EP1212043A2 - Composition to be administered through mucous membrane - Google Patents

Abstract

The invention concerns a novel use of onion-structured multilamellar vesicles having a liquid-crystal internal structure formed by stacked concentric double layers based on amphiphilic agents with layers of water, aqueous solution or solution of polar liquid and wherein is incorporated at least an antigen for making a composition, in particular a pharmaceutical composition, and more particularly a vaccine composition, to be administered through a mucous membrane to induce a mucous and/or serumal systemic response and/or for protecting the system against infection caused by said antigen. The invention also concerns a vaccine method through a mucous membrane and a method for producing antibodies, in particular, IgA. The invention is particularly applicable for intranasal administration.

Description

Compositions for mucosal administration.

The present invention relates to novel compositions for administration via the mucosa. More particularly, it relates to a pharmaceutical composition, in particular a vaccine composition intended for administration by the mucous route.

It also relates to a method for producing antibodies, in particular IgA.

Some definitions are given below: Mucosal administration: non-invasive administration of the antigen to a mucous site, for example

- nasopharyngeal area

- oral area

- bronchial tree - intestine

- urogenital tract

- inner ear

- conjunctiva

- mammary, salivary and lacrimal glands. Among these various constituents of the lymphoid tissue associated with the mucous membranes (MALT), certain routes of introduction are easier to access and accept: nasal, buccal, gastrointestinal, rectal and vaginal. Mucosal response: response of the immune system to the mucous membranes characterized by production of IgA (isotype in high concentration in the mucous membranes) and of IgG and / or a cellular response within the mucous site and the draining glands.

Svstemic response: generalized immune system response resulting in the presence of circulating antibodies (IgG and also IgA) and / or a cellular response (Thelper or CTL) in secondary lymphoid organs (spleen, lymph nodes).

Dissemination of the response: installation of a mucosal response in other compartments than that of induction, due to the circulation and relocation of the induced lymphocytes to the site of administration (vaginal IgA after nasal administration for example). Adjuvant: substance added to the antigen so as to amplify and orient the specific immune response of this antigen. Synthetic vector / vectorization: inco oration of the antigen in or on the surface of a particle or vesicle, so as to protect the antigen and / or facilitate its capture by competent cells of the immune system and / or facilitate the preparation of the antigen by said cells, and thus to boost the immune response. The addition of an empty vector to the free antigen provides little or no amplification.

Until recently, vaccines were obtained from killed, attenuated or less virulent microorganisms. The pharmaceutical industry today seeks to avoid this approach for reasons of limitation of side effects, ease of production, safety of administration and efficiency.

Advances in molecular biology have enabled the industrial production of subunits of these microorganisms, in particular so-called recombinant proteins, whether they are membrane, nuclear or cytoplasmic. Unfortunately these subunits are not immunogenic enough by themselves to provide a sufficient response from the point of view of vaccination.

They need to be added or vectorized to induce a sufficient response.

Today, the only adjuvants accepted in human medicine are aluminum hydroxide or phosphate as well as calcium phosphate. These salts are in the form of a suspension of grains of aluminum or calcium salt, on the surface of which the antigen is adsorbed. They have several drawbacks: they induce local inflammatory reactions and the production of IgE. they are not effective for all antigens and they are unable to generate cell-mediated CTL-type reactions. In fact, they have not been used so far in the case of administration by mucosa.

However, the importance of the mucous surfaces is considerable, on the one hand because they are present at the level of all the tracts and on the other hand because they are the first line of defense against the invasion of pathogenic agents. The protection of the mucous surfaces is ensured both by innate or non-adaptive defense mechanisms (peristalsis, ciliated movements and mucus) and by the implementation of an adaptive cellular and humoral immune response specific to the pathogen which can generalize to other lymphoid organs. It is the lymphoid tissue associated with the mucous membranes (MALT) which is responsible for the specific component. In this respect, mucosal vaccination represents a considerable _challenge .

Today, all vaccines except that against polio are injectable, which implies: - A medicalization

- A bad welcome

- Discomfort (fever, pain at the injection site ...)

- Risk of contamination (HIV, hepatitis)

- High cost in developing countries Mucosal administration would present an advantage for various reasons:

- lower production costs linked to less restrictive preparation conditions than those for an injectable vaccine

- its non-invasive nature which eliminates the injection-related problems described above

To do this, it should:

- provide immunity to mucosal administration sites, making it possible to strengthen the mucosal barrier by a specific response, hence an interest in infections transmissible by mucosa (air, genital, etc.)

- provide systemic immunity and a generalized effectπce response following an easy administration

- do not cause side effects (local irritation).

For a fairly complete review of mucosal immunology, reference may be made to the "Handbook of mucosal immunology", edited by Pearay Ogra et al, 1994, Académie press, Inc. san Diego California, USA.

Numerous trials and studies have been carried out in order to develop new adjuvants and vectors capable of inducing an effective mucosal and systemic response in terms of protection against the pathogenic agent. Different works relate to the use of recombinant living vectors (attenuated bacteria or viruses), synthetic vectors (immunostimulating complexes also called iscoms, which corresponds to an abbreviation of the English expression "immunostimulating complexes", liposomes, microspheres ... ) or toxins from microorganisms (choleπque toxin -CT- or thermolabile toxin from E. Coli -LT-) However, a large part of these studies are still in the development stage and the majority of formulations have disadvantages: - high toxicity (CT and LT) and therefore impossibility of use in humans,

- preparation difficulties (microspheres),

- low stability (liposomes), - low efficiency (liposomes).

Several patents relate to the mucosal administration of antigens, vectorized or adjuvanted by different systems. We can cite :

- EP 0 440 289 (Duphar) which describes the use of liposomes in mixture with an antigen for vaccination against influenza. This is an adjuvant effect of lipids, the same result being obtained when the antigen is mixed extemporaneously with empty liposomes.

- WO 98/10748 (The School of Pharmacy) using cationic liposomes, in the case of an injection or a mucosal administration, - US 5,679,355 (Proteus) describing nonionic vesicles, which can be administered by the parenteral or mucosa (especially oral).

On the other hand, a very abundant scientific literature describes the use of polymer microparticles as a vector of antigen by mucous route. A good review of this literature can be found in: D. T. O'HAGAN, Adv. Drug Deliv. Rev., 34 (1,198) 305-320.

However, none of these technologies has resulted in a product on the market today. Antigen presentation methods therefore still need to be improved and the development of new adjuvants or antigen vectors remains an urgent need in the vaccine industry. Among the vesicles, spherical objects formed from molecular arrangements of amphiphilic molecules, the multilamellar vesicles with onion structure have been the subject of considerable 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 with thermodynamic equilibrium

- their internal crystal-liquid structure formed by a regular stack of concentric bilayers of amphiphiles alternating with layers of water or aqueous solution or solution of a polar liquid (glycerol, for example) - the varied nature of the molecules amphiphiles which can be used to constitute them, alone or in mixture. International application WO 99/16468 detects vesicles with an onion structure incorporating within them an antigen and making it possible to amplify the immune response of this antigen. This international application which for the first time detects the incorporation (also designated in this document by encapsulation) of antigens within a multilamellar vesicle with onion structure clearly shows in its examples that such encapsulation allows very clearly boost the immune response during parenteral administration of an antigen encapsulated in a multilamellar vesicle with an onion structure. The inventors of the present invention have now discovered that the administration by mucous route, in particular nasal, of the compositions of the type of those described in application WO 99/16468, generates an immune response not only within different mucous sites but also in the system compartment This response is characterized by the induction of immunoglobulin A (IgA). Such a result was by no means evident in view of the application WO 99/16468 since under the conditions in which the injection was made in the examples of this application, the antigen, even in encapsulated form, did not induce an IgA response detectable.

Immunoglobulins (Ig) or antibodies (Ac), associated with cellular components, are essential actors in the wild immune response against a pathogen. Ig are highly specific proteins of the antigen

Different classes of antibodies have been listed, which are distinguished by their mode of induction, their location and their function (recognition, neutralization of toxic or enzymatic activity)

In the blood compartment, the majority of the Ig produced are circulating IgG (subdivided into different subclasses), IgA and IgE remaining very minority. Conversely, the major isotype in the mucous membranes is IgA which is produced by the IgA plasma cells of the mucous lymphoid system and actively secreted by the epithehum of the mucous membrane.

IgA are specialized antibodies adapted to the defense of mucous surfaces constantly exposed to pathogens Adapts because, because of their biochemical structure (glycosylation, polymerization, secretory), they resist the effects of proteases secreted by microorganisms and because, unlike other isotypes, they limit the inflammatory reactions in these compartments in a state of constant activation Specialized, because they act several secreted levels, they agglutinate pathogens, limit by neutralizing the effects of toxins and the entry of pathogens; they can also act within the epithelium during their transcytosis or in the lamina propπa. They therefore represent an essential active barrier which, associated with non-specific mechanisms, limit invasion by pathogens. Naturally, with the antibody response, there are associated cytotoxic and specific cellular responses which aim to eliminate the pathogen. If the local response is not sufficient, a systemic response is triggered.

One of the very interesting characteristics of the mucosal immune system is the recirculation of B and T lymphocytes induced at a mucous site and their possible domiciliation in sites other than those of induction, thus propagating the specific response to other tracts. This phenomenon is very interesting in terms of vaccination

While the mechanisms of induction of a mucosal immune response are being elucidated, it often remains extremely difficult to induce a mucosal response by parenteral administration of an antigen (adjuvant or not) and even by mucosal administration. of the antigen. In fact, parenteral administration of an antigen leads to the induction of specific circulating antibodies, of the IgG type. This type of injection does not make it possible to induce IgA within the mucous (or systemic) compartments. It is very clear that parenteral vaccination will not strengthen the local natural defenses necessary to fight against many respiratory or genital infections for example

For these various reasons, it is entirely surprising, even in view of the teaching of WO 99/16468, that the same antigen incorporated into the same vesicles, administered by mucous (nasal) route not only induces a systemic response (IgG in sera) confirming the previous results in another way, but also a response in the mucous sites The incorporation of the antigen in the onion-structured vesicles generates the production of IgA in the hey compartment at the administration site ( bronchopulmonary) but also in other mucous compartments with a predominance in genital secretions In addition, it should be noted that human serum albumin (HSA). used by way of example, is a very weakly immunogenic antigen, which, administered by the nasal route, alone, without being incorporated into vesicles, is incapable of inducing any response, whether mucous or systemic In addition, it can be noted that the administration by mucous membrane requires, in relation to parenteral administration, the management of the antigen in the mucosa and therefore the penetration of the antigen, which implies that the antigen must be optimally presented in order to overcome non-specific defense mechanisms (ciliated movements, mucus) and to resist enzymes present in the mucosa or on its surface. It appears that these parameters are optimized by the structure of the vesicles during mucosal administration. These functions of the vesicles in mucosal administration were not necessarily present during parenteral administration, where the injection makes it possible to directly reach areas more favorable for capture by the cells of the immune system.

The result observed by mucosal administration is therefore not a simple extrapolation of the result obtained by parenteral administration, but the demonstration of a new functionality of onion-structured vesicles.

The inventors of the present invention have now discovered that vesicles identical to those found in international application WO 99/16468 incorporating an antigen provoke an immune response much stronger than the administration of the free antigen In addition, for certain antigens which do not induce any response by mucous administration, their administration, incorporated within multilamellar vesicles with onion structure, makes it possible to induce a quite remarkable response Finally, the induced response is found not only locally in the mucous compartment (response mucosa) but also in the blood circulation (systemic response)

On the other hand, these multilamellar vesicles are prepared from biocompatible constituents known for their harmlessness. In addition, the preparation process is simple to implement and only uses common chemical devices. The fact that the process uses an initial lamellar phase at thermodynamic equilibrium gives it excellent reproducibility and allows great stability of the vesicles obtained

Thus, according to one of its essential characteristics, the invention relates to the use of multilamellar vesicles with an onion structure having an internal liquid-crystal structure formed by a stack of concentπque bilayers based on amphiphilic agents alternating with layers of water, aqueous solution or solution of a polar liquid and within which is found incoφor at least one antigen, for the manufacture of a composition, more particularly pharmaceutical, and in particular vaccine, intended for administration by mucous route.

By the term "incoφorated" whose use seems preferable to that of the term "encapsulated", we mean that the antigen (s) are an integral part of the entity formed by the vesicle. Indeed, one can find molecules of antigen (s) in any layer between the center and the periphery of said vesicle.

As emerges from the detailed description which follows as well as from the examples, the pharmaceutical compositions used according to the invention allow the preparation of a mucous vaccine intended to induce a serum mucosal and or systemic response.

As is clear from the examples, the onion-structured vesicles as defined above incoφorating an antigen induce, when administered by mucosa, in humans or animals, the production of anticoφs.

As has been indicated, one of the advantages of the invention is to induce a very large production of anticoφs characterized by the presence of IgA and IgG. This implies an increase in the frequency of lymphocytes carrying IgA or IgG specific for the antigen. The preparation can therefore be used for activation and differentiation of antigen-specific B lymphocytes which can then be used in cell fusion in order to produce monoclonal antibodies. Specifically, the specific lymphocytes present in large quantities in the ganglia draining the administration site can be immortalized by fusion with a non-secreting myeloma and lead to hybridomas secreting monoclonal anticoφs.

The invention, by its capacity to amplify and increase the anticoφs responses, can also be used for the purpose of producing anticoφs in particular polyclonal IgA, isotype difficult to generate in conventional procedures, or polyclonal IgG . These anticoφs can be used for non-therapeutic purposes, for example for research, more particularly for research in biology or immunology.

The methods for collecting and purifying the anticoφs are known to those skilled in the art. Thus, the invention also relates to a process for producing anticoφs. and more particularly of IgA. Furthermore, it has been demonstrated that the composition of the invention, when administered via the mucous membrane, induces protection of the organism with regard to the infection for which the antigen incorporated in said is responsible. composition. Thus, according to another of its essential characteristics, the invention relates to a method of treatment of human or animal coφs by vaccination by mucosa, according to which a composition containing multilamellar vesicles with onion structure having an internal cπstalquid structure formed is administered. of a stack of concentric bilayers based on amphiphilic agents alternating with layers of water, aqueous solution or solution of a polar liquid and within which there is incoφorated at least one antigen

As is apparent from the description and from the examples which follow, it turns out that the compositions of the invention in which the antigen is found incoφorated in a vesicle with onion structure, as defined above, allow, when 'They are administered via the mucosa, to elicit a mucosal immune response and to stimulate the lymphoi ^' tissue of common mucosa. Such capacities prove to be of particular importance when dealing with antigens with vaccinating potential, in the face of mvasive pathogens with mucous tropism.

The double 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 mucosal levels to defend the pathways to microorganisms and systems. for more disseminated or generalized infections

Furthermore, the results obtained in the context of the invention demonstrate that the invention not only makes it possible to amplify the anticoφs response, but that it also generates an immune response with protective efficacy against infection. The invention is therefore applicable both to the production of anticoφs, and to vaccination

The results obtained make it possible to affirm that the vesicles administered by mucous route, in particular by nasal route, can be used in order to induce or amplify the antico ants response in the mucous compartments, to disseminate this response to other sites more away from the administration site and generate a systemic response The vesicles used according to the invention generally have diameters between 0.1 and 25 μm, preferably between 0.2 and 15 μm.

More specifically, the vesicles used according to the invention preferably 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, we therefore obtain a compπs diameter between 0.1 μm and a few tens of micrometers. This is what is observed experimentally, the vesicles being observable by optical microscopy (in polarized light in order to have a better contrast linked to their birefringence), either as unresolved points for the smallest of them, or as birefringent spheres for larger ones. The size profile can be studied using a laser granulometer (using the static scattering of a laser beam, analyzed from several angles). One generally obtains a Gaussian profile centered on a value ranging between 0.1 and 25 μm showing a small heterogeneity of the size for a given formulation, under given operating conditions of preparation.

The vesicles in which the antigen is found have, as previously explained, a multilamellar onion structure and are made up, from their center to their periphery, of a succession of lamellar layers separated by a liquid medium. These vesicles can be obtained by a process comprising the preparation of a crystalline lamellar phase and its transformation by application of a shear. Such a method is in particular described in patent WO 93/19735 from French patent FR-2 689 418 or WO 95/18601 introduced here by reference.

According to French patent FR-2 689 418, this transformation can be made during a homogeneous shearing step of the cπstal-hquide phase. which leads to vesicles also called microcapsules of controlled size. However, by playing on the formulation of the liquid-crystal lamellar phase, in particular on the nature of the surfactants entering into its composition, the transformation of this cπstalquide phase into vesicles can be obtained by simple mechanical stress, in particular during mixing. constituents.

Such vesicles have, among other things, the advantage of being able to be prepared by a particularly simple preparation process making it possible to use a large variety of surfactants. Another advantage, hey also essentially to the process used to prepare the onion-structure vesicles used according to the invention, lies in the fact that the ingredients and the additives are incorporated prior to the formation of the vesicles, which allows a excellent encapsulation yield, hence better efficiency and very significant savings for extremely expensive molecules.

Such structures are advantageously obtained by incoφoration of at least one antigen in a cπstalquide lamellar phase comprising at least one surfactant, then transformation of this lamellar cπstalquide 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 crystal-liquid phase is incorporated which incoφor at least one antigen and the said crystal-liquid phase is reacted in multilamellar vesicles by application of a shear This shear pouπa be a homogeneous shear, which has the advantage of leading to vesicles of perfectly homogeneous size. However, simple mechanical stirring may prove to be sufficient to lead to the formation of the multilamellar vesicles of the invention.

The antigen may be any molecule for which it is desired to generate an immune response, whether it has an exogenous source such as an infectious pathogenic organism, parasites or microorganisms (yeasts, fungi, bacteria or viruses) or an intrinsic natural origin. (case of autoimmune diseases or canceπsation) It can be of different biochemical natures. It can, in particular, be an antigen chosen from the group consisting

- proteins, in particular extracted or recombinant proteins, glycosylated or not,

- peptides, - popeptides,

- polysacchaπdes, or representing a mixture of several of these components

The multilamellar vesicles with onion structure are prepared according to the methods previously described, in particular in international application WO 99/16468 The amphiphilic molecules used for their preparation will be chosen, without this being an obligation, from among the molecules described in the pharmacopoeia, or already used in drugs applied to the mucous membranes.

According to an advantageous variant, the membranes 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,

- Cg to C30 fatty acids, saturated or mono- or polyunsaturated, linear or branched, in the form of the acid or salt of an alkali, alkaline earth metal or of an amine, - esters, ethoxylated or not , of these same fatty acids and

. sucrose,

. sorbitan,

. from mannitoi,

. glycerol or polyglycerol,. glycol,

- mono-, di- or triglycerides or mixtures of glycerides of these same fatty acids,

- C 1 to C 30 fatty alcohols, saturated or mono- or polyunsaturated, linear or branched, ethoxylated or not, - ethers, ethoxylated or not, of these same fatty alcohols and

. sucrose,

. sorbitan,

. from mannitoi,

. glycerol or polyglycerol,. glycol,

- polyethoxylated vegetable oils, hydrogenated or non-hydrogenated,

- block polymers of polyoxyethylene and polyoxypropylene (poloxamers),

- polyethylene glycol hydroxystearate, - alcohols with a sterol skeleton such as cholesterol, sistosterol,

- sphyngolipids,

- polyalkylglucosides,

- copolymers of polyethylene glycol and of alkyl glycol (for example the family of ELFACOS from AKZO NOBEL), - di- or triblock copolymers of ethers of polyethylene glycol and of polyalkylene glycol (for example the family of ARLACELL from ICI). Co-surfactants may be added to these surfactants which can be used alone or as a mixture in order to improve the viscosity and the tightness of the membranes forming the vesicle. Among these molecules, there may be mentioned: - cholesterol and its derivatives, in particular charged or neutral cholesterol esters such as cholesterol sulfate

- other deπvés with a sterol skeleton, in particular those of plant origin (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 scales, which makes it possible to continuously adjust the properties of the bilayers and thus to control the appearance of the instability which governs the formation of multilamellar vesicles. Thus, one will advantageously choose from the above surfactants, two surfactants having relatively different properties, in particular a different hydrophilic-lipophilic balance (HLB). The first surfactant will advantageously have a hydrophilic-lipophilic balance compπse between 1 and 6, preferably between 1 and 4, while the second surfactant will have a hydrophilic-lipophilic balance between 3 and 15, preferably between 5 and 15

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

The encapsulation technique used according to the present invention makes it possible to easily achieve very high encapsulation yields, or even close to 100%. However, such yields are not always essential depending on the intended applications. Thus, the encapsulation yield of the antigen (s) in the compositions of the invention is advantageously greater than 50%, preferably greater than 80%

It seems that the structure of the vesicles is responsible for the particularly advantageous results obtained, and that the multilamellar vesicles of the invention allow the antigen to arrive intact up to the antigen presenting cells (APC) and to promote its capture. by these cells. So it looks good 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 we can add to this formulation natural or artificial polymers such as polysacchaπdes (alginates, chitosan, etc.) in order to strengthen the solidity of the vesicle, and allow it to stay longer on the site of administration or in the organism, thus delivering the antigen over a longer time. These polymers can be incorporated in the vesicle as well as deposited around it in the form of a coating. In this case, the vesicle or the particle formed from vesicles coated in the polymeric matπce has a diameter greater than that of the vesicles alone. These polymers can optionally be crosslinked to further strengthen their solidity.

In addition, and this is one of the other advantages of the technology, the formulation can be supplemented by the addition of immunomodulating molecules (chitosan, interleukmes ...) which will reinforce by their intrinsic properties the amplification and the orientation of the immune response.

The vesicles incoφorating the antigens are advantageously prepared in a process consisting in preparing, firstly, the lamellar phase. This is obtained by simple mixing of the ingredients, in an order determined by the experimenter according to the miscibihtees of each of the constituents. It may be necessary to heat certain pasty or solid constituents in order to facilitate their incoφoration. In this case, the antigen is added preferably at the end of the mixture in order to avoid it being subjected to an excessively high temperature. It is also possible to prepare a mixture of all the constituents except for the antigen or its aqueous solution in the form of a “stock” mixture which will be used as necessary to prepare the lamellar phase. The aqueous solution can contain different constituents intended to ensure its biological compatibility and in particular buffer mixtures but also different antigens. The lamellar phase thus prepared is then subjected to moderate shearing (from 0 to 1000 s ⁻¹ ) for a limited time (from 0 to 60 minutes).

In most cases, this shearing 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 using a microspatula in an Eppendorf type tube. The sheared lamellar phase is then dispersed in a final medium, in the form of water. or a stamp, identical or different from that used during of the preparation of the lamellar phase. This dispersion is advantageously carried out at room temperature (20-25 ° C) by slow addition of the medium to the lamellar phase with constant stirring.

A preservative and possibly other additives intended to complete the galenical formulation can be added to the product.

All of the compositions previously determined comprising at least one incoφorated antigen within the vesicles with a lamellar structure onion have the advantage of being able to be used for administration by mucous route and very particularly by nasal route and of inducing a mucosal response and / or systemic.

Example I given below clearly demonstrates such an effect with HSA

Figures 1 and 2 given with reference to this example bring together the responses obtained on the one hand in different mucous samples (Figure 1) and on the other hand in the sera of animals (Figure 2) after nasal administration of compositions according to the invention (group I) in comparison with those obtained by administration either of compositions in which the same antigen is free (group II), or of compositions containing the same but empty vesicles (group III). Example II illustrates a method of immunization with FHA. The results obtained in this example are illustrated by Figures 3, 4 and 5 which respectively show

- Figure 3 the anticoφs responses in different mucous sites.

- Figure 4. the anticoφs responses in the sera, - Figure 5: the pulmonary bacterial load of the mice infected with

Bordetella pertussis

EXAMPLES

EXAMPLE I PRODUCTION OF SPECIFIC HSA ANTIBODIES

I - Preparation of vesicles containing human serum albumin (HSA)

Formulation (in percentage by weight)

O Potassium oleate (FLUKA) 05.0% θ Lauπque alcohol ethoxylated with 4 ethylene oxides (SEPPIC) 02.0% θ Cholesterol of lanolin (FLUKA) 05.0% O Cholesterol 3-sulfate (SIGMA): 02.5%

© PBS lx stéπle (LIFE TECHNOLOGIES): 20.0%

© Phospholipon 90G soy lecithm (NATTERMANN): 45.5%

© Human albumin (SIGMA) at 30 mg / ml in 1x PBS: 20.0%

Procedure

The components are sterilized by UV irradiation for 60 min. Containers and accessories (spatulas, stirrers ...) are sterilized with the flame _just prior use. The constituents O to θ are introduced into a piluher, in an indifferent order, then heated at 80 ° C. for 60 min with very strong magnetic stirring. The total dissolution of the constituents © and © is checked by microscopic observation.

Is introduced at room temperature into an Eppendorf tube of 1.5 ml stéπle the desired quantity of the mixture © to θ then the constituents © and © are added. The whole is homogenized using a stéπle needle, then left to stand

The preparation is then dispersed at 33.33% in sterile 1x PBS.

II - Immunization protocol

In order to test the effect of the vesicles according to the invention during mucosal administration, female BALB / c souπs of 6-8 weeks of age received, nasally, twice (on D0 and D30), various preparations described below. - below. Administration by the nasal route requires the anesthesia of the animals with a solution, mixture of Ketamme, Valium and Atropine, injected by intra-petoneal route. One month after the last immunization, the animals are sacrificed, the sera are collected and the mucous secretions are taken, with the exception of vaginal washes which are taken from the live mouse during the three days preceding the end of the experiment.

Immunization groups

The souπs were divided into 4 groups noted I to IV, group IV constituting a control group (unimmunized mice also called naive mice) and the other groups being subjected to an immunization protocol as defined above using the following products ^• - group I:

* Encapsulated HSA: 20 μl per nostril of vesicles according to the invention incoφorating HSA which corresponds to 80 μg of HSA per mouse

- group II: * HSA: 20 μl per nostril of an HSA solution corresponding to

80 μg per mouse

- group III:

* Empty vesicles: 20 μl per nostril of empty vesicles according to the invention

Secretion samples

All samples are taken with cold solutions (4 ° C) and are stored on ice as and when to limit degradation by proteolytic enzymes.

Bronchoalveolar lavage:

The trachea of the mice is cannulated using a probe and 750 μl of PBS are injected slowly so as not to create hemorrhage, the lungs are thus washed 3 times with the same solution, the sample is then centrifuged in order to remove lung cells and separate into aliquots kept at -20 ° C until assay.

Vaginal washings

These samples are taken from the non-anesthetized live mouse, by injection of 50 μl of PBS into the opening of the vagina, the vagina is washed 3 times with the same solution. This type of sample is renewed for 3 consecutive days to cover variations due to the hormonal cycle of the mouse. Secretions are pooled and stored at -20 ° C.

Intestinal washes The intestine is removed, released from the mesentery and rinsed in water to remove outside blood. It is then cut longitudinally and incubated on ice in 1 ml of a washing solution enriched in protease inhibitor. The whole is centrifuged and the supernatant is collected and frozen at -20 ° C. Antibody determination

The specific HSA antico danss present in the sera and secretions are assayed by ELISA technique in which the IgA and IgG specific for HSA are determined (biotinylated anti-IgA / Streptavidin peroxidase, anti-IgG peroxidase). The results are expressed in mean titer determined relative to a reference serum of naive mouse and corresponding to the inverse of the dilution equal to the reference threshold.

III - Results The results of the specific antico spécifiquess assay are presented in the form of 2 figures (Figure 1 and Figure 2) respectively illustrating the anticoφs responses obtained in the mucous samples for the response associated with the mucous membranes and in the sera of the animals (systemic response ).

In these figures, the number of mice having reacted to the immunization protocol relative to the number of mice subjected to this protocol has also been indicated in each case (the mention "n / m" signifying that n mice reacted to m mice subjected immunization protocol).

It is clear from these two figures that the nasal administration of vesicles according to the invention incoφorant HSA results in a significant production of anticoφs in the lungs (FIG. 1). Only immunization in the encapsulated form generates a pulmonary immune response of isotype IgA and IgG (the existence at the pulmonary level of these two isotypes is reported in various publications). Although this route of administration is not the easiest in animals, all animals immunized by nasal route with the vesicles according to the invention are responders.

Analysis of the other mucosal samples reveals the presence of HSA-specific IgA in the vagina and intestine of animals immunized by the vesicles according to the invention incoφorating HSA, mucous membranes very distant from the site of administration with a predilection for the mucosa. vaginally. These responses indicate a generalization of the induced response in the pulmonary or nasal mucous lymphoid tissue and the circulation and redistribution of activated and differentiated lymphocytes near the site of administration.

Unlike the pulmonary mucosa, the specific isotype response of HSA predominant in the intestine and the vagina is IgA. Although titration of IgG _H + _L could not be performed in vaginal lavage, experiments similar tests performed with another antigen indicate the predominance of IgA in vaginal secretions.

Only the vectoπsee form of the antigen leads to an intense and specific response. The few animals with an detectable IgA response in groups immunized with free HSA (titer of 3 against 650 in vaginal secretions with the vesicles according to the invention containing HSA) cannot be distinguished from the empty vesicle group according to the invention.

The study of animal sera (Figure 2) indicates that the vesicles according to the invention incoφorant HSA administered by nasal route are capable of generating a systemic response characterized by the majority presence of IgG but also by a significant level of IgA seric Only the vectoπsee form leads to this production (IgA HSA alone = 35, IgA vesicles according to the invention HSA = 17,620) It is remarkable to obtain a systemic response of the vectoπsé antigen Recall that it is extremely difficult to induce a serum IgA response by systemic immunization with an antigen alone or accompanied by an adjuvant autoπsé for human use.

In conclusion of these results, not only does the so-called encapsulated form generate a pulmonary immune response but also, only this form allows the dissemination of the response induced in the respiratory tract to other mucous membranes indicating that the vesicles according to the invention are powerful vectors to induce mucosal immunity, by promoting the charge and the induction of the response within a site and by redistributing it to other sites

EXAMPLE II PRODUCTION OF SPECIFIC FHA ANTIBODIES AND PROTECTION AGAINST BORDETELLA PERTUSSIS

The FHA protein is a filamentous adhesin from Bo detella pertussis. pertussis bacteria Unlike HSA previously used, FHA is more immunogenic and therefore capable of inducing an anticoφs response by itself This protein is one of the protective antigens contained in commercial pertussis vaccines Finally, the muπn model can be infects with Bordetella pertussis and it is thus possible to carry out an infection test following mucosal immunization and to show the protective character of the response induced by the immunization A - Induction of antibodies

I - Preparation of vesicles containing FHA

Formulation

© Potassium oleate (FLUKA): 5.0% θ PEG-4 lauric ether (Simulsol P4, SEPPIC): 2.0% θ Lanolin cholesterol (FLUKA): 5.0%

O Cholesterol 3-sulfate (SIGMA): 2.5% θ PBS lx stéπle (LIFE TECHNOLOGIES): 20.0%

© Soy lecithm (Phosphohpon 90G, NATTERMANN): 45.5% θ FHA of Bordetella pertussis in PBS (1 mg / ml): 20.0%

The operating procedure for the preparation is similar to that of Example 1. The finished product has an FHA concentration of 3 μg per 40 μl.

II - Immunization protocol For the study of the anticoφs response, the mucosal immunization and sampling protocol is identical to what was described in Example 1. Two groups of 5 animals were formed, the group I receiving the antigen encapsulated in the vesicles, and group II receiving the non-encapsulated antigen, in solution in PBS. At each immunization, each animal receives 3 μg of FHA, divided into two instillations, one in each nostril.

Secretion samples

The samples are taken strictly identical to what is described in Example 1.

Antibody determination The specific FHA anticoφs present in the serums and secretions are doses by ELISA technique according to a protocol identical to that decπt in Example 1, with the reagents specific to the FHA antigen.

III - Results

The results are presented in FIGS. 3 and 4, where the mean titers of IgA and IgG anticoφs in the mucous secretions (FIG. 3) and in the serum (FIG. 4) are given for the two groups I and II. The mucous secretions are respectively studied as in Example I, from broncho-alveolar washes (A), intestinal washes (B) and vaginal washes (C).

In group I of mice immunized with the antigen incorporated in the vesicles of the invention (FIG. 3) all of the animals respond to the antigen whereas when the free antigen is administered, only 1/5 of the animals are responders and this in a very weak way.

As in the case of HSA in Example 1, a very strong amplification of the antico ants response is observed in the mucosal sites. Immunization by nasal route with the incoφorated antigen in the vesicles of the invention makes it possible to induce a mucosal response (IgA) in the lungs, close to the site of administration (IgA titer for the incoφorated antigen in the vesicles of the invention 8,925 compared to 4.25 for the free antigen) but also a disseminated mucosal response to the other secretory compartments (intestine and vagina) (Title in vaginal IgA 13,500 for the incoφorated antigen in the vesicles of l invention compared to 2.5 for free antigen). In addition, IgGs were detected at significant levels in the three mucosal compartments studied.

As in the case of HSA, high titers of anticoφs IgA and IgG in the circulation are observed (FIG. 4) following the nasal administration of the incoφorated antigen into the vesicles (IgA titer for the antigen in the vesicles of the invention 1,300 compared to 39.2 for the free antigen; IgG titer

1,265,000 compared to 3,000).

The results of this example 2 completely confirm those obtained on HSA. They make it possible to affirm that the vesicles administered by the nasal route can be used in order to induce or amplify the anticoφs response in the mucous compartments. disseminate this response to other sites further away from the administration site and generate a systemic response.

B - Protection test In order to check whether the immunization obtained is protective, a protection test, known as a "challenge test", was carried out. In this experiment, mice previously immunized were infected by the nasal route with a defined number of Bordetella pertussis, then the animals were sacrificed at different times, to observe the progression of the infection. The measurement is made by counting the colonies in the lungs. I - Preparation of vesicles containing FHA

The formulation and the preparation of the vesicles are stnctement identical to that previously used for the caractéπsation of the response anticoφs.

II - Immunization protocol

The immunization protocol and the doses of antigens administered are identical to those used for the induction of antibodies. Three groups of mice are used:

I. Souπs immunized with the incoφorated antigen in the vesicles of the invention IL Mice immunized with free antigen in solution in PBS III. Unimmunized mice

Each group consists of 4 mice, immunized at the same time, with the same batch of antigen, in order to be able to carry out all the infection measurements under the same conditions.

III - Infection protocol

Four weeks after the second immunization, the mice are anesthetized and infected with Bordetella pertussis by the nasal route (5.10 ⁷ bacteria / mice administered in 20 μl) and the initial infectious load is checked as early as 3 hours after infection.

IV - Protocol for measuring the infectious load

The mice are sacrificed and their lungs are removed at various times after infection and diluted in PBS until a homogeneous suspension is obtained. Different dilutions of the suspension are spread on a specific nutet medium of Bordetella and the colonies are counted after three days of growth.

V - Results

The results are presented in FIG. 5 or the number of colonies of Bordetella pertussis in the lungs of the souπs is indicated, 3 days (D3, full baπe) and 5 days (D5, hollow baπe) after infection in millions of bacterial colonies for the three groups of mice below: Group I: mice immunized with the inco incorated antigen in vesicles of the invention.

Group II: mice immunized with free antigen in solution in

PBS.

Group III: non-immunized mice.

It is observed that, from the ^3rd day after infection, the mice immunized with the antigen incoφoré in the vesicles of the invention have a lower bacterial load (factor of 2) than that of mice immunized with the free antigen, and much lower than that of unimmunized mice (factor 8). Remarkably, while 5 days after infection, the mice immunized with the free antigen as well as the non-immunized mice see their bacterial load increase, the mice immunized with the incororated antigen in the vesicles of the invention have fewer bacteria on D5 than on D3 indicating a favorable evolution of the disease. In addition, at this stage, the number of bacterial colonies in the group immunized with the incoφorated antigen in the vesicles of the invention is 6 times lower than in the group immunized with free FHA and 13 times lower than in the group not immune indicating good protection.

These results demonstrate that the invention not only amplifies the anticoφs response, but also generates an immune response with protective efficacy against infection. The invention is therefore applicable both to the production of anticoφs, and to vaccination.

Claims

1. Use of multilamellar vesicles with onion structure having an internal crystal-liquid structure formed by a stack of concentric bilayers based on amphiphilic agents alternating with layers of water, aqueous solution or solution of a polar liquid and in which there is incoφor at least one antigen, for the manufacture of a composition intended for administration by mucous route.

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

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

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

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

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

7. Use according to one of claims 1 or 6, characterized in that said antigen is chosen from the group consisting of: - proteins, in particular proteins extracted or recombinant, glycosylated or not,

- peptides,

- lipopeptides,

- polysaccharides, or represents a mixture of several of these components.

8. Use according to one of claims 1 to 7, characterized in that said vesicles contain at least one surfactant chosen from the group consisting of:

- hydrogenated or non-hydrogenated phospholipids, - Cg to C30 fatty acids, saturated or mono- or polyunsaturated, linear or branched, in the form of the acid or salt of an alkali, alkaline-earth metal or an amine,

- esters, ethoxylated or not, of these same fatty acids and. sucrose,

. sorbitan,. from mannitoi,

. glycerol or polyglycerol,. glycol, - mono-, di- or triglycerides or mixtures of glycerides of these same fatty acids,

- fatty alcohols in C to C30, saturated or mono- or polyunsaturated, linear or branched, ethoxylated or not,

- ethers, ethoxylated or not, of these same fatty alcohols and. sucrose,

. sorbitan,. from mannitoi,

. glycerol or polyglycerol,. glycol, - polyethoxylated vegetable oils, hydrogenated or non-hydrogenated,

- block polymers of polyoxyethylene and polyoxypropylene (poloxamers),

- polyethylene glycol hydroxystearate,

- sterol skeleton alcohols such as cholesterol, sistosterol, - sphyngolipids,

- polyalkylglucosides,

- copolymers of polyethylene glycol and of alkyl glycol,

- di- or triblock copolymers of polyethylene glycol ethers and of polyalkylene glycol.

9. Use according to 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 the tightness of the membranes of said vesicles.

10. Use according to claim 9. characterized in that said co-surfactant is chosen from the group consisting of: - cholesterol and its derivatives, in particular charged or neutral cholesterol esters such as cholesterol sulfate - derivatives with a sterol skeleton, in particular those of vegetable origin such as sitosterol or sigmasterol,

- ceramides.

11. Use according to one of claims 1 to 10, characterized in that said vesicles also contain an immunomodulating substance.

12. Use according to one of claims 1 to 1 1, characterized in that said vesicles have a diameter between 0, 1 and 25 microns, preferably between 0.2 and 15 microns.

13. Use according to 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) of between 1 and 6, preferably between 1 and 4, and the other a hydrophilic-lipophilic balance (HLB) of between 3 and 15, preferably between 5 and 15.

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

15. Use according to one of claims 1 to 14, characterized in that said mucosal administration is a nasal administration.

16. A method of treating human or animal coφs by vaccination, characterized in that it comprises the administration by mucous route of a composition as defined in one of claims 1 to 15.

17. Method according to claim 16, characterized in that said administration is carried out by the nasal route.

18. A method of producing anticoφs, characterized in that it comprises the introduction into a host organism, by mucous route, of lamellar vesicles with onion structure having an internal crystal-liquid structure formed of a stack of concentric bilayers based on amphiphilic agents alternating with layers of water, aqueous solution or solution of a polar liquid and within which there is incoφor at least one antigen and, the collection and purification of these anticoφs.

19. Method for producing IgA, characterized in that it comprises the introduction into a host organism, via the mucosa, of multilamellar vesicles with onion structure having an internal crystal-liquid structure formed by a stack of concentric bilayers based on amphiphilic agents alternating with layers of water, aqueous solution or solution of a polar liquid and within which is found the appropriate antigen and, the collection and purification of these immunoglobulins.