draining 25 the administration site can be immortalized by fusion with a non secreting myeloma and result in hybridomas secreting monoclonal antibodies. Its capacity to simplify and increase the antibody response means that the invention can also be employed 30 for the production of antibodies, in particular polyclonal IgA, an isotype that is difficult to generate in conventional operational modes, or polyclonal IgG. These antibodies can be used for non therapeutic purposes, for example for research, more particularly for 35 biological or immunological research. Antibody sampling and purification methods are known to the skilled person.

11 Thus, the invention also concerns a method for producing antibodies, more particularly IgA. Further, it has been shown that the composition of the invention, when administered via the mucosa, induces 5 a protection of the organism to the infection for which the antigen incorporated into said composition is responsible. In a further essential feature, the invention concerns a method for treating a human or animal body by 10 vaccination via the mucosa, in which a composition is administered that contains multilamellar vesicles with an onion-like structure having an internal liquid crystal structure formed by a stack of concentric bilayers based on amphiphilic agents alternating with layers of water, 15 an aqueous solution or a solution of a polar liquid and into which at least one antigen is incorporated. As will become apparent from the following description and examples, it has been shown that when administered via the mucosa, the compositions of the 20 invention in which the antigen is incorporated into a vesicle with an onion-like structure as defined above can cause a mucous immune response and stimulate the common mucous lymphoid tissue. Such capacities have been shown to be of particular importance regarding antigens with a 25 vaccinating potential as opposed to invasive pathogens with mucous tropism. The dual possibility of inducing both a mucous response and a systemic response is of huge interest in vaccination as it simplifies administration while 30 offering immunity on a number of fronts: mucous to defend against micro-organism access, and systemic to defend against more disseminated or generalized infections. Further, the results obtained in the context of the invention demonstrate that the invention can not only 35 amplify the antibody response but can also generate an immune response with a protective efficacy against 12 infection. Therefore, the invention is applicable both to the production of antibodies and to vaccination. The results obtained confirm that vesicles administered via the mucosa, in particular nasally, can 5 be used to induce or amplify the antibody response in the mucous compartments, to disseminate this response to other sites more distant from the administration site, and generate a systemic response. The diameter of the vesicles used in the invention 10 is generally in the range 0.1 micrometers (gm) to 25 jm, preferably in the range 0.2 gm to 15 gm. More precisely, the vesicles used in accordance with the invention are preferably constituted by a plurality of layers of amphiphilic agents alternating with layers 15 of aqueous or polar phase. The thickness of each of these layers is molecular, typically of the order of 5 nanometers (nm) to 10 nm. For a stack of ten to a few tens of layers, then, a diameter is obtained that is in the range from 0.1 gm to a few tens of micrometers. This 20 has been observed experimentally, the vesicles being observable under optical microscopes (in polarized light to provide a better contrast because of their birefringence), either as unresolved points for the smallest layers, or as birefringent spheres for the 25 largest. The size profile can be studied using a laser granulometer (using static laser beam diffusion, analyzed under a plurality of angles). In general, a gaussian profile is obtained centered on a value of 0.1 jm to 25 jm, showing a slight heterogeneity of size for a given 30 formulation, under the given operating conditions for the preparation. As described above, vesicles into which the antigen is incorporated have a multilamellar onion-like structure and are constituted, from their center to their 35 periphery, by a succession of lamellar layers separated by a liquid medium. These vesicles can be obtained by a method comprising preparing a lamellar liquid crystal 13 phase and its transformation by applying shear. Such a method has been described in particular in WO-A-93/19735 claiming priority from French patent FR-A-2 689 418 or WO-A-95/18601, hereby incorporated by reference. 5 In French patent FR-A-2 689 418, this transformation can be carried out during a homogeneous shear step for the liquid crystal phase, which produces vesicles that are also known as microcapsules, with a controlled size. However, adjusting the formulation of the lamellar liquid 10 crystal phase, in particular the nature of the surfactants forming part of its composition, can enable to transform this liquid crystal phase into vesicles by simple mechanical stress, in particular when mixing the constituents. 15 Such vesicles also have the advantage, inter alia, of being capable of being prepared by a particularly simple preparation method that can employ a wide variety of surfactants. A further advantage, also essentially linked to the 20 method used to prepare the vesicles with an onion-like structure used in accordance with the invention, lies in the fact that the active ingredients and additives are incorporated prior to forming the vesicles, which provides an excellent encapsulation yield, and therefore 25 better efficacy, and also economizes on very expensive molecules. Such structures are advantageously obtained by incorporating at least one antigen into a lamellar liquid crystal phase comprising at least one surfactant then 30 transforming this lamellar liquid crystal phase into a dense phase of small multilamellar vesicles. Thus, the vesicles used in the invention can be obtained using a process in which a liquid crystal lamellar phase incorporating at least one antigen is 35 prepared and said liquid crystal. phase is caused to rearrange itself into multilamellar vesicles by applying shear.

14 This shear can be homogeneous shear, with the advantage of resulting in perfectly homogeneous vesicles. However, simple mechanical agitation can prove sufficient to result in the formation of the multilamellar vesicles 5 of the invention. The antigen can be any molecule against which an immune response is desired, whether exogenous such as an infectious pathogenic organism, parasite or micro organism (yeast, fungus, bacteria or virus), or of 10 intrinsic natural origin (auto-immune disease or cancer). It may have different biochemical natures. In particular, it may be an antigen selected from the group consisting of : * proteins, in particular extracted or recombinant 15 proteins, which may or may not be glycosylated; " peptides; e lipopeptides; e polysaccharides; or is a mixture of a plurality of these components. The multilamellar vesicles with an onion-like structure are prepared using methods described above, in particular in International patent application WO-A-99/16468. The amphiphilic molecules used to prepare 25 them will be selected, but this is not obligatory, from molecules that are described in the pharmacopeia, or are already used in drugs applied to the mucosa. According to an advantageous embodiment, the membranes of the vesicles contained in the compositions 30 of the invention contain at least one surfactant selected from the group consisting of : e phospholipids, which may or may not be hydrogenated; 35 e linear or branched, saturated or mono- or poly unsaturated C 6 to C3o fatty acids, in the form of 15 an acid or of a salt of an alkali or alkaline earth metal, or of an amine; e esters, which may or may not be ethoxylated, of said fatty acids and 5 e saccharose; e sorbitan; " mannitol; " glycerol or polyglycerol; e glycol; 10 * mono-, di- or tri-glycerides or mixtures of glycerides of said fatty acids; * linear or branched, saturated or mono- or poly unsaturated C 6 to C 30 fatty alcohols, which may or may not be ethoxylated; 15 e ethers, which may or may not be ethoxylated, of said fatty alcohols and: * saccharose; e sorbitan; e mannitol; 20 e glycerol or polyglycerol; e glycol; e polyethoxylated vegetable oils, which may or may not be hydrogenated; * block polymers of polyoxyethylene and 25 polyoxypropylene (poloxamers); * polyethyleneglycol hydroxystearate; e alcohols with a sterol skeleton such as cholesterol or sistosterol; e sphyngolipids; 30 e polyalkylglucosides; e copolymers of polyethylene glycol and alkylglycol (for example the ELFACOS family from AKZO NOBEL); e di- or tri-block copolymers of ethers of polyethyleneglycol and polyalkyleneglycol (for 35 example the ARLACELL family from ICI). Optional co-surfactants can be added to these surfactants (which can be used alone or as a mixture), to 16 improve the rigidity and tightness of the membranes forming the vesicle. Examples of such molecules that can be cited are: * cholesterol and its derivatives, in particular 5 charged or neutral cholesterol esters such as cholesterol sulfate; e other derivatives with a sterol skeleton, in particular those of plant origin (sitosterol, sigmasterol,...); 10 e ceramides. The formulation can advantageously involve a mixture of surfactant molecules. In general, at least two different surfactants are used with different hydrophilic-lipophilic balances, which allows continuous 15 adjustment of the bilayer properties and can control the appearance of an instability, which governs the formation of the multilamellar vesicles. Advantageously, from the above surfactants, two surfactants are selected with relatively different 20 properties, in particular a different hydrophilic lipophilic balance (HLB). The first surfactant will advantageously have a hydrophilic-lipophilic balance in the range 1 to 6, preferably in the range 1 to 4, while the second surfactant will have a hydrophilic-lipophilic 25 balance in the range 3 to 15, preferably in the range 5 to 15. The preparation obtained after transformation of the lamellar liquid crystal phase into multilamellar vesicles can then be diluted, in particular with an aqueous 30 solvent such as a buffer solution, a saline solution or a physiological solution, for example, to obtain an aqueous suspension of vesicles. The encapsulation technique used in the present invention can readily achieve very high encapsulation 35 yields, which may be as high as 100%. However, such yields are not always vital to the function of the envisaged applications.

17 The encapsulation yield of the antigen(s) in the compositions of the invention is advantageously more than 50%, preferably more than 80%. It appears that the structure of the vesicles is 5 responsible for the particularly advantageous results obtained, and that the multilamellar vesicles of the invention allow the antigen to arrive intact at the antigen presenting cells (APC) and assist its capture by these cells. It thus appears that the function of the 10 vesicles of the invention is to vectorize, protect and improve capture of the antigen by the immune system. A further advantage of the technology is that natural or artificial polymers can be added to this 15 formulation, such as polysaccharides (alginates, chitosan, etc.) to reinforce the solidity of the vesicle, and to enable it to remain longer at the administration site or in the organism, thereby delivering the antigen over a longer period. These polymers can either be 20 incorporated into the vesicle, or be deposited around it in the form of a coating. In this case, the diameter of the vesicle or particle formed from vesicles coated with the polymer matrix is greater than that of the vesicles alone. These polymers can optionally be cross-linked to 25 further reinforce their solidity. Furthermore, and this constitutes a further advantage of the technology, the formulation can be completed by adding immuno-modulating molecules (chitosan, interleukines, ...), which have intrinsic 30 properties that reinforce amplification and orientation of the immune response. Vesicles incorporating antigens are advantageously prepared in a process consisting of preparing the lamellar phase in a first stage. This is obtained simply 35 by mixing the ingredients, in an order determined by the scientist according to the miscibilities of each of the constituents. It may be necessary to heat certain pasty 18 or solid constituents to facilitate their incorporation. In that case, the antigen is preferably added when mixing is complete to avoid subjecting it to too high a temperature. It is also possible to prepare a mixture of 5 all of the constituents except for the antigen or its aqueous solution in the form of a stock mixture, which is used as required to prepare the lamellar phase. The aqueous solution can contain different constituents that ensure its biological compatibility, in particular the 10 buffer mixtures but also the different antigens. The lamellar phase so prepared is then subjected to moderate shear (0 to 1000 s1) for a limited period (0 to 60 minutes). In the majority of cases, shear is obtained directly 15 by the action of the device performing the mixing. For very small quantities, it can be obtained by hand by mixing the preparation using a microspatula in an Eppendorf type tube. The sheared lamellar phase is then dispersed in a 20 final medium, in general water or a buffer, which may be identical to that used during the preparation of the lamellar phase. This dispersion is advantageously produced at ambient temperature (20*C-25*C) by slowly adding the medium to the lamellar phase with constant 25 stirring. A preservative and optional other additives intended to complete the galenical formulation can be added to the product. All of the compositions described above comprising 30 at least one antigen incorporated into vesicles with a lamellar onion-like structure have the advantage of being capable of being used for administration via the mucosa and in particular nasally and induce a mucous and/or seric systemic response. 35 Example I below clearly demonstrates such an effect with HSA.

19 Figures 1 and 2 accompanying this example summarize the results obtained from different mucous samples (Figure 1) and from animal serum (Figure 2) after nasal administration of the compositions of the invention 5 (group I) compared with those obtained by administration either of compositions in which the same antigen is free (group II) or compositions containing the same vesicles, but empty (group III). Example II illustrates a process for immunization 10 with FHA. The results obtained in this example are illustrated in Figures 3, 4 and 5, which respectively show: e Figure 3: antibody responses in different mucous sites; 15 * Figure 4: antibody responses in serum; e Figure 5: the pulmonary bacterial load in mice infected with Bordetella pertussis. EXAMPLES EXAMPLE 1: PRODUCTION OF HSA-SPECIFIC ANTIBODIES 20 I - Preparation of vesicles containing human serum albumin (HSA) Formulation (percentage by weight) 1. Potassium oleate (FLUKA): 5.0% 2. Lauric alcohol, ethoxylated with 4 ethylene 2.0% oxides (SEPPIC): 3. Lanolin cholesterol (FLUKA): 5.0% 4. Cholesterol 3-sulfate (SIGMA): 2.5% 5. PBS 1x, sterile (LIFE TECHNOLOGIES): 20.0% 6. Phospholipon 90G soya lecithin (NATTERMANN): 45.5% 7. Human albumin (SIGMA), with 30 mg/ml in PBS 1x: 20.0% Operating procedure 25 The components were sterilized with UV irradiation for 60 minutes. The containers and accessories (spatulas, stirrers...) were flame sterilized immediately prior to use. Constituents 1 to 5 are introduced into a pill maker 30 in no particular order then heated to 80 0 C for 60 minutes 20 with very vigorous magnetic stirring. Total dissolution of constituents 3 and 4 was verified microscopically. The desired quantities of mixture 1 to 5 were introduced into a sterile 1.5 ml Eppendorf tube at 5 ambient temperature then constituents 6 and 7 were added. The ensemble was homogenized using a sterile needle then left overnight at 4 0 C. The preparation was then dispersed to 33.33% in sterile PBS 1x. 10 II- Immunization protocol To test the effect of the vesicles of the invention during administration via the mucosa, 6-8 week old BALB/c female mice received, nasally, twice (at DO and D30), the different preparations described below. Nasal 15 administration necessitated anaesthesia of the animals by a solution, a mixture of Ketamine, Valium and Atropine, injected intraperitoneally. One month after the last immunization, the animals were sacrificed, the serum was collected and samples were taken of the mucous secretions 20 with the exception of the vaginal lavages, which were taken from the live mouse over the three days preceding the end of the experiment. Immunization groups The mice were divided into 4 groups, I to IV, group 25 IV constituting a control group (non immunized mice, also known as naive mice) and the other groups being subjected to an immunization protocol as defined above using the following products: e Group I: 30 0 Encapsulated HSA: 20 pl, per nostril, of the vesicles of the invention incorporating HSA, corresponding to 80 jg of HSA per mouse; e Group II: e HSA: 20 g1, per nostril, of the vesicles of the 35 invention incorporating HSA, corresponding to 80 gg of HSA per mouse; 21 * Group III: e Empty vesicles: 20 l, per nostril, of empty vesicles of the invention. 5 Secretion sampling All samples were taken with cold solutions (4*C) and were placed in ice as they were taken to limit degradation by proteolytic enzymes. Bronchio-alveolar lavages: 10 The mouse trachea was cannulated using a probe and 750 gl of PBS was injected slowly to prevent a hemorrhage, the lungs were washed 3 times with the same solution, and the sample was then centrifuged to eliminate pulmonary cells and separated into aliquots 15 that were stored at -20 0 C until assay. Vaginal lavages These samples were taken from the non-anaesthetized live mouse by injecting 50 g1 of PBS into the vaginal orifice and washing the vagina three times with the same 20 solution. This sample was taken over three consecutive days to cover variations in the hormonal cycle of the mouse. The secretions were combined and stored at -20 0 C. Intestinal lavages The intestine was removed, freed from the mesentera 25 and rinsed with water to eliminate external blood. It was then cut longitudinally and incubated in ice in 1 ml of a lavage solution enriched with protease inhibitor. The whole was centrifuged and the supernatant was recovered and frozen at -20 0 C. 30 Antibody assay HSA-specific antibodies present in the serum and secretions were assayed using ELISA to determine the HSA specific IgA and IgG (biotinylated anti-IgA/streptavidine peroxidase, anti-IgG peroxidase). The results are 35 expressed as the mean titer determined with respect to a reference serum from the naive mouse and corresponding to 22 the inverse of the dilution equal to a reference threshold. III - Results The results of the specific antibody assay are shown 5 in the form of two figures (Figure 1 and Figure 2) respectively illustrating the antibody responses obtained in mucous samples for the response associated with the mucosa and in animal serum (systemic response). In these figures, in each case we also indicated the 10 number of mice that reacted to the immunization protocol with respect to the number of mice subjected to this protocol (the mention "n/m" signifying that n mice reacted out of m mice subjected to the immunization protocol). 15 It is clear from these two figures that nasal administration of the vesicles of the invention incorporating HSA cause a substantial production of antibodies in the lungs (Figure 1). only immunization in the encapsulated form causes a pulmonary immune response 20 of isotype IgA and IgG (the pulmonary existence of these two isotypes has been reported in various publications). While this administration route is not very easy in an animal, all of the animals immunized nasally with the vesicles of the invention responded. 25 Analysis of the other mucous samples revealed the presence of HSA specific IgA in the vagina and intestine of animals immunized by the vesicles of the invention incorporating HSA, which mucosa were very distant from the administration site, with a predilection for vaginal 30 mucosa. These responses indicate a generalization of the response induced in the pulmonary or nasal mucous lymphoid tissue and the circulation and re-distribution of activated and differentiated lymphocytes close to the administration site. 35 In contrast to the pulmonary mucosa, the predominant HSA specific isotype in the intestine and vagina was IgA. While IgGH 4 L titers could not be carried out for the 23 vaginal lavages, similar experiments carried out with another antigen indicated the predominance of IgA in vaginal secretions. Only the vectorized form of the antigen resulted in 5 an intense and specific response. The few animals presenting a detectable IgA response in the groups immunized with free HSA (titer of 3 as opposed to 650 in the vaginal secretions with vesicles of the invention containing HSA) could not be differentiated from the 10 group using empty vesicles of the invention. Studying the animal serum (Figure 2) indicates that the vesicles of the invention incorporating nasally administered HSA are capable of causing a systemic response characterized by the major presence of IgG but 15 also a large quantity of seric IgA. Only the vectorized form led to this production (IgA, HSA alone = 35; IgA, vesicles of the invention HSA = 17620). It is remarkable to obtain a systemic response to the vectorized antigen. It should be remembered that it is extremely difficult to 20 induce a seric IgA response by systemic immunization with an antigen alone or accompanied by an adjuvant authorized for human use. In conclusion, not only does the encapsulated form cause a pulmonary immune response but also, only this 25 form permits dissemination of the induced response in the respiratory tract to other mucosa, indicating that the vesicles of the invention are powerful vectors for inducing mucous immunity, encouraging loading and induction of the response at a site and re-distributing 30 it to other sites. EXAMPLE II: PRODUCTION OF FHA-SPECIFIC ANTIBODIES AND PROTECTION AGAINST BORDETELLA PERTUSSIS FHA protein is a filamentous adhesin of Bordetella pertussis, the whooping-cough bacterium. In contrast to 35 the HSA used above, FHA is more immunogenic and is thus susceptible of itself inducing an antibody response. This protein forms part of protective antigens contained 24 in commercial vaccines against whooping-cough. Finally, the murine model can be infected with Bordetella pertussis and it is thereby possible to carry out an infection test following mucous administration and to 5 demonstrate the protective nature of the response induced by immunization. A - Antibody induction I - Preparation of vesicles containing FHA Formulation 1. Potassium oleate (FLUKA): 5.0% 2. Lauryl ether of PEG-4 (Simulsol P4, SEPPIC): 2.0% 3. Lanolin cholesterol (FLUKA): 5.0% 4. Cholesterol 3-sulfate (SIGMA): 2.5% 5. PBS 1x, sterile (LIFE TECHNOLOGIES): 20.0% 6. Soya lecithin (Phospholipon 90G NATTERMANN): 45.5% 7. FHA from Bordetella pertussis in PBS (1 mg/ml): 20.0% 10 The operating protocol for the preparation was similar to that of Example 1. The FHA concentration of the finished product was 3 pg for 40 l. II - Immunization protocol 15 To study the antibody response, the mucous immunization and sampling protocol was identical to that described in Example 1. Two groups of 5 animals were constituted, group I receiving the antigen encapsulated in the vesicles, and group II receiving the non 20 encapsulated antigen in solution in PBS. For each immunization, each animal received 3 jg of FHA, distributed into two instillations, one into each nostril. Secretion sampling 25 The samples were taken in a manner that was strictly identical to that described for Example 1. Antibody assay The FHA specific antibodies present in the serum and secretions were assayed using ELISA using a protocol 30 identical to that described in Example 1, with reagents specific to the FHA antigen.

25 III - Results The results are presented in Figures 3 and 4, showing the mean titers of IgA and IgG antibodies in mucous secretions (Figure 3) and in the serum (Figure 4) 5 for the two groups I and II. The mucous secretions were respectively studied as described in Example 1, from bronchio-alveolar lavages (A), intestinal lavages (B) and vaginal lavages (C). In group I, mice immunized by the antigen 10 incorporated into the vesicles of the invention (Figure 3), all of the animals responded to the antigen while when the free antigen was administered, only 1/5 of the animals responded, and then very weakly. As was the case with HSA in Example 1, a very strong 15 amplification of the antibody response in the mucous sites was observed. Nasal immunization with the antigen incorporated into the vesicles of the invention enables to induce a mucous response (IgA) in the lungs, close to the administration site (IgA titer for an antigen 20 incorporated into the vesicles of the invention, 8925, compared with 4.25 for the free antigen), but also a mucous response disseminated into other secretory compartments (intestine and vagina) (titer of vaginal IgA, 13500 for the antigen incorporated into the vesicles 25 of the invention compared with 2.5 for the free antigen). Further, IgG were detected in significant amounts in the three mucous compartments being studied. As was the case with HSA, we observed large titers of IgA and IgG antibodies in the circulation (Figure 4) 30 following nasal administration of the antigen incorporated into the vesicles (IgA titer for the antigen in the vesicles of the invention, 1300 compared with 39.2 for the free antigen; IgG titer 1 265 000 compared with 3 000). 35 The results of this Example 2 completely confirm those obtained for HSA. They confirm that administered nasally vesicles can be used to induce or amplify the 26 antibody response in mucous compartments, to disseminate this response to other sites more distant from the administration site and to generate a systemic response. B - Protection test 5 To check whether the immunization obtained was protective, a protection test known as a challenge test was carried out. In this experiment, immunized mice were infected nasally with a fixed amount of Bordetella pertussis, then the animals were sacrificed at different 10 times, to observe the progress of infection. The measurement was made by counting the colonies in the lungs. I - Preparation of vesicles containing FHA 15 The formulation for and preparation of the vesicles were strictly identical to that used to characterize the antibody response. II - Immunization protocol The immunization protocol and the doses of 20 administered antigens were identical to those used to induce antibody. Three groups of mice were used: I. mice immunized by the antigen incorporated into the vesicles of the invention; 25 II. mice immunized by the free antigen in solution in PBS; III. non-immunized mice. Each group was composed of 4 mice, immunized at the same time, with the same batch of antigen, in order to 30 carry out the same infection measurements under the same conditions. III - Infection protocol Four weeks after the second immunization, the mice were anaesthetized and infected with Bordetella pertussis 35 nasally (5 x 107 bacteria/mouse, administered in 20 gl) and the initial infectious load was verified from 3 hours after infection.

27 IV - Protocol for measuring the infectious charge The mice were sacrificed and their lungs were removed at different times after infection and dilacerated in PBS to obtain a homogeneous suspension. 5 Different dilutions of the suspension were spread on specific nutrient medium for Bordetella and the colonies were counted after growing for 3 days. V - Results The results are shown in Figure 5, which indicates 10 the number of colonies of Bordetella pertussis in the lungs of mice 3 days (D3, solid bar) and 5 days (D5, open bar) after infection, in millions of bacteria colonies for the three groups of mice shown below: Group I: mice immunized with antigen incorporated 15 into the vesicles of the invention. Group II: mice immunized with free antigen in solution in PBS. Group III: non-immunized mice. It can be seen that, from the third day following 20 infection, the bacterial load for mice immunized with the antigen incorporated into the vesicles of the invention was lower (factor 2) than that for non immunized mice (factor 8). Remarkably, while 5 days after infection, the mice immunized with the free antigen and the non 25 immunized mice experienced the bacterial load increase, the mice immunized with the antigen incorporated into the vesicles of the invention had fewer bacteria on D5 than on D3, indicating favorable evolution of the disease. Further, at this stage, the number of bacterial colonies 30 in the group immunized with the antigen incorporated into the vesicles of the invention was 6 times lower than the group immunized with free FHA and 13 times lower than in the non immunized group, indicating good protection. These results demonstrate that the invention can not 35 only amplify the antibody response, but that it also generates an immune response with protective efficacy 28 against infection. The invention is thus also applicable to the production of antibodies and to vaccination.

Claims (19)

1. Use of multilamellar vesicles with an onion-like structure having an internal liquid crystal structure formed by a stack of concentric bilayers based on 5 amphiphilic agents alternating with layers of water, an aqueous solution or a solution of a polar liquid and into which at least one antigen is incorporated, for the production of a composition for administration via the mucosa. 10

2. Use according to claim 1, characterized in that said composition is a composition intended to induce a mucous response. 15

3. Use according to claim 1 or claim 2, characterized in that said composition is a composition intended to induce a systemic seric response.

4. Use according to any one of claims 1 to 3, 20 characterized in that said composition is a composition intended to induce the production of antibodies.

5. Use according to any one of claims 1 to 4, characterized in that said composition is a 25 pharmaceutical composition, in particular a vaccine, intended to induce protection of the organism against the infection for which said antigen is responsible.

6. Use according to any one of claims 1 to 5, 30 characterized in that said antigen is of exogenous or intrinsic natural origin.

7. Use according to any one of claims 1 to 6, characterized in that said antigen is selected from the 35 group consisting of : * proteins, in particular extracted or recombinant proteins, which may or may not be glycosylated; 30 e peptides; e lipopeptides; e polysaccharides; or is a mixture of a plurality of these components.

8. Use according to any one of claims 1 to 7, characterized in that said vesicles contain at least one surfactant selected from the group consisting of " phospholipids, which may or may not be hydrogenated; e linear or branched, saturated or mono- or poly unsaturated C 6 to C 30 fatty acids, in the form of 15 an acid or of a salt of an alkali or alkaline earth metal, or of an amine; e esters, which may or may not be ethoxylated, of said fatty acids and " saccharose; 20 e sorbitan; " mannitol; e glycerol or polyglycerol; " glycol; " mono-, di- or triglycerides or mixtures of 25 glycerides of said fatty acids; e linear or branched, saturated or mono- or poly unsaturated C 6 to C 30 fatty alcohols, which may or may not be ethoxylated; " ethers, which may or may not be ethoxylated, of 30 said fatty alcohols and: e saccharose; e sorbitan; e mannitol; e glycerol or polyglycerol; 35 e glycol; e polyethoxylated vegetable oils, which may or may not be hydrogenated; 31 " block polymers of polyoxyethylene and polyoxypropylene (poloxamers); e polyethyleneglycol hydroxystearate; e alcohols with a sterol skeleton such as 5 cholesterol or sistosterol; e sphyngolipids; " polyalkylglucosides; e copolymers of polyethylene glycol and alkylglycol; e di- or tri-block copolymers of ethers of 10 polyethyleneglycol and polyalkyleneglycol.

9. Use according to any one of claims 1 to 8, characterized in that said vesicles also contain at least one co-surfactant intended to improve the rigidity and/or 15 tightness of the membranes of said vesicles.

10. Use according to claim 9, characterized in that said co-surfactant is selected from the group consisting of formed by: 20 e cholesterol and its derivatives, in particular charged or neutral cholesterol esters such as cholesterol sulfate; e derivatives with a sterol skeleton, in particular those of plant origin such as sitosterol or 25 sigmasterol; e ceramides.

11. Use according to any one of claims 1 to 10, characterized in that said vesicles also contain an 30 immuno-modulating substance.

12. Use according to any one of claims 1 to 11, characterized in that the diameter of said vesicles is in the range 0.1 gm to 25 gm, preferably in the range 0.2 im 35 to 15 im. 32

13. Use according to any one of claims 1 to 12, characterized in that the bilayers of said vesicles comprise at least two surfactants, one of which has a hydrophilic-lipophilic balance (HLB) in the range 1 to 6, 5 preferably in the range 1 to 4, and the other has a hydrophilic-lipophilic balance (HLB) in the range 3 to 15, preferably in the range 5 to 15.

14. Use according to any one of claims 1 to 13, 10 characterized in that the encapsulation yield of the antigen (or antigens) in said vesicles is more than 50%, preferably more than 80%.

15. Use according to any one of claims 1 to 14, 15 characterized in that said administration via the mucosa is nasal administration.

16. A method of treating a human or animal body by vaccination, characterized in that it comprises 20 administration via the mucosa of a composition as defined in any one of claims 1 to 15.

17. A method according to claim 16, characterized in that said administration is carried out nasally. 25

18. A method of producing antibodies, characterized in that it comprises introducing into a host organism, via the mucosa, lamellar vesicles with an onion-like structure having an internal liquid crystal structure 30 formed by a stack of concentric bilayers based on amphiphilic agents alternating with layers of water, an aqueous solution or a solution of a polar liquid and into which at least one antigen is incorporated, then removing and purifying said antibodies. 35

19. A method of producing IgA, characterized in that it comprises introducing into a host organism, via the 33 mucosa, lamellar vesicles with a multilamellar onion-like structure with an internal liquid crystal structure formed by a stack of concentric bilayers based on amphiphilic agents alternating with layers of water, an 5 aqueous solution or a solution of a polar liquid and into which the appropriate antigen is incorporated, and removing and purifying said immunoglobulins.