MXPA99004452A - Adjuvant for transcutaneous immunization - Google Patents

Abstract

A transcutaneous immunization system delivers antigen to immune cells without perforation of the skin, and induces an immune response in an animal or human. The system uses an adjuvant, preferably an ADP-ribosylating exotoxin, to induce an antigen-specific immune response (e.g., humoral and/or cellular effectors) after transcutaneous application of a formulation containing antigen and adjuvant to intact skin of the animal or human. The efficiency of immunization may be enhanced by adding hydrating agents (e.g., liposomes), penetration enhancers, or occlusive dressings to the transcutaneous delivery system. This system may allow activation of Langerhans cells in the skin, migration of the Langerhans cells to lymph nodes, and antigen presentation.

Description

ADJUVANT FOR TRANSCUTANEOUS IMMUNIZATION BACKGROUND OF THE INVENTION The present invention relates to transcutaneous immunization, and to adjuvants useful therein, to induce an antigen-specific immune response. Transcutaneous immunization requires the passage of an antigen through the outer barriers of the skin, which are normally impervious to such a step, and an immune response to the antigen. In U.S. Patent Application No. 08 / 749,164, it was shown that the use of the cholera toxin as an antigen causes a strong antibody response that is highly reproducible; The antigen could be applied in a saline solution to the skin, with or without liposomes. In the present application, transcutaneous immunization is shown using adjuvants such as, for example, bacterial endotoxins, their subunits, and related toxins. There is a transdermal immunization report with trans-ferosomes by Paul et al. (1995). In this publication, REF, 30311 transferosomes are used as a carrier for proteins (bovine serum albumin and binding proteins) against which complement-mediated lysis of the sensitized liposomes with the antigen is directed. An immune response was not induced when the solution containing the protein was "placed on the skin, only the transferosomes were able to transport the antigen through the skin and achieve immunization." As discussed in US Patent Application No. 08 / 749,164, the transferosomes are not liposomes Figure 1 of Paul et al (1995) showed that only one formulation of the antigen and the transferosomes induced an immune response, evaluated by the lysis of the liposomes sensitized with the antigen. Antigen in solution, antigen and mixed micelles, and antigen and liposomes (for example, mesophasic smears), applied to the skin did not induce an immune response equivalent to that induced by subcutaneous injection. example, antigen and trans-ferosomes), to validate their negative conclusion that a formulation of antigen and liposomes did not cause transdermal immunization. Paul and colleagues (1995) stated on page 3521 that the skin is an effective barrier that is "impenetrable to substances with a molecular mass of at most 750 DA", excluding non-invasive immunization with large immunogen through the skin. intact Therefore, the reference could be far from teaching the use of a molecule such as cholera toxin (which is 85,000 daltons) because one would expect such molecules not to penetrate the skin and, therefore, could be hope they did not achieve immunization. In this way, the skin represents a barrier that could make the penetration by an adjuvant or antigen such as cholera toxin, unexpected without the description of the present invention. Paul and Cevc (1995) state on page 145, "Large molecules do not normally cross the intact skin of mammals, thus it is impossible to immunize epicutaneously with the simple peptide or the protein solutions. They concluded" the liposomal micellar immunogens or mixed dermally applied are biologically as inactive as single protein solutions, whether or not they are combined with the lipid A immunoadjuvant. "Wang and colleagues (1996) placed a solution of ovalbumin (OVA) in water on the skin of mice shaved to induce an allergic-type response as a model for atopic dermatitis.The mice were anesthetized and covered with an occlusive patch containing up to 10 mg of OVA, which was placed on the skin continuously for four days.This procedure was repeated after of two weeks.In Figure 2 of Wang et al. (1996), an ELISA assay performed to determine the response of the anti IgG2a body showed no response of the IgG2a antibody to OVA. However, IgE antibodies that are associated with allergic responses could be detected. In a further experiment, the mice were more extensively placed with OV patch in solution for four days every two weeks. This was repeated five times, for example, the mice carried the patches for a total of 20 days. Again, the high dose of OVA did not produce significant IgG2a antibodies. Significant levels of IgE antibodies were produced.

The authors state on page 4079 that "an animal model was established to show that epicutaneous exposure to the Ag protein, in the absence of adjuvant, can sensitize animals and induce a dominant response similar to Th2 with high levels of IgE." Intense epicutaneous exposure to high doses of protein antigen could not produce significant IgG antibodies, but could induce IgE antibodies, the hallmark of an allergic type reaction. Thus, Wang et al. (1996) teach that exposure to OVA as described is a model for atopic dermatitis and not a mode of immunization. Therefore, after teaching the reference, it could have been expected that transcutaneous immunization with the antigen could induce high levels of IgE antibodies if it were to pass through the skin and induce an immune response. Instead, we have unexpectedly found that the antigen placed on the skin in an adjuvanted saline solution induces high levels of IgG and some IgA but no IgE. In contrast to the cited references, the inventors have found that the application to the skin of the antigen and adjuvant provides a transcutaneous distribution system for the antigen, which can induce a specific immune response of the antigen, of IgG or IgA. The adjuvant is preferably an ADP ribosyla exotoxin. Optionally, hydration, penetration enhancer, or occlusive dressings can be used in the transcutaneous distribution system.

BRIEF DESCRIPTION OF THE INVENTION An object of the invention is to provide a system for transcutaneous immunization that induces an immune response (eg, humoral and / or cellular effectors) in an animal or human. The system provides simple application to the intact skin of an organism of a formulation comprised of antigen and adjuvant, to induce a specific immune response against the antigen. In particular, the adjuvant can activate antigen-presen cells of the immune system (eg, Langerhans cells in the epidermis, dermal dendritic cells, dendritic cells, macrophages, B lymphocytes) and / or induce antigen-presen cells. for phagocytosis of the antigen. The antigen-presen cells then present the antigen to the T and B cells. In the case of Langerhans cells, the antigen-presen cells can then migrate from the skin to the lymph nodes and present the antigen to the lymphocytes (e.g. B and / or T cells), whereby a specific immune response of the antigen is induced. In addition to causing immune reactions that lead to acceleration of a B lymphocyte and / or T lymphocyte specific for an antigen, including a cytotoxic T lymphocyte (CTL), another objective of the invention is to regulate positively and / or negatively the components of the immune system by using the transcutaneous immunization system to affect antigen-specific T helper cells (Th1, Th2, or both). In a first embodiment of the invention, a formulation containing antigen and adjuvant is applied to the intact skin of an organism, the antigen is presented to the immune cells, and a specific immune response is induced for the antigen without piercing the skin. The formulation may include additional antigens, such that transcutaneous application of the formulation induces an immune response for multiple antigens. In such a case, the antigens may or may not be derived from the same source, but the antigens will have different chemical structures, to induce specific immune responses for the different antigens. Antigen-specific lymphocytes can participate in the immune response and, in the case of involvement by B lymphocytes, antigen-specific antibodies can be part of the immune response. In a second embodiment of the invention, the above method is used to treat an organism. If the antigen is derived from a pathogen, the treatment vaccinates the organism against infection by the pathogen or against its pathogenic effects, such as those caused by - the secretion of toxins. A formulation that includes a tumor antigen can provide a cancer treatment, a formulation that includes an autoantigen can provide a treatment for a disease caused by the body's own immune system (e.g., autoimmune disease), and a formulation that includes a Allergen can be used in immunotherapy to treat an allergic disease.

In a third embodiment of the invention, a patch is provided for use in the above methods. The patch comprises a bandage, and effective amounts of antigen and adjuvant. The bandage may be occlusive or non-occlusive. The patch may include additional antigens such that application of the patch induces an immune response for multiple antigens. In such a case, the antigens may or may not be derived from the same source, but the antigens will have different chemical structures to induce a specific immune response for different antigens. For effective treatment, multiple patches can be applied at frequent intervals or constantly over a period of time. Furthermore, in a fourth embodiment of the invention, the formulation is applied to the intact skin by overlaying more than one field of draining lymph nodes, using either single or multiple applications. The formulation may include additional antigens, such that application to intact skin induces an immune response for multiple antigens. In such a case, the antigens may or may not be derived from the same source, but the antigens will have different chemical structures to induce a specific immune response for the different antigens. The products and methods of the invention can be used to treat the existing disease, to prevent the disease, or to reduce the severity and / or duration of the disease. However, the induction of allergy, atopic disease, dermatitis, or contact hypersensitivity is not preferred. In addition to the antigen and the adjuvant, the formulation may further comprise a hydration agent (eg, liposomes), a penetration enhancer, or both. For example, the antigen-adjuvant formulation may further comprise an emulsion made with AQUAPHOR (petrolatum, mineral oil, mineral wax, wool wax, panthenol, bisabol, and glycerin), emulsions (eg, aqueous creams), water emulsions. in oil (for example, oily creams), anhydrous lipids and oil-in-water emulsions, anhydrous lipids and water-in-oil emulsions, fats, waxes, oil, silicones, humectants (for example, glycerol), a jelly (for example, SURGILUBE, KY jelly), or a combination thereof. The formulation can be provided as an aqueous solution.

The formulation preferably does not include an organic solvent. The formulation can be applied after the skin has been rubbed with alcohol. However, removal of the keratinocyte layer prior to the application of the formulation, to the extent achieved with a depilating agent, is not preferred. The antigen can be derived from a pathogen that can infect the organism (e.g., bacterium, virus, fungus, or parasite), or a cell (e.g., tumor cell or normal cell). The antigen can be a tumor antigen or an autoantigen. Chemically, the antigen can be a carbohydrate, glycolipid, glycoprotein, lipid, lipoprotein, phospholipid, polypeptide, or chemical conjugate or recombinant of the foregoing. The molecular weight of the antigen can be greater than 500 daltons, preferably greater than 800 daltons, and more preferably greater than 1000 daltons. The antigen can be obtained by recombinant means, chemical synthesis, or purification from a natural source. Preferred are the protein antigen or conjugates with polysaccharide. The antigen can be at least partially purified in cell-free form.

Alternatively, the antigen can be provided in the form of a live virus, an attenuated live virus, or an inactivated virus. The inclusion of an adjuvant can allow the enhancement or modulation of the immune response. In addition, the selection of a suitable antigen or suitable adjuvant may allow the preferred induction of a humoral or cellular immune response, specific antibody isotypes (eg, IgM, IgD, IgAl, I gA2, IgE, IgG1, IgG2, IgG3, IgG4, or a combination thereof), and / or subsets of specific T cells (eg, CTL, Th1, Th2, TDTH, or a combination thereof). Preferably, the adjuvant is an ADP-ribosylating exotoxin or a subunit thereof. Optionally, a Langerhans activator can be used. Optionally, the antigen, the adjuvant, or both can be provided in the formulation by means of a nucleic acid (e.g., DNA, RNA, cDNA, cRNA) encoding the antigen or adjuvant, as appropriate. This technique is called genetic immunization. The term "antigen" as used in the invention, is understood to describe a substance that induces a specific immune response when presented to the immune cells of an organism. An antigen can comprise a unique in uniogenic epitope, or a plurality of immunogenic epitopes recognized by a B cell receptor (e.g., the antibody on the B cell membrane) or a T cell receptor. A molecule can be an antigen and an adjuvant (e.g., cholera toxin) and, thus, the formulation may contain only one component. The term "adjuvant" as used in the invention, is understood to describe a substance added to the formulation, to help induce an immune response to the antigen. A substance can act as an adjuvant and antigen by inducing immunostimulation and a specific antibody or T cell response. The term "effective amount" as used in the invention is understood to describe that amount of antigen that induces a specific immune response. for the antigen. * Such induction of an immune response can provide a treatment such as, for example, immunoprotection, desensitization, immunosuppression, modulation of autoimmune disease, enhancement of immunosurvival to cancer, or therapeutic vaccination against an established infectious disease. The term "field of draining lymph nodes" as used in the invention, means an anatomical area over which the lymph collected is filtered through a defined group of lymph nodes (eg, cervical, axillary, inguinal, epitrochear, popliteos, those of the abdomen and thorax).

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the cholera toxin (CT) that induces the expression of class II of the increased complex of increased stoichiometry (MHC), on Langerhans cells (LC), the changes in LC morphology, and the loss of LCs (presumably through migration). BALB / c mice (H-2d) were transcutaneously immunized with 250 μg of cholera CT or its B subunit (CTB) in saline on the ear. Previous experiments had established that mice were easily immunized when the skin of the ear was used (7000 units of anti-CT ELISA after a simple immunization). After 16 hours, epidermal sheets were prepared and stained for MHC class II molecules (the scale is 50 μm). The panels indicate (A) saline alone as a negative control, (B) transcutaneous immunization with CT in saline, (C) transcutaneous immunization with CTB in saline, and (D) intradermal injection with tumor necrosis factor alpha ( 10 μg) as a positive control.

DETAILED DESCRIPTION OF THE INVENTION A transcutaneous immunization system distributes agents to specialized cells (eg, antigen-presenting cells, lymphocytes) that produce an immune response (Bos, 1997). These agents as a class are called antigens. The antigen can be composed of chemicals such as, for example, carbohydrate, glycolipid, glycoprotein, lipid, lipoprotein, phospholipid, polypeptide, protein, conjugates thereof, or any other material known to induce an immune response. The antigen can be provided as a complete organism such as, for example, a bacterium or virion; the antigen can be obtained from an extract or lysate,. either from whole cells or from the membrane alone; or the antigen can be chemically synthesized or produced by recombinant means, or by inactivation of a virus. Processes for the preparation of a pharmaceutical formulation are well known in the art, whereby the antigen and the adjuvant are combined with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their preparation are described, for example, in Remington's Pharmaceutical Sciences by E.W. Martin. Such formulations will contain an effective amount of the antigen and the adjuvant together with a suitable amount of carrier in order to prepare pharmaceutically acceptable compositions, suitable for administration to a human or animal. The formulation can be applied in the form of a cream, emulsion, gel, lotion, ointment, paste, solution, suspension, or other forms known in the art. In particular, formulations that improve skin hydration, penetration, or both, are preferred. Other pharmaceutically acceptable additives may also be incorporated, including, for example, diluents, binders, stabilizers, preservatives, and colorants. Increased hydration of the stratum corneum will increase the rate of percutaneous absorption of a given solute (Roberts and Wal er, 1993). As used in the present invention "penetration enhancer" does not include substances such as, for example: water, physiological buffers, salt solutions, and alcohols, which may not puncture the skin. It is an object of the present invention to provide a novel means for immunization from intact skin, without the need to pierce the skin. The transcutaneous immunization system provides a method by which the antigens and the adjuvant can be distributed to the immune system, especially the specialized antigen-presenting cells underlying the skin such as, for example, Langerhans cells. Without being bound by any particular theory but only to provide an explanation of our observations, it is presumed that the transcutaneous immunization distribution system carries the antigen to the cells of the immune system where an immune response is induced. The antigen can pass through the normal protective outer layers of the skin (for example, the stratum corneum) and induce the immune response directly, or through an antigen presenting cell (eg, macrophage, tissue macrophage, Langerhans cells, dendritic cell, dermal dendritic cell, B lymphocyte, or Kupffer cell) present the processed antigen to a T lymphocyte. Optionally, the antigen can pass through the stratum corneum by means of the hair follicle or an organelle of the skin (for example, the sweat gland, cebaceous gland). Transcutaneous immunization with bacterial ribosylating exotoxins by ADP (bAREs) can be directed to epidermal Langerhans cells, which are known to be among the most efficient of antigen-presenting cells (APCs) (Udey, 1997). bAREs activate Langerhans cells when they are applied epicutaneously to the skin in saline, Langerhans cells direct specific immune responses through phagocytosis of antigens, and migration to lymph nodes when they act as APCs for present the antigen to the lymphocytes (Udey, 1997), and with this they induce a powerful antibody response.Although it is generally considered that the skin is a barrier against the invasion of organisms, the imperfection of this barrier is attested by the numerous cells of Langerhans distributed throughout the epidermis, which are designed to orchestrate the immune response ra organisms that invade via the skin (Udey, 1997). According to Udey (1997): "Langerhans cells are cells derived from the bone marrow that are present in all stratified mammalian squamous epithelia, which include all the activity of accessory cells that are present in the non-inflamed epidermis. , and in the current paradigm are essential for the initiation and propagation of immune responses directed against epicutaneously applied antigens.Langerhans cells are members of a family of potent accessory cells ('dendritic cells') that are widely distributed. but infrequently represented, in epithelia and solid organs, as well as in lymphoid tissue ... "" It is now recognized that Langerhans cells (and presumably other dendritic cells) have a life cycle with at least two distinct stages. Langerhans that are located in the epidermis constitute a regular network of "sentinel" cells that They trap antigens.The epidermal Langerhans cells can ingest particulate materials, including microorganisms, and are efficient processors of complex antigens. However, they express only low levels of MHC class I and II antigens and costimulatory molecules (ICAM-1, B7-1 and B7-2) and are poor stimulators of non-primed T cells. After contact with the antigen, some Langerhans cells become activated, leave the epidermis and migrate to the T-cell dependent regions of the regional lymph nodes, where they are located as mature dentritic cells. In the course of the epidermis leaving and migrating to the lymph nodes, the epidermal Langerhans cells carrying antigen (now the 'mensa j eras') show dramatic changes in morphology, superficial phenotype and function. In contrast to epidermal Langerhans cells, lymphoid dendritic cells are essentially non-phagocytic and inefficiently process protein antigens, but express high levels of MHC class I and class II antigens and various ry costimulatory molecules and are the most potent stimulators of intact T cells that have been identified. "It is considered that the potent antigen presentation capacity of epidermal Langerhans cells can be exploited for transdermally administered transdermal vaccines.A transcutaneous immune response using the immune system - of the Skin may require the distribution of vaccine antigen only to Langerhans cells in the stratum corneum (the outermost layer of the skin consisting of cornified cells and lipids) via passive diffusion and the subsequent activation of Langerhans cells to capture the antigen, migrate to the follicles of cé B cells and / or the T cell regions, and present the antigen to B and / or T cells (Stingl et al., 1989). If the antigens different from the bAREs (for example BSA) were to be. phagocytosed by Langerhans cells, then these antigens could also be carried to the lymph node for presentation to T cells, and subsequently induce a specific immune response for that antigen (e.g., BSA). Thus, a characteristic of transcutaneous immunization is the activation of Langerhans cells, presumably by a bacterial exotoxin of ADP ribosylation, subunits binding to the ADP-ribosylating exotoxin (e.g., B subunit of the cholera), or another activating substance of the Langerhans cells. The mechanism of transcutaneous immunization via Langerhans cell activation, migration and antigen presentation is clearly shown by the upregulation of MHC class II expression in the epidermal Langerhans cells, from the epidermal leaves transcutaneously immunized with CT or CTB. In addition, the magnitude of the antibody response induced by transcutaneous immunization and by the isotype change predominantly to IgG, is generally achieved with the help of T cells stimulated with antigen-presenting cells such as Langerhans cells or dendritic cells ( Janeway and Travers, 1996), and activation of the Thl and Th2 pathways as suggested by the production of IgG1 and IgG2a (Paul and Seder, 1994; Seder and Paul, 1994). In addition, the proliferation of T cells towards the OVA antigen is shown in mice immunized with CT + OVA. Alternatively, a large antibody response can be induced by a thymus-independent antigen type 1 (TI-1) which directly activates the B cell (Janeway and Travers, 1996). The spectrum of immune responses of the most commonly known skin is represented by contact dermatitis and atopy. Contact dermatitis, a pathogenic manifestation of the activation of LC, is directed by the Langerhans cells which phagocytose the antigen, migrate to the lymph nodes, present the antigen, and sensitize the T cells to the intense destructive cellular response, which occurs at the site of the affected skin (Dahl, 1996; Leung, 1997). Atopic dermatitis can use the Langerhans cell in a similar manner, but it is identified with Th2 cells and is generally associated with high levels of IgE antibody (Dahl, 1996, Leung, 1997). Transcutaneous immunization with cholera toxin and related BAREs on the other hand, is a novel immune response with absence of superficial and microscopic skin findings after immunization (eg, non-inflamed skin) shown by the absence of lymphocyte infiltration 24, 48 and 120 hours after immunization with cholera toxin. This indicates that the Langerhans cells "comprise all the accessory cellular activity that is present in the non-inflamed epidermis, and in the current paradigm they are essential for the initiation and propagation of immune responses directed against epicutaneously applied antigens "(Udey, 1997) .The uniqueness of the transcutaneous immune response here is also indicated by the high levels of antigen-specific IgG antibody. and the type of antibody produced (for example, IgM, IgG1, IgG2a, IgG2b, IgG3 and IgA) and the absence of anti-CT IgE antibodies Thus, it has been found that the toxins derived from bacteria, applied in the The surface of the skin can activate the Langerhans cells or other antigen-presenting cells, and induce a strong immune response manifested as high levels of circulating antigen-specific IgG antibodies, such adjuvants can be used in transcutaneous immunization to improve the response of IgG antibody to otherwise non-immunogenic proteins by themselves, when placed on the skin. Transcutaneous to Langerhans cells can also be used to deactivate its antigen-presenting function, thereby preventing immunization or sensitization. Techniques for deactivating Langerhans cells include, for example, the use of interleukin-10 (Peguet-Navarro et al., 1995), monoclonal antibody for interleukin-1 (Enk et al., 1993), or exhaustion or decrease via superantigens such as through the decrease of epidermal Langerhans cells induced by enterotoxin A (SEA) is tafilocócica (Shankar et al., 1996). Transcutaneous immunization can be induced via the binding activity to GM1 ganglioside of CT, LT or subunits such as CTB (Craig and Cuatrecasas, 1975) .Ganglioside GM1 is a ubiquitous cell membrane glycolipid found in all cells of the cell. mammal (Plotkin and Mortimer, 1994) When the B subunit of CT, pentameric is bound to the cell surface, a hydrophilic pore is formed which allows the A subunit to penetrate through the lipid bilayer (Ribi et al., 1988) It has been shown that transcutaneous immunization by CT or CTB may require binding activity to GM1 ganglioside When mice were transcutaneously immunized with CT, CTA and CTB, only CT and CTB resulted in an immune response CTA contains the exotoxin activity of ADP ribosylation, but only CT and CTB containing the binding activity were able to induce an immune response indicating that the B subunit was necessary and enough to immunize through the skin. It was concluded that Langerhans cells and other antigen-presenting cells can be activated by CTB that binds to their cell surface.

ANTIGEN: The antigen of the invention can be expressed by recombinant means, preferably as a fusion with an affinity or epitope tag (Summers and Smith, 1987; Goeddel, 1990; Ausubel et al., 1996); The chemical synthesis of an oligopeptide, either free or conjugated to the carrier proteins, can be used to obtain the antigen of the invention (Bodanszky, 1993, Wisdo, 1994). Oligopeptides are considered a type of polypeptide. Oligopeptide lengths of 6 residues to 20-residues are preferred. The polypeptides can also be synthesized as branched structures such as those described in U.S. Patent Nos. 5,229,490 and 5,390,111. Antigenic polypeptides include, for example, synthetic or recombinant epitopes of B cells and T cells, universal epitopes of T cells, and mixed epitopes of T cells from one organism or disease, and epitopes of B cells from another. The antigen obtained through recombinant means or peptide synthesis, as well as the antigen of the invention obtained from natural sources or extracts, can be purified by means of the physical and chemical characteristics of the antigen, preferably by fractionation or chromatography (Janson and Ryden, 1989; Deutscher, 1990; Scopes, 1993). A multivalent antigen formulation can be used to induce an immune response for more than one antigen at the same time. The conjugates can be used to induce an immune response for multiple antigens, to boost the immune response or both. In addition, toxins can be triggered by the use of toxoids, or toxoids reinforced by the use of toxins. Transcutaneous immunization can be used to reinforce responses initially induced by other immunization routes such as by injection, or oral or intranasal routes. The antigen includes, for example, toxins, toxoids, subunits thereof, or combinations thereof (e.g., cholera toxin, tetanus toxoid). The antigen can be solubilized in water, a solvent such as methanol, or a buffer. Buffers include, but are not limited to, saline solution buffered with Cai + / Mg ++ free phosphate (PBS), normal saline (150 mM NaCl in water), and Tris buffer. The antigen not soluble in neutral buffer can be solubilized in 10 mM acetic acid and then diluted to the desired volume with a neutral buffer such as PBS. In the case of soluble antigen only at acid pH, acetate-PBS can be used at acid pH as a diluent after solubilization in dilute acetic acid. The glycerol can be a non-aqueous buffer suitable for use in the present invention. If an antigen such as, for example, hepatitis A virus is not soluble per se, the antigen may be present in the formulation in a suspension, or even as an aggregate. The hydrophobic antigen can be solubilized in a detergent, for example a polypeptide containing a membrane extension domain. In addition, for formulations containing liposomes, an antigen in a detergent solution (eg, a cell membrane extract) can be mixed with lipids, and the liposomes can then be formed by removing the detergent by dilution, dialysis, or column chromatography. Certain antigens such as, for example, those of a virus (e.g., hepatitis A) need not be soluble per se, but can be incorporated directly into a liposome in the form of a virosome (Morein and Simons, 1985). Plotkin and Mortimer (1994) provide antigens that can be used to vaccinate animals or humans to induce a specific immune response for particular pathogens, as well as methods for the preparation of the antigen, determining an adequate dose of antigen, evaluating the induction of a response immune, and treating the infection by a pathogen (eg, bacteria, virus, fungus, or parasite). Bacteria include, for example: anthrax, campylobacter, cholera, diphtheria, enterotoxic E. coli, giardia, gonococci, Hel i coba ct er pyl ori (Lee and Chen, 1994), Hemophilus infl uenza B, Hemophilus infl uenza no typifiable, meningococcus, whooping cough, pneumonococcus, salmonella, shigella, S trept ococcus B, S trept ococcus group A, tetanus, Vibri or chol era e, yersinia, Staphyl ococcus, Pseudomona species and species of Cl di tri di a. Viruses include, for example: adenovirus, dengue serotypes 1 to 4 (Delenda et al., 1994; Fonseca et al., 1994; Smucny et al., 1995), Ebola (Jahrling et al., 1996), enterovirus, hepatitis serotypes A to E (Blum, 1995, Katkov, 1996, Lieberman and Greenberg, 1996, Mast, 1996, Shafara and colleagues, 1995, Smedila and collaborators, 1994; US Patents Nos. 5,314,808 and 5,436,126), herpes simplex virus 1 or 2, human immunodeficiency virus (Deprez et al., 1996), influenza, Japanese equine encephalitis, measles, Norwalk, papilloma virus, parvovirus B19, polio, rabies, rotavirus, rubella, rubella, vaccinia, vaccinia constructs that contain genes that code for other antigens such as malaria antigens, varicella, and yellow fever. Parasites include, for example: In tamoeba hi s t olyti ca (Zhang et al., 1995); Pl a smodi um (Bathurst et al., 1993, Chang et al., 1989, 1992, 1994, Fries et al., 1992a, 1992b, Herrington et al., 1991, Khus ith et al., 1991, Malik et al., 1991; Migliorini et al. collaborators, 1993, Pessi et al, 1991, Tam 1988, Vreden et al, 1991, White et al, 1993, Wiesmueller et al., 1991), Lei shmani a (Frankenburg et al., 1996), Toxoplasmosis, and Helminths. The antigens can also comprise those used in biological warfare such as ricin, for which protection can be achieved by means of antibodies.

ADJUVANT: The formulation also contains an adjuvant, although one. Simple molecule may contain adjuvant and antigen properties (eg, cholera toxin) (Elson and Dertzbaugh, 1994). Adjuvants are substances that are used to specifically or non-specifically boost an antigen-specific immune response. Usually, the adjuvant and the formulation are mixed before the presentation of the antigen but, alternatively, these can be separately presented within a short time interval. Adjuvants include, for example, an oil emulsion (for example, complete or incomplete Freund's adjuvant), a chemokine (for example, defensins 1 or "2, RANTES, MlPl-a, MIP-2, int erleucine-8) or a cytokine (e.g., interleukin-l, -2, -6, -10 or -12;? -interferon; tumor necrosis factor a: or a granulocyte-monocyte colony stimulation factor (reviewed in Nohria and Rubin, 1994), a muramyl dipeptide derivative (eg, raurabutide, thonyl-MDP or muramyl tripeptide), a heat shock protein or a derivative, a derivative of Lei shmani to maj or LelF ( Skeiky et al., 1995), cholera toxin or cholera toxin B, a lipopolysaccharide derivative (LPS) (for example, lipid A or monophosphoryl lipid A), or a superantigen (Saloga et al., 1996). Also, see Richards et al. (1995) for adjuvants useful in immunization. An adjuvant can be chosen to preferentially induce the antibody or cellular effectors, specific antibody isotypes (eg, IgM, IgD, IgAl, I gA2, secretory IgA, IgE, IgGl, IgG2, IgG3, and / or IgG4), or subgroups specific T cells (eg, CTL, Thl, Th2 and / or TDTH) (Glenn et al., 1995). Cholera toxin is a bacterial exotoxin from the family of ADP ribosylating exotoxins (called bAREs). Most bAREs are organized as dimer A: B with a B subunit of linkage and a subunit A containing ADP-ribosyl trans ferase. Such toxins include diphtheria toxin, Pseudon as exotoxin A, cholera toxin (TC), thermally labile enterotoxin E. coli (LT), pertussis toxin, C2 toxin from C. bo t ul i num, C3 toxin from C. bo t ul i num, exoenzyme from C. l imosum, exoenzyme from B. cereus, S exotoxin from Pseudomonas, Staphyl ococcus to ureus EDIN, and B toxin. sphaeri cus. Cholera toxin is an example of bARE that is organized with subunits A and B. Subunit B is the binding subunit and consists of a subunit B pentamer that is non-covalently bound to subunit A. Subunit B pentamer is accommodated in a symmetrical structure in the form of a donut that binds to ganglioside GMi on the target cell. Subunit A serves for ADP-ribosylating the alpha subunit "of a subset of the heterotrimeric GTP proteins (G proteins) including the Gs protein which results in high intracellular levels of cyclic AMP.This stimulates the release of ions and fluid from intestinal cells in the case of cholera Cholera toxin (CT) and its subunit B (CTB) have adjuvant properties when used as either an intramuscular or oral immunogen (Elson and Dertzbaugh, 1994; Trach et al., 1997) Another antigen, the heat-labile enterotoxin from E. coli (LT) is 80% homologous to the amino acid level with CT and has similar binding properties; it also appears to bind to the GM ganglioside receptor in the intestine, and has similar ADP ribosylating exotoxin activities. Another bARE, Pseudomonas exotoxin A (ETA), binds to the protein related to the low density lipoprotein receptor, macroglobulin receptor a2 (Kounnas et al., 1992). The bAREs are reviewed by Krueger and Barbieri (1995). The toxicity of CT by the oral, nasal, and intramuscular routes limits the dose that can be used as an adjuvant. In a comparative CT test injected intramuscularly, extensive swelling at the site of the injection was caused. In contrast, equivalent or greater doses of CT placed on the skin did not cause toxicity. The following examples show that cholera toxin (CT), its B subunit (CTB), the enterotoxin of E. Heat-labile coli (LT), and pertussis toxin are potent adjuvants for transcutaneous immunization, inducing high levels of IgG antibodies but not IgE antibodies. It is also shown that CTB without CT can also induce high levels of IgG antibodies. In this way, bAREs and a derivative thereof can immunize effectively when applied epicutaneously to the skin in a simple solution. In addition, these examples demonstrate that CT, CTB and bAREs can act as an adjuvant and as an antigen. When an adjuvant such as CT is mixed with BSA, a protein not usually immunogenic when applied to the skin, anti-BSA antibodies are induced. An immune response for diphtheria toxoid was induced using pertussis toxin as an adjuvant, but not with diphtheria toxoid alone. In this way, the bAREs can act as adjuvants for non-immunogenic proteins in a transcutaneous immunization system. Other proteins can also act as an adjuvant and as an antigen. For example FLUZONE (Lederle), the divided virion vaccine of influenza "A and B contains neuraminidase and aglut inina which are highly immunogenic, confers protection and can immunize effectively through the skin acting as its own adjuvant and antigen. Toxins such as diphtheria toxoid that have been toxoids using formalin, pertussis toxoid that has been toxoid using hydrogen peroxide, or mutant toxins such as cholera enterotoxin or heat labile E. coli, which have been toxoids using genetic techniques to destroy ribosyltransferase activity, can continue to harbor adjuvant qualities and act as antigen and adjuvant.Protection against life-threatening infections such as diphtheria, pertussis, and tetanus (DPT) can be achieved By inducing high levels of circulating anti-toxin antibodies, whooping cough may be an exception, that some researchers feel that these antibodies directed to other portions of the invading organism are necessary for protection, although this is controversial (see Schneerson et al., 1996) and most vaccines against acellular acellular cough of new generation have PT as a component of the vaccine (Krueger and Barbieri, 1995). Pathologies in diseases caused by DPT are directly related to the effects of their toxins, and antitoxin antibodies most certainly play a role in protection (Schneerson et al., 1996). In general, toxins can be chemically inactivated to form toxoids that are less toxic but remain immunogenic. It is considered that the transcutaneous immunization system that uses toxin-based immunogens and adjuvants, can achieve anti-toxin levels suitable for protection against these diseases. Anti-toxin antibodies can be induced through immunization with the toxins, or genetically detoxified toxoids themselves, or with toxoids and adjuvants such as CT or by toxoids alone. Genetically toxoid toxins that have ADP ribosylation endotoxin activity, altered but not binding activity, are considered especially useful as non-toxic activators of antigen-presenting cells used in transcutaneous immunization. It is considered that CT can also act as an adjuvant to induce antigen-specific CTLs through transcutaneous immunization (see 'Bowen et al., 1994; Porgador et al., 1997 for the use of CT as an adjuvant in oral immunization). The bARE adjuvant can be chemically conjugated to other antigens including, for example, carbohydrates, polypeptides, glycolipids, and glycoprotein antigens. Chemical conjugation with toxins, their subunits, or toxoids with these antigens, could be expected to improve the immune response to these antigens when applied epicutaneously. To overcome the problem of toxicity of toxins, (for example, it is known that diphtheria toxin is so toxic that a molecule can kill a cell) and to overcome the difficulty of working with such potent toxins as tetanus, several workers have taken a recombinant procedure to produce genetically produced toxoids. This is based on the inactivation of the catalytic activity of ADP-ribosiltrans ferase by genetic suppression. These toxins retain the binding capacities, but lack the toxicity, of the natural toxins. This procedure is described by Burnette et al. (1994), Rappuoli et al. (1995), and Rappuoli et al. (1996). Such genetically toxoid exotoxins could be useful for the transcutaneous immunization system, since they could not create a safety problem, since the toxoids could not be considered toxic. These can act as antigens and as adjuvants, improving the immune response to themselves or to aggregated antigens. In addition, there are several techniques to produce chemically toxoid toxins, which can face the same problem (Schneerson et al., 1996). Alternatively, fragments of the toxin or toxoids can be used such as fragment C of Tetanus. These techniques may be important for certain applications, especially pediatric applications, in which ingested toxins (eg, diphtheria toxin), may possibly create adverse reactions. Optionally, a Langerhans-cell activator can be used as an adjuvant. Examples of such activators include: heat shock protein inducers; contact sensitizers (for example, trinit roclorobenzene, dinitrof luorobenzene, nitrogen mustard, pentadecylcatechol); toxins (e.g., Shiga toxin, staphylococcal enterotoxin B); lipopolysaccharides, lipid A, or derivatives thereof; Bacterial DNA (Stacey et al., 1996); cytokines (for example, tumor necrosis factor a, interleukin-Iß, -10, -12); and chemokines _ (for example, defensins 1 or 2, RANTES, MlP-la, MIP-2, interleukin-8). A combination of different adjuvants can be used in the present invention. For example, a combination of bacterial DNA containing the nucleotide sequences of CpG and an ADP ribosylating exotoxin could be used to direct the response of helper T cells to antigens administered transcutaneously. Thus, Thl or Th2-like responses for antigens adjuvanted with CT could be changed by the use of unmethylated CpG bacterial DNA, or other proteins such as LelF or calcium channel blockers. CpGs are among a class of structures that have patterns that allow the immune system to recognize its pathogenic origins to stimulate the innate immune response, leading to adaptive immune responses. (Medzhitov and Janewy, Curr Opin. Im unol., 9: 4-9, 1997). These structures are called molecular patterns associated with the pathogen (PAMPs), and include lipopolysaccharides, teichoic acids, non-methylated CpG portions, double-stranded RNA and mannins.

PAMPs induce endogenous signals that can mediate the inflammatory response, act as co-stimulators of T cell function and control effector function. The ability of PAMPs to induce these responses plays a role in their potential as adjuvants and their targets are APCs, such as macrophages and dendritic cells. Antigen presenting cells of the skin could likewise be stimulated by PAMPs through the skin. For example, the Langerhans cells, a type of dendritic cell, could be activated by a PAMP in solution on the skin, with a transcutaneously poorly immunogenic molecule, and be induced to migrate and present this poorly immunogenic molecule to the T cells in the cells. lymph nodes, inducing an antibody response to the poorly immunogenic molecule. The PAMPs could also be used in conjunction with other skin adjuvants such as cholera toxin to induce different co-stimulatory molecules and control different effector functions, to guide the immune response, for example from a Th2 response to a Thl response. If an immunization antigen has sufficient Langerhans cell activation capabilities, then the separate adjuvant may not be required, as is the case with CT which is both antigen and adjuvant. It is considered that the preparations of whole cells, live viruses, attenuated viruses, DNA plasmids, and bacterial DNA could be sufficient to immunize transcutaneously. It may be possible to use low concentrations of contact sensitizers or other activators of Langerhans cells to induce an immune response without inducing skin lesions.

LIPOSOMES AND THEIR PREPARATION Liposomes are closed vesicles that surround an internal aqueous space. The internal compartment is separated from the external environment by a lipid bilayer composed of discrete lipid molecules. In the present invention, the antigen can be distributed through contact with the skin to specialized cells of the immune system, whereby a specific immune response of the antigen is induced. Transcutaneous immunization can be achieved through the use of liposomes; however, as shown in the examples, the liposomes are not required to elicit a specific immune response of the antigen. Liposomes can. be prepared using a variety of membrane techniques and lipids (reviewed in Gregoriadis, 1993). The liposomes can be preformed and then mixed with the antigen. The antigen can be dissolved or suspended, and then added to (a) the preformed liposomes in a lyophilized state, (b) the dry lipids as a swelling solution or suspension, or (c) the soXution of lipids used to form the liposomes . These can also be formed from lipids extracted from the stratum corneum including, for example, ceramide and cholesterol derivatives (Wertz, 1992). Chloroform is a preferred solvent for lipids, but it can deteriorate after storage. Therefore, at intervals of one to three months, the chloroform is redistilled before its use as the solvent in the formation of liposomes. After distillation, 0.7% ethanol can be added as a preservative. Ethanol and methanol are other suitable solvents. The lipid solution used to form liposomes is placed in a round bottom flask.

Pear-shaped boiling flasks are preferred, particularly those flasks sold by Lures Scientific (Vineland, NJ, Cat. No. JM-5490). The volume of the flask should be more than ten times greater than the volume of the anticipated aqueous suspension of the liposomes, to allow adequate agitation during the formation of liposomes. Using a rotary evaporator, the solvent is removed at 37 ° C under negative pressure for 10 minutes with a filter aspirator attached to a water tap. The flask is also dried under low vacuum (for example, less than 50 mm Hg) for 1 hour in a desiccator. To encapsulate the antigen within liposomes, an aqueous solution containing antigen can be added to the lyophilized liposomal lipids, in a volume that results in a concentration of approximately 200 mM with respect to the liposomal lipid, and agitated or vortexed until all dry liposomal lipids are moist. The liposome-antigen mixture can then be incubated for 18 hours up to 72 hours at 4 ° C. The formulation of 1 iposoma-antigen can be used immediately or stored for several years. It is preferred to employ such a formulation directly in the transcutaneous immunization system without removing the unencapsulated antigen. Techniques such as bath sonication can be used to decrease the size of liposomes, which can increase transcutaneous immunization. Liposomes can be formed as described above but without the addition of antigen to the aqueous solution. The antigen can then be added to the preformed liposomes and, therefore, the antigen could be in solution and / or associated with, but not encapsulated by, the liposomes. This process of making the formulation containing liposomes is preferred due to its simplicity. Techniques such as bath sonication can be employed to alter the size and / or laminar character of liposomes to improve immunization. Although not required to practice the present invention, hydration of the stratum corneum can be improved by the addition of liposomes to the formulation. Liposomes have been used as carriers with adjuvants to improve the immune response to antigens mixed with, encapsulated in, coupled to, or associated with liposomes.

TRANSCUTANEOUS DISTRIBUTION OF ANTIGEN Efficient immunization can be achieved with the present invention because the transcutaneous distribution of the antigen can be directed to the Langerhans cells. These cells are found abundantly in the skin and are efficient antigen presenting cells that lead to the memory of T cells and powerful immune responses (Udey, 1997). Due to the presence of large numbers of Langerhans cells in the skin, the efficiency of the transcutaneous distribution may be related to the surface area exposed to the antigen and the adjuvant. In fact, the reason that transcutaneous immunization is so effective may be that it targets a larger number of these efficiently antigen presenting cells than intramuscular immunization. However, even a small number of Langerhans cells or dendritic cells may be sufficient for immunization. It is considered that the present invention will improve access to immunization, while inducing a potent immune response. Because transcutaneous immunization does not involve the penetration of the skin and the complications and difficulties of it, the requirements of trained personnel, sterile technique, and sterile equipment are reduced. In addition, barriers to multiple site immunization or multiple immunizations are diminished. Immunization through a simple application of the formulation is also considered. The immunization can be carried out using epicutaneous application of a simple solution of antigen and adjuvant impregnated in gauze under an occlusive patch, or by the use of other patch techniques; creams, immersion, ointments and sprays, are other possible methods of application. Immunization could be given by untrained personnel, and is adapted for self-application. Large-scale field immunization could occur given the easy accessibility to immunization. In addition, a simple immunization procedure could improve access to immunization by pediatric and adult patients, and populations in Third World countries. Similarly, the animals could be immunized using the present invention. The application to anatomical sites such as the ear, the lower abdomen, the legs, the conjunctiva, the intertriginous regions, or the anal region, or by means of immersion, could also be used. For previous vaccines, their formulations were injected through the skin with needles. The injection of vaccines using needles involves certain drawbacks, including the pain associated with injections, the need for sterile needles and syringes, trained medical personnel to administer the vaccine, discomfort from injection, and potential complications caused by puncture to the skin with the needle. Immunization through the skin without the use of needles (for example, transcutaneous immunization) represents a major advance for the distribution of vaccines by avoiding the aforementioned drawbacks. The transcutaneous delivery system of the invention is also not related to the penetration of intact skin by sound or electrical energy. Such a system that uses an electric field to induce dielectric breakdown of the stratum corneum is described in U.S. Patent No. 5,464,386.

In addition, transcutaneous immunization may be superior to immunization using needles since more immune cells could be reached by the use of various sites targeting large surface areas of the skin. A therapeutically effective amount of the antigen, sufficient to induce an immune response, can be distributed transcutaneously either in a single cutaneous site, or over an area of intact skin covering multiple fields of draining lymph nodes (eg, cervical, axillary nodes , inguinal, epitroquelares, poplíteos, those of the abdomen and thorax). Such sites near numerous different lymph nodes at sites throughout the body will provide a more widespread stimulus to the immune system than when a small amount of antigen is injected into a single site by intradermal, subcutaneous or intramuscular injection. The antigen that passes through or to the skin can find the antigen-presenting cells, which process the antigen in a way that induces an immune response. Multiple immunization sites can recruit a larger number of antigen-presenting cells and the larger population of antigen-presenting cells that were recruited could result in greater induction of the immune response. Transcutaneous immunization may allow application in close proximity to a lymphatic node drainage site, and thereby improve the efficiency or potency of immunization. It is conceivable that absorption through the skin can distribute the antigen to phagocytic skin cells such as, for example, dermal dendritic cells, macrophages, and other skin cells presenting antigen.; the antigen can also be distributed to the phagocytic cells of the liver, spleen and bone marrow, which are known to serve as antigen-presenting cells through the bloodstream or the lymphatic system. The result could be the very widespread distribution of the antigen to the antigen-presenting cells, to a degree that is rarely, even if achieved, by current immunization practices. The transcutaneous immunization system can be applied directly to the skin and allowed to air dry; rubs on the skin or on the scalp; stays in place with a bandage, patch or absorbent material; otherwise it is maintained by a device such as a stocking, slipper, glove, or T-shirt; or sprayed on the skin to maximize contact with the skin. The formulation can be applied in an absorbent bandage or gauze. The formulation can be covered with an occlusion bandage such as, for example, in an emulsion of antigenic solution and AQUAPHOR (petrolatum, mineral oil, wool wax, panthenol, bisabol, and Beiersdorf glycerin), plastic film, impregnated polymer, COMFEEL (Coloplast) or Vaseline; or a non-occluder bandage such as, for example, DUODERM (3M) or OPSITE (Smith and Napheu). An occlusion bandage completely excludes the passage of water. Alternatively, a partially occluder bandage such as TEGADERM may be applied to provide hydration and may allow the longer application of the patch or may prevent maceration of the skin. The formulation can be applied in single or multiple sites, in single or multiple extremities, or in large surface areas of the skin by complete immersion. The formulation can be applied directly to the skin. Genetic immunization has been described in U.S. Patent Nos. 5,589,466 and 5,593,972. The nucleic acid (s) contained in the formulation can code for the antigen, the adjuvant, or both. The nucleic acid may or may not be able to replicate; This can be non-integrative and non-infectious. The nucleic acid may further comprise a regulatory region (eg, promoter, enhancer, silencer, transcription termination and start site, acceptor sites and RNA splice sites, polyadenylation signal, internal ribosome binding site, sites of start and end of the translation) operably linked to the sequence encoding the antigen or the adjuvant. The nucleic acid can be formed in complex with a transfection promoting agent, such as cationic lipid, calcium phosphate, DEAE-dexthan, polybrene-DMSO, or a combination thereof. The nucleic acid may comprise regions derived from the viral genomes. Such materials and techniques are described in Kriegler (1990) and Murray (1991). An immune response may comprise humoral effector arms (e.g., antigen-specific antibody), and / or cellular (e.g., lymphocytes specific for the antigen such as B cells, CD4 + T cells, CD8 + T cells, CTL, Thl cells, Th2 cells, and / or TDTH cells). In addition, the immune response may comprise NK cells that mediate antibody-dependent cell-mediated cytotoxicity (ADCC). The immune response induced by the formulation of the invention may include the production of antibodies specific for the antigen and / or Cytotoxic lymphocytes (CTL, reviewed in Alving and Wassef, 1994). The antibody can be detected by immunoassay techniques, and the detection of various isotypes (for example, IgM, IgD, IgAl, IgA2, secretory IgA, IgE, IgGl, IgG2, IgG3, or IgG4) may be expected.

An immune response can also be detected by neutralization assay. "Antibodies are protective proteins produced by B lymphocytes." Antibodies are highly specific and generally target an epitope of an antigen.Antibodies often play a role in protecting against disease by reacting specifically with antigens derived from pathogens. The immunization can induce antibodies specific for the immunization antigen, such as cholera toxin.These antigen-specific antibodies are induced when the antigen is distributed through the skin by liposomes.The CTLs are immune protective cells. Individuals, produced to protect against infection by a pathogen, are also highly specific Immunization can induce antigen-specific CTLs, such as a synthetic oligopeptide based on a malaria protein, in association with the antigen autohis tocompatibility The CTLs in Carried out by immunization with transcutaneous distribution systems can kill cells infected with the pathogen. Immunization can also produce a memory response as indicated by the booster responses in antibodies and CTLs, the proliferation of lymphocytes by the culture of lymphocytes stimulated with the antigen, and the delayed type hypersensitivity responses to the intradermal challenge in the skin. , of the antigen alone. It is considered that the response of helper T cells, induced by transcutaneous immunization, can be manipulated by the use of calcium channel blockers (eg, nifedipine, verpamil) which suppress the contact hypersensitivity reaction by inhibiting catabolism of the antigen and the subsequent presentation by the epidermal Langerhans cells. The transcutaneous application of a calcium channel blocker could be expected to affect the surface expression of co-stimulatory molecules (eg, family related to B7) and the generation of a subsequent response of helper T cells. It is also considered that the addition of the calcium channel blocker can inhibit delayed-type hypersensitivity responses, and could be used to select an immune response that is predominantly a cellular or humoral response. In a viral neutralization assay, serial dilutions of sera are added to the host cells, which are then observed for infection after challenge with infectious virus. Alternatively, serial dilutions of sera can be incubated with infectious titers of virus prior to inoculation of an animal, and the inoculated animals are then observed for signs of infection. The transcutaneous immunization system of the invention can be evaluated using challenge models in either animals or humans, which evaluate the ability of immunization with the antigen to protect the subject from the disease. Such protection could demonstrate a specific immune response of the antigen. Instead of challenge, achieving anti-diphtheria antibody titres of 5 IU / ml or greater is generally assumed to be an indicator of optimal protection, and serves as a surrogate marker for protection (Plotkin and Mortimer, 1994). In addition, the challenge model with Pl asmodi um fa ciparum can be used to induce a human immune response specific for the antigen. ~ Human volunteers can be immunized using the transcutaneous immunization system containing oligopeptide, idos or proteins (polypeptides) derived from the malaria parasite, and then exposed to malaria experimentally or in a natural setting. The challenge model with mouse malaria Pl asmodi um yoell i, it can be used to evaluate the protection in mice against malaria (Wang et al., 1995). Alving et al. (1986) injected liposomes comprising lipid A as an adjuvant, to induce an immune response to cholera toxin (CT) in rabbits and to a synthetic protein consisting of a maculo oligopeptide containing four tetrapeptides ( Asn-Ala-Asn-Pro) conjugated to BSA. The authors found that the immune response to cholera toxin or synthetic malaria protein was notoriously enhanced by encapsulation of the antigen with liposomes containing lipid A, in comparison to similar liposomes lacking lipid A. Several antigens already derived either the circumsporozoite protein (CSP) or the superficial merozoite proteins of Pl asmodi um fal ciparum have been encapsulated in liposomes containing the lipid A. All the antigens of malaria that have been encapsulated in liposomes containing the lipid A they have been shown to induce humoral effects (e.g., antigen-specific antibodies), and some have been shown to induce also cell-mediated responses. The generation of an immune response and immunoprotection in an animal vaccinated with a malaria antigen can be evaluated by an immunofluorescence to the complete sporozoites of malaria, fixed or CTLs, killing the transfected target cells with CSP. Mice transcutaneously immunized with cholera toxin can be protected against intranasal challenge with a dose of 20 μg of cholera toxin. Mallet et al. (Personal communication) have found that C57B1 / 6 mice develop a fatal orrhagic pneumonia - in response to intranasal challenge with CT. Alternatively, mice can be challenged with an intraperitoneal dose of CT (Dragunsky et al., 1992). IgG or IgA antibodies specific to cholera toxin can provide protection against challenge with cholera toxin (Pierce, 1978; Pierce and Reynolds, 1974). Similar protective effects could be expected in humans immunized with LT or CT, and challenged with E. col i that secretes LT or Vi -ri or chol was e that secretes CT, respectively. In addition, cross protection has been demonstrated between subjects immune to CT_ and LT and CT-mediated disease and LT. As shown in the following examples, mucosal immunity can be achieved by the transcutaneous route. Mucosal IgG and IgA can be detected in mice immunized with CT transcutaneously. This may be important for protection in diseases where the pathology occurs in mucosal sites such as LT or CT-mediated disease, where the entry of the pathogen occurs at a mucosal site, or where mucosal infection is important for pathogenesis. One would expect that transcutaneous immunization against diseases such as influenza could be effective either through the induction of mucosal immunity or by systemic immunity, or by a combination of humoral, cellular or mucosal immunity. Vaccines can be effective against host effects such as the binding of erythrocytes to the vascular endothelium in malaria, through the induction of anti-sequester antibodies. Protective antibodies such as anti-hepatitis A, B or hepatitis E antibodies can be induced by the transcutaneous route using the complete inactivated virus, subunits derived from the virus or recombinant products. Protection against tetanus, diphtheria and other toxin-mediated diseases can be conferred by transcutaneously induced anti-toxin antibodies. A "reinforcer" patch of tetanus could be considered to contain an adjuvant such as CT and toxoids such as tetanus and diphtheria, or fragments such as fragment C of tetanus. The reinforcement could be achieved after the primary immunization by injection or transcutaneous immunization with the same or similar antigens. For injectable immunizations that induce immunity but have potential side effects after reinforcement, transcutaneous reinforcement may be preferable. Oral or nasal immunization can be conceivably enhanced using the transcutaneous route. The simultaneous use of injectable and transcutaneous immunizations could also be used. Vaccination has also been used as a treatment for cancer and autoimmune diseases. For example, vaccination with a tumor antigen (for example, prostate-specific antigen) can induce an immune response in the form of antibodies, CTLs and lymphocyte proliferation, which allows the body's immune system to recognize and kill the cells. tumor cells. The targeting of dendritic cells, of which Langerhans cells are a specific subgroup, has been shown to be an important strategy in immunotherapy against cancer. Tumor antibodies useful for vaccination have been described for melanoma (U.S. Patent Nos. 5,102,663, 5,141,742, and 5,262,177), prostate carcinoma (U.S. Patent No. 5,538,866) and lymphoma (U.S. Patent Nos. 4,816,249, 5,068,177, and 5,227,159) . Vaccination with the oligopeptide T cell receptor can induce an immune response that interrupts the progression of the autoimmune disease (US Patent No. 5,612,035 and 5,614,192; Antel et al., 1996; Vanderbark et al., 1996). U.S. Patent No. 5,552,300 also describes antigens suitable for the treatment of autoimmune disease. It is understood that the following is illustrative of the present invention; however, the practice of the invention is not limited or restricted in any way by the examples.

EXAMPLES Immunization Procedure BALB / c mice from 6 to 8 weeks were shaved with a # 40 shaver. This shaving could be performed without any sign of trauma to the skin. The shaving was performed from the middle thorax until just below the nape of the neck. The mice were then left to rest for 24 hours. Before this the mice had been labeled in the ear for identification, and pre-bled to obtain a sample of pre-immune serum. The mice were also transcutaneously immunized without shaving by applying 500 μl of immunization solution to each ear. The mice were then immunized in the following manner. The mice were anesthetized with 0.03 to 0.06 ml of a 20 mg / ml solution of xylazine and 0.5 ml of 100 mg / ml ketamine; the mice were immobilized by this dose of anesthesia for approximately one hour. The mice were placed from the ventral side down on a heating blanket. The immunization solution was then placed on the shaved dorsal skin of a mouse as follows: a 1.2 cm x 1.6 cm stencil made of polystyrene was gently placed on the back and a sterile gauze moistened with saline was used to partially moisturize the skin (this allowed the uniform application of the immunization solution), the immunization solution was then applied with a pipette to the area circumscribed by the stencil to produce a patch of 2 cm2 of immunization solution. Alternatively, a fixed volume of immunization solution was uniformly applied to the shaved area or to the ear. Care was taken not to scratch or rub the skin with the tip of the pipette. The immunization solution was dispersed around the area to be covered with the soft side of the tip of the pipette. The immunization solution (between about 100 μl to about 200 μl) was left on the back of the mouse for 60 to 180 minutes. At the end of 60 minutes, the mouse was gently held by the neck and tail under a copious stream of lukewarm water from the tap, and washed for 10 seconds. The mouse was then gently beaten to dry with a piece of sterile gauze and a second wash was performed for 10 seconds; the mouse was then tapped to dry it a second time and left in the cage. The mouse did not appear to show adverse effects due to anesthesia, immunization, washing procedure or exotoxin toxicity. No skin irritation, swelling or redness was observed after immunization, and the mice appeared to thrive. Immunization using the ear was performed as described above except that the coat was not removed prior to immunization.

Antigen The following antigens were used for immunization and ELISA, and mixed using PBS or normal saline. Cholera toxin or CT (List Biologicals, Cat. # 101B, lot # 10149CB), subunit B of CT (List Biologicals, Cat. # BT01, lot # CVXG-14E), subunit A of CT (List Biologicals, Cat. # 102A, lot # CVXA-17B), subunit A of CT (Calbiochem, Cat. # 608562); pertussis toxin, salt-free (List biologicals, lot # 181120a); Tetanus toxoid (List Biologicals, lots # 1913a and # 1915a); Pseudomonas exotoxin A (List Biologicals, lot # ETA25a); Diphtheria toxoid (List Biologicals, lot # 15151); heat-labile enterotoxin of E. col i (Sigma, lot # 9640625); bovine serum albumin or BSA (Sigma, Cat. # 3A-4503, lot # 31F-0116); and conjugate B from Hemophi l us infl uenza (Connaught, lot # 6J81401).

ELISA - IgG (H + L) Antibodies specific for CT, LT, ETA, pertussis toxin, diphtheria toxoid, tetanus toxoid, Hemophi B conjugate, infl uenza, influenza, sequestrin, and BSA were determined using ELISA in a technique similar to Glenn et al. (1995). All antigens were dissolved in sterile saline at a concentration of 2 μg / ml. Fifty microliters of this solution (0.1 μg) per well were placed on IMMULON-2 polystyrene plates (Dynatech Laboratories, Chantilly, VA) and incubated at room temperature overnight. The plates were then blocked with a blocking buffer of 0.5% casein / 0.05% Tween 20 in solution, for one hour. The sera were diluted with 0.5% casein diluent / 0.05% Tween 20; The dilution series were made in columns on the plate. The incubation was for 2 hours at room temperature. The plates were then washed in a wash solution of PBS-0.05% Tween 20 four times, and the secondary antibody bound to goat anti-mouse horseradish peroxidase (HRP), IgG (H + L) (Bio-Rad Laboratories , Richmond, CA, Cat. # 170-6516) was diluted in casein diluent at a dilution of 1/500 and left on plates for one hour at room temperature. The plates were then washed four times in the PBS-Tween wash solution. One hundred microliters of the 2,2'-azino-di (3-ethyl-benzothiazolon) sulfonic acid substrate (Kirkegaard and Perry) was added to each well and the plates were read at 405 nm after 20-40 minutes of development. The results are reported as the geometric mean of the individual sera and the standard error of the mean of the ELISA units (the dilution of the serum in which the absorbance is equal to 1.0) or as individual antibody responses in ELISA units.

ELISA - IgG (?), IgM (μ) and IgA (a) The levels of IgG antibody (?), IgM (μ) and IgA (a) anti-CT were determined using ELISA with a technique similar to Glenn et al. ( nineteen ninety five) . CT was dissolved in sterile saline at a concentration of 2 μg / ml. Fifty microliters of this solution (0.1 μg) per well were placed on IMMULON-2 polystyrene plates (Dynatech Laboratories, Chantilly, VA) and incubated at room temperature overnight. The plates were then blocked with a blocking buffer of 0.5% casein-Tween 20 for one hour. The sera were diluted and the casein diluent and serial dilutions were made on the plate. This was incubated for two hours at room temperature. The plates were then washed in PBS-Tween wash solution four times and goat anti-mouse secondary antibody IgG (y) bound to HRP (Bio-Rad Laboratories, Richmond, CA, Cat. # 172-1038), goat anti-mouse IgM (μ) bound to HRP (BioRad Laboratories, Richmond, CA, Cat. # 172-1030), or goat anti-mouse IgA bound to HRP (Sigma, Saint Louis, MO, Cat. # 1158985), it was diluted in casein diluent at a 1/1000 dilution and left on the plates for one hour at room temperature. The plates were then washed four times in a PBS-Tween wash solution. One hundred microliters of the substrate 2, 2 '-azino-di (3-ethyl-benzothiazolon) -sulfonic acid substrate (Kirkegaard and Perry, Gaithersburg, MD), were added to the wells, and the plates were read at 405 nm. The results are reported as the geometric mean of the individual sera and the standard error of the mean of the ELISA units (the dilution of the serum at which the absorbance is equal to 1.0).

ELISA - IgG subclass IgG subclass antibody (IgGl, IgG2a, IgG2b, and IgG3) antigen-specific, against CT, LT, ETA and BSA was carried out as described by Glenn. Et al. (1995). The solid phase ELISA was performed on IMMULON-2 polystyrene plates (Dynatec Laboratories, Chantilly, VA). The wells were incubated with the respective antigens in saline overnight (0.1 μg / 50 μl) and blocked with 0.5% casein-Tween 20. Sera from individual mice diluted in 0.5% casein were serially diluted, and they were incubated at room temperature for four hours. The secondary antibody consisted of goat anti-mouse antibody, isotype-specific, conjugated to horseradish peroxidase (IgG1, IgG2a, IgG2b, IgG3, The Binding Site, San Diego CA). A standard curve for each subclass was determined using IgGl, IgG2a, IgG2b, and mouse myeloma IgG3 (The Binding Site, San Diego, CA). The standard wells were coated with goat anti-mouse IgG (H + L) (Bio-Rad Laboratories, Richmond, CA, Cat. # 172-1054) to capture the standards of the myeloma IgG subclass which were added in dilutions serially. The myeloma IgG subclass was also detected using the anti-mouse subclass specific antibody, conjugated to peroxidase. Test sera and myeloma standards were detected using 2, 2'-azino-di (3-ethyl-benzothiazolon) sulfonic acid (Kirkegaard and Perry, Gaithersburg, MD) as a substrate. The absorbances were read at 405 nm. Individual antigen-specific subclasses were quantified using values from the linear titration curve computed against the standard myeloma curve and reported as μg / ml.

ELISA-IgE Quantitation of the antigen-specific IgE antibody was performed using a protocol of Pharmingen Technical Protocols, page 541 of Research Products Catalog, 1996-1997 (Pharmigengen, San Diego, CA). Fifteen microliters of 2 μg / ml anti-mouse IgE capture monoclonal antibody, purified (Pharmingen Cat. # 02111D) in 0.1 M NaHCO 3 (pH 8.2) were added to IMMUNO plates (Nunc, Cat. # 12-565-136). Plates were incubated overnight at room temperature, washed three times with PBS-Tween 20, blocked with 3% BSA in PBS for two hours, and washed three times with PBS-Tween. The sera were diluted in 1% BSA in PBS, added at 1/100 dilutions, and serially diluted in columns (eg, 1/100, 1/200, etc.). Purified mouse IgE standards (Pharmingen, Cat. # 0312D) were added with an initial dilution of 0.25 μg / ml and serially diluted in the columns. The plates were incubated for two hours and washed five times with PBS-Tween. Biotinylated anti-mouse mAB IgE (Pharmingen, Cat. # 02122D) at 2 μg / ml in 1% BSA in PBS was incubated for 45 minutes and washed five times with PBS-Tween. Avidin-peroxidase (Sigma A3151, 1: 400 of a 1 mg / ml solution) was added for 30 minutes and the plates were washed six times with PBS-Tween. Test sera and IgE standards were detected using 2,2'-azino-di (3-e-t-yl-benzothiazolon) sulphonic acid (Kirkegaard and Perry, Gaithersburg, MD) as a substrate. The absorbances were read at 405 nm. The individual antigen-specific subclasses were quantified using the values from the linear titration curve computed against the standard IgE curve and reported as μg / ml.

Liposomal Preparation Where the liposomes were included in the formulation for transcutaneous immunization, the multilamellar liposomes composed of dimyristoyl-phosphatidyl-choline, dimyristoyl-phosphatidyl-glycerol, cholesterol, were prepared according to Alving et al. (1993). Dimyristoyl-fos fat idylcholine, dimyristoyl-phosphatidylglycerol and cholesterol were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). The reserve solutions of the lipids in chloroform were removed from the freezer at -20 ° C where they were stored. The lipids were mixed in a molar ratio of 0.9: 0.1: 0.75 of dimyristoyl-fos fatidylcholine, dimiristoi-1-phosphatidylglycerol, and cholesterol in a pear-shaped flask. Using a rotary evaporator, the solvent was removed at 37 ° C under negative pressure for 10 minutes. The flask was further dried under low vacuum for two hours in a desiccator to remove the residual solvent. The liposomes were swollen in 37 mM phospholipid using sterile water, lyophilized and stored at -20 ° C. These liposomes were mixed in their lyophilized state with normal saline (pH 7.0) to achieve a designated concentration of phospholipid in the saline. Alternatively, the dry lipids were swollen to make liposomes with normal saline (pH 7.0) and were not lyophilized.

EXAMPLE 1 BALB / c mice 6 to 8 weeks old were "transcutaneously immunized as described above for the" Immunization Procedure "in groups of five mice.The mice were immunized using 100 μl of immunization solution which was prepared as follows: liposomes prepared as described above for "Liposomal Preparation" were mixed with saline to form the liposomes.The preformed liposomes were then diluted either in saline (liposomal group alone) or with CT in saline to produce a solution of immunization containing 10-150 mM phospholipid liposomes with 100 μg of CT per 100 μl of immunization solution.CT was mixed in saline to make an immunization solution containing 100 μg of CT per 100 μg of solution for the group receiving CT ONLY The solutions were vortexed for 10 seconds before immunization. They were immunized transcutaneously at 0 and 3 weeks. Antibody levels were determined using ELISA as described above for "ELISA IgG (H + L)" 3 weeks after the booster immunization, and compared against the pre-immune sera. As shown in Table 1, the level of CT-induced anti-CT antibodies without liposomes was not different from the level of anti-CT antibodies generated using liposomes, except in mice where 150 mM liposomes were used. CT in saline alone was able to immunize mice against CT to produce high titers of antibody.

Table 1. Anti-CT antibodies Group Units of SEM ELISA CT only 27,482 (16, 635-48,051) CT + Liposomes 150 mM 4,064 * (2,845-5,072) CT + 100 mM Liposomes 35, 055 (25,932-44,269) CT + 50 mM Liposomes 9,168 (4,283-12,395) CT + 25 mM Liposomes 18,855 (12,294-40,374) CT + 10 mM Liposomes 28,660 (18,208-31,498) 50 mM Liposomes 0 * Significantly different from the CT Group alone (P <0.05) Example 2 BALB / c mice 6 to 8 weeks old were transcutaneously immunized as described above for the "Immunization Procedure", in groups of five mice. Mice were immunized at 0 and 3 weeks using 100 μl of the immunization solution prepared as follows: BSA was mixed in saline to make an immunization solution containing 200 μg of BSA per 100 μl of saline for the group that received BSA alone; BSA and CT were mixed in saline to make an immunization solution containing 200 μg of BSA and 100 μg of CT per 100 μl of saline for the group that received BSA and CT. Where the liposomes were used, the liposomes were prepared as described above for "Liposomal Preparation", and were first mixed with saline to form the liposomes. These were then diluted in BSA or BSA and CT in saline to produce an immunization solution containing liposomes at 50 mM phospholipid with 200 μg of BSA per 100 μl of the immunization solution, or 200 μg of BSA + 100 μg. of CT per 100 μl of the immunization solution. The solutions were vortexed for 10 seconds before immunization. The antibodies were determined using ELISA as described above for "ELISA IgG (H * L)" in sera 3 weeks after the second immunization. The results are shown in Table 2. BSA alone, with or without liposomes, was not able to elicit an antibody response. However, the addition of CT stimulated an immune response to BSA. CT acted as an adjuvant for the immune response to BSA, and high-titre anti-BSA antibodies were produced.

Table 2. Anti-BSA antibodies Group Units of SEM ELISA BSA in saline solution 0 BSA + 50 mM Liposomes 0 CT + BSA in saline solution 8,198 (5,533-11,932) CT + BSA + 50 mM 3,244 (128-3,242) Example 3 BALB / c mice 6 to 8 weeks of age were transcutaneously immunized as described above for the "Immunization Procedure" in the groups of five mice. Mice were immunized at 0 and 3 weeks using 100 μl of the immunization solution prepared as follows: LT was mixed in saline to make an immunization solution containing 100 μg of LT per 100 μl of saline for the group that received LT alone. Where liposomes were used, the liposomes were prepared as described above for the "Liposomal Preparation", and were first mixed with saline to form the liposomes. The preformed liposomes were then diluted in LT in saline to produce an immunization solution containing liposomes at 50 mM phospholipid with 100 μg of LT per 100 μl of immunization solution. The solutions were vortexed for 10 seconds before immunization. Anti-LT antibodies were determined using ELISA as described above for "ELISA IgG (H + L)" 3 weeks after the second immunization. The results are shown in Table 3. LT was clearly immunogenic with and without liposomes, and there was no significant difference between the groups. LT and CT are members of the bacterial exotoxin family of ADP ribosylation (bAREs). These are recognized as A: B proenzymes with the activity of ADP-ribosyl transferase, contained in the A subunit and the target cell that binds to a function of the B subunits. LT is 80% homologous with CT at the amino acid level and has an organization of non-covalently linked subunits, similar, is similar tequiome (A: B5), the same binding target, ganglioside GM1, and is similar in size (MW approximately 80,000). The similarities of LT and CT appear to influence their immunogenicity by the transcutaneous route, as reflected by the similar magnitude of the antibody response to CT and LT (Tables 1 and 3).

Table 3. Anti-LT Antibodies Group Units ELISA SEM LT in saline 23,461 (20,262-27,167) LT + 50 mM Liposomes 27,247 (19,430-38,211) EXAMPLE 4 C57B1 / 6 mice 6 to 8 weeks of age were transcutaneously immunized as described above for the "Immunization Procedure" in groups of five mice. Mice were immunized once using 100 μl of the immunization solution prepared as follows: LT was mixed in saline to make an immunization solution containing 100 μg of LT per 100 μl of saline. The solution was vortexed for 10 seconds before immunization. Anti-LT antibodies were determined using ELISA as described above for "ELISA IgG (H + L)" 3 weeks after the single immunization. The results are shown in Table 4. LT was clearly immunogenic with a simple immunization and antibodies were produced at 3 weeks. The rapid increase in antibody titers and responses to simple immunization could be a useful aspect of the transcutaneous immunization method. It is conceivable that a simple rapid immunization could be useful in epidemic issues, for travelers, and where access to medical care is poor.

Table 4. Anti-LT antibodies Mouse Number ELISA Units 5141 6, 582 5142 198 5143 229 5144 6, 115 5145 17, 542 Media Geo 2, 000 Example 5 C57B1 / 6 mice from 8 to 12 weeks of age were transcutaneously immunized as described above for "Immunization Procedure", in groups of five mice. Mice were immunized once using 100 μl of the immunization solution prepared as follows: CT was mixed in saline _ to make an immunization solution containing 100 μg of CT per 100 μl of saline. The solution was vortexed for 10 seconds before immunization. Anti-CT antibodies were determined using ELISA as described above for "ELISA IgG (H + L)" 3 weeks after the single immunization. The results are shown in Table 5. CT was highly immunogenic with a simple immunization. Rapidly increasing antibody titers and responses to simple immunization may be a useful aspect of the transcutaneous immunization method. It is conceivable that a simple rapid immunization could be useful in epidemic issues, for travelers, and where access to medical care is poor.

Table 5. Anti-CT antibodies Mouse Number ELISA Units 2932 18, 310 2933 30, 878 2934 48, 691 2935 7, 824 Media Geo 21, 543 Example 6 BALB / c mice 6 to 8 weeks old were immunized transcutaneously as described above for the "Immunization Procedure" in groups of five mice. Mice were immunized at 0 and 3 weeks using 100 μl of immunization solution prepared as follows: ETA was mixed in saline to make an immunization solution containing 100 μg of ETA per 100 μl of saline for the group that received ETA only. Where the liposomes were used, the liposomes were prepared as described above for the "Liposomal Preparation", and were first mixed with saline to form the liposomes. The preformed liposomes were then diluted with ETA in saline to produce an immunization solution containing the liposomes at 50 mM phospholipid with 100 μg of ETA per 100 μl of immunization solution. The solutions were vortexed for 10 seconds before immunization. The antibodies were determined using ELISA as described above for "ELISA IgG (H + L)" in the sera 3 weeks after the second immunization. The results are shown in Table 6. ETA was clearly immunogenic with and without liposomes, and no significant difference between the groups could be detected. ETA differs from CT and LT in that ETA is a simple peptide of 613 amino acids with A and B domains on the same peptide and binds to a completely different receptor, the macroglobulin receptor-related protein a2 / low density lipoprotein receptor ( Kounnas et al., 1992). Despite the lack of similarity between ETA and CT in size, structure and binding target, ETA also induced a transcutaneous antibody response.

Table 6. Anti-ETA antibodies Group Units of ELISA SEM ETA in saline solution 3,756 (1,926-7,326) ETA + Liposomes 50 mM 857 (588-1,251) Example 7 BALB / c mice 6 to 8 weeks old were transcutaneously immunized as described above for the "Immunization Procedure", in groups of five mice. The mice were immunized using 100 μl of immunization solution which was prepared as follows: CT was mixed in solution. saline to make 100 μg of CT per 100 μl of immunization solution, LT was mixed in saline to make 100 μg of LT per 100 μl of immunization solution, ETA was mixed in saline to make 100 μg of ETA per 100 μl of immunization solution, and CT and BSA were mixed in saline to make 100 μg of CT per 100 μl of immunization solution and 200 μg of BSA per 100 μl of immunization solution. The solutions were vortexed for 10 seconds before immunization. Mice were transcutaneously immunized at 0 and 3 weeks and antibody levels were determined using ELISA as described above for the "ELISA Subclass IgG", three weeks after the booster immunization and compared against the pre-immune sera. The response of the subclass of IgG to CT, BSA and LT had similar levels of IgG1 and IgG2a reflecting the activation of helper T cells from Th1 and Th2 lymphocytes (Seder and Paul, 1994), while the IgG subclass response ETA consisted almost exclusively of IgG1 and IgG3, consistent with the response similar to Th2 (Table 7). Thus, it appears that all subclasses of IgG can be produced using transcutaneous immunization.

Table 7. IgG subclasses of the induced antibodies Antibody IgG1 IgG2a IgG2b IgG2 specificity Inm. of Antibody (μg / μl) (μg / μl) (μg / μl) (μg / μl) CT CT 13 4 25 27 CT + BSA BSA 10 8 17 12 LT LT 155 28 10 ETA ETA 50 0 1 1 0 E JEMPLO 8 BALB / c mice 6 to 8 weeks old were transcutaneously immunized as described above for the "Immunization Procedure", in groups of five mice. Mice were immunized using 100 μl of the immunization solution which was prepared as follows: LT was mixed in saline to make an immunization solution containing 100 μg of LT per 100 μl of saline for the group that received LT alone , CT was mixed in saline to make an immunization solution containing 100 μg of CT per 100 μl of saline for the group that received CT alone, ETA was mixed in saline to make an immunization solution containing 100 μg of ETA per 100 μl of saline for the group that received ETA alone and BSA and CT were mixed in saline to make an immunization solution containing 100 μg of BSA and 100 μg of CT per 100 μl of saline for the group that He received BSA and CT. Mice were transcutaneously immunized at 0 and 3 weeks and antibody levels were determined using ELISA as described above for "ELISA IgE" one week after booster immunization and compared against pre-immune sera. As shown in Table 8, no IgE antibodies were found although the sensitivity of the detection was 0.003 μg / ml. The IgG antibodies were determined in the same mice using "ELISA IgG (H + L)" on the sera 3 weeks after the second immunization. The IgG antibody response to LT, ETA, CT and BSA are shown to indicate that the animals were successfully immunized and responded with high antibody titers to the respective antigens.

Table 8. IgE antibodies for LT, ETA, CT and BSA IgE Specificity Group (μ IgG (ELISA Antibody Units) LT Ant i -LT 0 23, 461 ETA Anti -ETA 0 3, 756 CT Ant i -CT 0 39, 828 CT + BSA Ant i -BSA 0 8, 198 Example 9 BALB / c mice 6 to 8 weeks old were transcutaneously immunized as described above for the "Immunization Procedure", in groups of five mice. Mice were immunized for 0 to 3 weeks using 100 ml of the immunization solution which was prepared as follows: CT was mixed in saline to make an immunization solution containing 100 mg of CT per 100 ml of immunization solution. The immunization solution was vortexed for 10 seconds before immunization.

Mice were transcutaneously immunized at 0 and 3 weeks and antibody levels were determined using ELISA as described above for "ELISA IgG (H + L)" and "ELISA IgG (y)". The determinations were made at 1 and 4 weeks after the initial immunization, and were compared against the pre-immune sera. As shown in Table 9, high levels of Anti-CT IgG (y) antibodies were induced by CT in saline. Small amounts of IgM could be detected by the use of IgM-specific secondary antibody (μ). By 4 weeks, the response to the antibody was mainly IgG. The data are reported in units of ELISA.

Table 9. IgG (y) and IgM (μ) Inm Group IgG (y) IgM week (μ) CT 1 72 168 CT 4 21, 336 38 L (] + CT 1 33 38 LC + CT 4 22, 239 70 Example 10 BALB / c mice 6 to 8 weeks old were transcutaneously immunized as described above for the "Procedure of Immunization ", in groups of five mice Mice were immunized once using 100 μl of saline immunization solution prepared as follows: CT was mixed in saline to make an immunization solution containing 100 μg of CT per 100 μl of saline The solution was vortexed for 10 seconds before the immunization.The mice were transcutaneously immunized at 0 and 3 weeks.The antibody levels were determined using ELISA as described above for "ELISA IgG (H + L) "5 weeks after the booster immunization, and were compared against the pre-immune sera." As shown in Table 10, anti-CT IgA antibodies were detected in serum.

Table 10. Anti-CT IgA Antibodies Mouse number IgA (ng / ml) 1501 232 1502 22 1503 41 1504 16 1505 17 EXAMPLE 11 BALB / c mice 6 to 8 weeks old, transcutaneously immunized as described above for the "Immunization Procedure", in groups of five mice. Mice were immunized using 100 μl of immunization solution which was prepared as follows: CT was mixed in saline to make an immunization solution containing 100 μg of CT per 100 μl of immunization solution. The immunization solution was vortexed for 10 seconds before immunization. Mice were immunized with 100 μl of immunization solution transcutaneously at 0 and 3 weeks and antibody levels were determined using ELISA as described above for "ELISA IgG (H + L)" and "ELISA IgG (y)". Antibody measurements were made at 8 weeks after the initial immunization and compared against the pre-immune sera. As shown in Table 11, high levels of anti-CT antibodies in serum were induced by CT in saline. The pulmonary lavage IgG could be detected by ELISA using specific antibody IgG (H + L) or IgG (y). The antibody found on the surface of the pulmonary mucosa is diluted by the washing method used to collect the mucosal antibody and, thus, the exact amounts of the antibody detected are not as significant as the mere presence of the detectable antibody. The lung washes were obtained after the sacrifice of the mice. The trachea and lungs were exposed by gentle dissection and the trachea transected above the bifurcation. A 22-gauge polypropylene tube was inserted and tied over the trachea to form a tight seal at the edges. Half a milliliter of PBS was infused using a 1 ml syringe attached to the tubing, and the lungs were gently infiltrated with the fluid. The fluid was removed and reinfused for a total of 3 rounds of washing. The pulmonary lavage was then frozen at -20 ° C. Table 11 shows the response of IgG (H + L) and IgG (y) antibodies for cholera toxin in sera and pulmonary lavages at 8 weeks. The data are expressed in the ELISA units. Antibodies were clearly detectable for all mice in lung lavage. The presence of antibodies in the mucosa may be important for protection against mucosally active diseases.

Table 11. Mucosal Antibody for CT Animal # Group Inm. IgG (H + L) IgG (y) Source 1501 CT 133 34 Lungs 1502 CT 75 12 Lungs 1503 CT 162 28 Lungs 1504 CT 144 18 Lungs 1505 CT 392 56 Lungs Media Geo 156 26 1501 CT 34,131 13,760 Serum 1502 CT 11,131 2,928 Serum 1503 CT 21,898 10,301 Serum 1504 CT 22,025 8,876 Serum 1505 CT 34,284 10,966 Medium Serum Geo 23,128 8,270 Example 12 BALB / c mice were transcutaneously immunized at 0 and 3 weeks as described above for the "Immunization Procedure", in groups of four mice. The liposomes were prepared as described above for the "Liposomal Preparation", and were first mixed with saline to form the liposomes. The preformed liposomes were then diluted either with CT, CTA, or CTB in saline to produce an immunization solution containing liposomes at 50 mM phospholipid with 50 μg of antigen (CT, CTA or CTB) per 100 μl of immunization. The solutions were vortexed for 10 seconds before immunization. Antibodies were determined using ELISA as described above for "ELISA IgG (H + L)", one week after immunization 'booster and were compared against pre-immune serum. The results are shown in Table 12. CT and CTB were clearly immunogenic whereas CTA was not. Thus, the B subunit of CT is necessary and sufficient to induce a strong antibody response.

Table 12. Antibodies for CT, CTA and CTB Anti-CT Anti-CTB Anti-CTB Group CT + 50 mM Liposomes 12,636 136 7,480 CTB + 50 mM Liposomes 757 20 1,986 CTA + 50 mM Liposomes 0 0 0 Example 13 BALB / c mice were transcutaneously immunized as described above for the "Immunization Procedure", in groups of five mice. Mice were immunized at 0 and 3 weeks with 100 μg of diphtheria toxoid and 100 μg of pertussis toxin per 100 μl of saline. The "solutions were vortexed for 10 seconds before immunization.Antibodies were quantified using ELISA as described for" ELISA IgG (H + L). "Anti-diphtheria toxoid antibodies were detected only in animals immunized with the toxin. of pertussis and diphtheria toxoid The highest responder had 1.038 ELISA units of the anti-diphtheria toxoid antibody.Thus, a small amount of pertussis toxin acts as an adjuvant for the diphtheria toxoid antigen. only did not induce an immune response, suggesting that the process of toxoidization has affected the portion of the molecule responsible for the adjuvant effects found in the ADP ribosylation exotoxin.

Table 13. Antibody for Diphtheria Mouse number Antigen of ELISA Units Immunization of IgG 4731 DT + PT 1, 039 4732 DT + PT 1 4733 DT + PT 28 4734 DT + PT 15 4735 DT + PT 20 4621 DT 0 4622 DT 0 4623 DT 0 4624 DT 0 4625 DT 0 Example 14 BALB / c mice were transcutaneously immunized as described above for the "Immunization Procedure", in groups of five mice. Mice were immunized once at 0, 8 and 20 weeks with 50 μg of pertussis toxin (List, Catalog # 181, lot # 181-20a) per 100 μl of saline. The antibodies were quantified using ELISA as described for "ELISA IgG (H + L). "Anti-pertussis toxin antibodies were detected one week after the last booster in animals immunized with whooping cough All five animals had high levels of pertussis anti-toxin antibody after the last immunization Thus, pertussis toxin acts as an adjuvant to itself and induces PT-specific IgG antibodies.The adjuvant effect of PT may be useful in combination vaccines such as Diphtheria / Pertussis / Tetuses / Hib to improve the response of the antibody to the co-administered antigens as well as to the PT itself.

Table 14. Antibody Response to Pertussis Toxin Antigen Number Mouse 2 weeks 21 weeks 5156 PT 14 256 5157 PT 22 330 5158 PT 17 303 5159 PT 33 237 5160 PT 75 418 Example 15 BALB / c mice were transcutaneously immunized as described above for the "Immunization Procedure", in groups of five mice. Mice were immunized once at 0 weeks with 50 μg of tetanus toxoid and with 100 μg of cholera toxin per 100 μl of saline. The antibodies were quantified using ELISA as described by "ELISA IgG (H + L)". Anti-tetanus toxoid antibodies were detected 8 weeks in animal 5173 to 443 ELISA units.

Example 16 The possibility that oral immunization occurred through grooming after epicutaneous application and subsequent washing of the application site was evaluated using 125-labeled CT to track the antigen / adjuvant target. The mice were anesthetized, transcutaneously immunized as described above for the "Immunization Procedure" with 100 μg of the 125I-labeled CT (150,000 cpm / μg CT). The control mice remained anesthetized for 6 hours to exclude -the grooming, and the experimental mice were anesthetized for one hour and then allowed to groom after washing. The mice were sacrificed at 6 hours and the organs were weighed and counted for 125I on a Packard gamma counter. A total of 2 to 3 μg of CT was detected on the shaved skin at the immunization site (14,600 cpm / μg of tissue) while a maximum of 0.5 μg of CT was detected in the stomach (661 cpm / μg of tissue. ) and intestine (9 cpm / μg of tissue). Oral immunization (n = 5) with 10 μg of CT in saline at 0 and 3 weeks (without NaHC02) induced a medium IgG antibody response at 6 weeks less than 1,000 units of ELISA while transcutaneous immunization with 100 μg of CT, shown above, resulted in less than 5 μg of CT retained in the skin after washing, resulting in an anti-CT response of 42,178 ELISA units at 6 weeks. The induction of an orally fed CT immune response requires the addition of NaHCO3 to the immunization solution (Piece, 1978, Lycke and Holmgren, 1986). Thus, oral immunization does not contribute significantly to the antibodies detected when CT is applied epicutaneously to the skin.

Example 17 ~ In evidence of the activation of Langerhans cells was obtained using cholera toxin (CT) in saline applied epicutaneously to the skin, specifically the mouse ears, where large populations of Langerhans cells can be easily visualized. (Enk et al., 1993; Bacci et al., 1997), and staining for class II molecules of the histocompatibility major complex (MHC) which is upregulated in activated Langerhans cells (Shimada et al., 1987). ).

The ears of BALB / c mice were covered on the dorsal side with either 100 μg of CT in saline, 100 μg of CTB in saline, saline alone, or an intradermal injection of the positive controls, 100 pg of LPS or 10 μg of TNFa, for one hour while the mouse was anesthetized. The ears were then thoroughly washed and, after 24 hours, the ears were removed, and epidermal leaves were harvested and stained for MHC class II expression as described by Caugh an et al. (1986). The epidermal sheets were stained with MKD6 (anti-I-Ad) or Y3P as a negative control (anti-I-Ak), and FITC F (ab) 2 goat anti-mouse was used as a second step reagent. It had previously been found that mice transcutaneously immunized on the ear (as described above without shaving) had anti-CT antibodies of 7,000 ELISA units three weeks after a single immunization. Improved expression of MCH molecules of class II as detected by staining intensity, the reduced number of Langerhans cells (especially with cholera toxin), and changes in the morphology of Langerhans cells were found in epidermal leaves and mice immunized with CT and CTB comparable to controls (Figure 1) , suggesting that Langerhans cells were activated by epicutaneously applied cholera toxin (Aiba and Katz, 1990; Enk et al., 1993).

Example 18 Langerhans cells represent the epidermal contingent of a family of potent accessory cells termed 'dendritic cellsX Langerhans cells (and perhaps related cells in the dermis) are thought to be required for immune responses directed against foreign antigens which are found in the skin. The 'life cycle' of the cells of Langerhans is characterized by at least two distinct stages. Langerhans cells in the epidermis (the 'sentinels') can ingest particulates and efficiently process antigens, but they are weak stimulators of non-primed T cells. In contrast, the Langerhans cells that have been induced to migrate to the lymph nodes after contact with the antigen in the epidermis (the 'messengers') are poorly phacocytic and have limited antigen processing capabilities, but are potent stimulators of the antigens. native T cells. If the Langerhans cells are to fulfill their 'sentinel' and 'messenger' roles, they must be able to persist in the epidermis, and also be able to leave the epidermis in a controlled manner after exposure to the antigen. Thus, the regulation of keratinocyte adhesion of Langerhans cells represents a key control point in the trafficking and function of Langerhans cells. Langerhans cells express E-cadherin (Blauvelt et al., 1995), a homophilic adhesion molecule that is prominently represented in the epithelium. Keratinocytes also express this adhesion molecule, and E-cadherin clearly mediates the adhesion of murine Langerhans cells to keratinocytes. It is known that E-cadherin is involved in the localization of Langerhans cells in the epidermis. See Stingl et al. (1989) for a review of the characterization and properties of Langerhans cells and keratinocytes.

The migration of the Langerhans epidermal cells (LC) and their transport of the antigen from the skin to the draining lymph nodes is known to be important in the induction of cutaneous immune responses, such as contact sensitization. While . are in transit to the lymph nodes, the Langerhans cells are subject to a number of phenotypic changes required for their movement from the skin, and acquisition of the capacity for antigen presentation. In addition to the up-regulation of MHC class II molecules, there are alterations in the expression of adhesion molecules that regulate interactions with the neighboring tissue matrix and with T lymphocytes. The migration of Langerhans cells is known to be associated with a marked reduction in the expression of E-cadherin (Schwar zenberger and Udey, 1996, and a parallel upregulation of ICAM-1 (Udey, 1997)). Transcutaneous immunization with the bacterial exotoxins of ADP ribosylation (bARE's) is directed to the Langerhans cells in the epidermis. BAREs activate the Langerhans cells, transforming them from their sentinel role to their messenger role. The ingested antigen is then taken to the lymph node where it is presented to B and T cells (Streilein and Grammer, 1989, Kripke et al, 1990, Tew et al., 1997). In the process, the epidermal Langerhans cells mature to an antigen-presenting dendritic cell in the lymph nodes (Schuler and Steinmna, 1985); the lymphocytes that enter a lymphatic node segregate towards the follicles of the B cells and towards the T cell regions. The activation of the Langerhans cells to become migratory Langerhans cells, is known to be associated not only with a remarkable increase in the MHC molecules of class II, but also with the marked reduction in the expression of E-cadherin, and the upregulation of ICAM-1. It is considered that cholera toxin (CT) and its B subunit (CTB) supraregulates the expression of ICAM-1 and downregulates the expression of E-cadherin on Langerhans cells, as well as upregulates the expression of MHC class II molecules on Langerhans cells. CT or CTB act as an adjuvant by releasing sentinel Langerhans cells, to present antigens such as BSA or diphtheria toxoid phagocytosed by Langerhans cells in the same site and at the same time of the CT or CTB encounter, when these They are acting as adjuvants. Activation of Langerhans cells to supregregulate ICAM-1 expression and downregulate E-cadherin expression can be mediated by cytokine release including TNFa and IL-1β from epidermal cells or Langerhans cells same. This adjuvant method for transcutaneous immunization is considered functional for any compound that activates Langerhans cells. Activation could occur in a manner such as to down-regulate the E-cadherin and supraregular ICAM-1. The Langerhans cells could then carry antigens made from mixtures of such Langerhans cell activating compounds and antigens (such as diphtheria toxoid or BSA) to the lymph nodes where the antigens are presented to the T cells and elicit an immune response. Thus, the activating substance such as bARE can be used as an adjuvant for an otherwise transcutaneous non-immunogenic antigen, such as diphtheria toxoid, by activating the Langerhans cells to phagocytose the antigen, such as diphtheria toxoid, to migrate to lymph nodes, mature to dendritic cells, and present the antigen to T cells. The response of T helper cells to antigens used in transcutaneous immunization, can be influenced by the application of cytokines and / or chemokines. For example, interleukin-10 (IL-10) may bias the antibody response towards an IgGl / IgE response of Th2, whereas anti-IL-10 may improve the production of IgG2a (Bellinghausen et al., 1996).

Example 19 Sequestrin is a molecule expressed on the surface of erythrocytes infected with malaria, which functions to anchor the red blood cells parasitized with malaria, to the vascular endothelium. This is essential for parasite survival and contributes directly to the pathogenesis of P malaria. fal ciparum in children dying of cerebral malaria. In cerebral malaria, cerebral capillaries become plugged with a large number of parasitized red blood cells, due to the specific interaction of the sequestrin molecule with the host endothelial receptor CD36. Ockenhouse et al. Identified the receptor CD36 and the parasite molecule (sequestrin) which mediates this receptor-ligand interaction. Ockenhouse et al. Have cloned and expressed as a recombinant protein produced by E. coli, the domain of the sequester molecule that interacts with the CD36 receptor. A truncated sequestrin product of 79 amino acids was used in the following example. Active immunization with recombinant sequestrin or DNA encoding the sequestrin gene could produce antibodies that block the adhesion of malaria-parasitized erythrocytes to host endothelial CD36, and thereby prevent the end of the parasite's life cycle, leading to to death of the parasite due to its inability to bind to the endothelium. The strategy is to develop an immunization method that elicits a high titer of blocking antibodies. One method of this type is the distribution of the vaccine transcutaneously. The measurement of total antibody titers as well as blocking activity and opsonization forms the basis for this procedure with transcutaneous immunization. The recombinant sequester protein used in the present experiments is 79 amino acids in length (approximately 18 kDa) and comprises the CD36 binding domain of the molecule. A naked DNA comprised of this domain has also been constructed and antibodies have been produced using the epidermal distribution of the gene gun. BALB / c mice (n = 3) were transcutaneously immunized as described above for the "Immunization Procedure". The mice were immunized at 0 and 8 weeks using 120 μl of the immunization solution prepared as follows: a plasmid encoded by the P sequesterine. fal ciparum was mixed in saline to make an immunization solution containing 80 μg of plasmid, 80 μg of CT (List Biologicals) per 100 μl of saline. One hundred twenty μl was applied to the unlabelled ear, then the ear was gently swabbed with an alcohol swab (Triad alcohol swab, isopropyl alcohol 70%). The immunization solution was not removed by washing.

Antibodies to sequestrin were determined using ELISA as described above for "ELISA IgG (H + L)" on sera collected from the tail vein at 3, 4, 7 and 9 weeks after primary immunization. The results are shown in Table 15. The DNA of the sequestrin with CT induced a detectable antibody response to the expressed protein, after the second booster immunization. For immunization to occur, the protein needs to be expressed and processed by the immune system. Thus, CT acted as an adjuvant for the immune response to the sequester protein, expressed by the plasmid encoding the sequestrin. It has been shown that DNA vaccines elicit neutralizing antibodies and CTLs in non-human primates, for diseases such as malaria (Gramzinski, Vaccine, 15: 913-915, 1997) and HIV (Shriver et al., Vaccine 15: 884-887, 1997) and have shown protection to varying degrees in several models (McClements et al., Vaccine, 15: 857-60, 1997). One might expect that immunization with DNA through the skin would elicit responses similar to that of the gene gun that targets the skin's immune system (Prayaga et al., Vaccine, 15: 1349-1352, 1997).

Table 15. Serum antibody against the sequester protein (Seq) in animals immunized with Seq DNA and cholera toxin (CT) ELISA units of IgG (H + L) Animal # Group Inm. Week 3 Week 4 Week 7 Week 9 8966 Seq DNA / CT 58 80 33 8967 Seq DNA / CT 76 81 41 146 8968 Seq DNA / CT 54 33 26 Media Geo 62 60 33 Bleeding 40 combined Example 20 BALB / c mice were transcutaneously immunized as described above for the "Immunization Procedure", in groups of five mice, using sequestrin. Mice were immunized at 0, 2 and 8 weeks using 100 μl of the immunization solution prepared as follows: at week 0 the mice were immunized with 59 μg of CT and 192 μg of sequestrin in 410 μl for the group that received sequestration and CT, 192 μg in 4 41100 μμll ppaarraa sseeccuueessttrriinnaa ssoollaa ,, yy 112200 μμgg of CCTTBB and and 225500 μμgg sseeccuueessttrriinnaa eenn 552200 μμll ppaarraa eell ggrruuppoo qqrererebiibrary kidnapping and CTB. Two weeks later the mice were reaffoorrzzaaddooss ccoonn 334455 μμll ddee ssoolluucci: saline that contained either 163 μg of sequestrin for the sequestran group alone, 345 μl of saline containing 163 μg of sequestrin with 60 μg of CT for CT plus the sequestraine group, 345 μl of saline which contains 163 μg of sequestrin and 120 μg of CTB for the sequestrin plus the group of CTB.In the second booster the mice were administered with 120 μg of sequestrin for the group of sequestrin alone, 120 μg of sequestrin and 120 μg of CT for CT plus the group of sequestrin and 120 μg of sequestrin and 120 μg of CTB for the group of sequestrin plus CTB. Antibodies were determined using ELISA as described above for "ELISA IgG (H + L)" in the sera at 3, 5, 7, 9, 10, 11 and 15 weeks after the first immunization. The results are shown in Table 16. Sequestrin alone induced a small but detectable antibody response. However, the addition of CT stimulated a much stronger immune response for the sequestrin, and CTB induced an immune response that was superior to the sequestrin alone. CT and CTB acted as adjuvants for the immune response to sequestrin, a recombinant protein.

Table 16 Seq, Seq + cholera toxin (CT), -Seg + - cholera toxin B (CTB) IgG ELISA Units (H + L) Units Detection Group Animal Immunization Anesthetics Bleeding Week 3 Week 5 Week 7 Week 8 Week 9 Week 11 Week 15 431 408 2861 Seq Seq 20 32 709 4 6 2862 Seq Seq 8 14 136 33 348 459 2863 Seq Seq 28 63 38 393 467 13 11 2864 Seq Seq 5 9 26 102 32 53 98 2865 Seq Seq 9 19 16 111 100 J 54 65 Media Geo 13 29 114 129 28963 42981 2866 Seq / CT Seq 923 1145 125 639 43679 20653 27403 2867 Seq / CT Seq 73 84 154 ND 9428 13169 7677 2868 Seq / CT Seq 805 370 1447 1105 ND 118989 270040 2869 Seq / CT Seq 175 760 1317 768 113792 ND 4277 2870 Seq / CT Seq 153 158 535 241 3245 Animal IgG (H + L) ELISA Unit Detection Group # Immunization Bleeding Antigen Week 3 Week 5 Week 7 Week 8 Week 9 Week 11 Week 15 Medium Geo 271 336 456 601 19747 31115 25279 2871 Seq / CTB Seq 8 3 87 40 22 29 192 2872 Seq / CTB Seq 4 6 24 22 35 24 34 2873 Seq / CTB Seq 107 138 128 51 2074 2283 2296 * - 2874 Seq / CTB Seq 6 7 22 18 41 40 457 2875 Seq / CTB Seq 515 504 1910 1744 ND 7148 5563 Media Geo 25 25 102 68 91 214 520 Combined Example 21 BALB / c mice were immunized transcutaneously as described above for the "Immunization Procedure", in groups of five mice. Mice were immunized at week 0 using 100 μl of immunization solution prepared as follows: FLUSHIELD (Wyeth-Ayerst, purified subvirion, formula 1997-98, batch # U0980-35-1) was lyophilized and mixed in saline for develop an immunization solution containing 90 μg of FLUSHIELD subvirion per 100 μl of saline for the group that received influenza alone; Influenza and CT were mixed in saline to make an immunization solution containing 90 μg of FLUSHIELD antigens and 100 μg of CT per 100 μl of saline, for the group that received influenza and CT. The antibodies were determined using ELISA as described above for "ELISA IgG (H + L)" in sera 3 weeks after the first immunization. The results are shown in Table 17. Influenza alone did not induce an antibody response. However, the addition of CT stimulated a much stronger immune response that was superior to that observed for influenza alone.

Thus, CT acted as an adjuvant to the immune response to FLUSHIELD, the subvirion influenza vaccine, a mixture of virally derived antigens.

Table 17. Serum antibody against influenza (Inf) types A and B in animals immunized with Inf alone or Inf + cholera toxin (CT) ELISA units of IgG (H + L) Animal # Inm Group Week 3 8601 CT / Inf 144 8602 CT / Inf 14 8603 CT / Inf 1325 8604 CT / Inf 36 8605 CT / Inf 29 Media Geo 77 606 Inf 17 607 Inf 16 608 Inf 20 609 Inf 23 610 Inf 23 Media Geo 20 Example 22 LT is in the family of ribosylating exotoxins of ADP, and is similar to CT in molecular weight, binds to ganglioside GM1, is 80% homologous with CT and has a similar stoichiometry of A: B5. Thus, LT was also used as an adjuvant for DT in transcutaneous immunization. BALB / c mice (n = 5) were immunized as described above at 0, 8 and 18 weeks with a saline solution containing 100 μg of LT (Sigma, Catalog # E-8015, lot 17hH12000) and 100 μg of CT (List Biologicals, Catalog # 101b) in 100 μl of saline. LT induced a modest response to DT as shown in Table 18. ETA (List Biologicals, lot #ETA 25A) is in the family of ADP ribosylating exotoxins, but it is a simple polypeptide that binds to a different receptor. One hundred μg of ETA were distributed in 100 μl of a saline solution containing 100 μg of CT to BALB / c mice on the back, as previously described at 0, 8 and 18 weeks. ETA reinforced the response to DT at 20 weeks. In this way, other ADP ribosylating exotoxins were able to act as adjuvants for the co-administered proteins (Table 18).

Table 18. Kinetics of antibody titers against diphtheria toxoid (DT) in animals immunized with Psy udomon exotoxin A and eru gin o sa (ETA) and DT or the heat-labile enterotoxin of E. c ol i (LT) and DT Group of Detection of Units of ELISA IgG (H + L) Animal # immunization Pre-Bleeding Antigen Week 20 5146 ETA / DT DT 31718 5147 ETA / DT DT 48815 5148 ETA / DT DT 135 5149 'ETA / DT DT 34 5150 ETA / DT DT 258 Media geo 1129 5136 LT / DT DT 519 5137 LT / DT DT 539 5138 LT / DT DT 38 5139 LT / DT DT 531 5140 LT / DT DT 901 Me: dia geo 348 Combined Example 23 BALB / c mice were transcutaneously immunized as described above for the "Immunization Procedure" in groups of five mice. Mice were immunized at 0 weeks, 8 weeks and 18 weeks with 100 μl of saline containing 100 μg of cholera toxin (List Biologicals, Catalog # 101B, lot # 10149CB), 50 μg of tetanus toxoid (List Biologicals, Catalog # 191B, lots # 1913a and 1915b) and 83 μg of diphtheria toxoid (List Biologicals, Catalog # 151, lot # 15151). The antibodies against CT, DT and TT were quantified using ELISA as described for "ELISA IgG (H + L)". Anti-CT, DT or TT antibodies were detected at 23 weeks after the first immunization. Anti-diphtheria toxoid and antibodies against cholera toxin were elevated in all immunized mice. The highest responders had 342 ELISA units of anti-tetanus toxoid antibody, approximately 80 times the level of antibody detected in sera from immunized animals. In this way, a combination of unrelated antigens (CT / TT / DT) can be used to immunize against inhibitory antigens. This shows that cholera toxin can be used as an adjuvant for multiple vaccines.

Table 19. Serum antibody in animals immunized simultaneously with cholera toxin, tetanus toxoid and diphtheria toxoid Group of Detection of Units of ELISA IgG (H + L) Animal # immunization Antigen Bleeding Week 23 5176 CT / TT / DT CT 7636 5177 CT / TT / DT CT 73105 5179 CT / TT / DT CT 126259 5216 CT / TT / DT CT 562251 5219 CT / TT / DT CT 66266 Combined < 3 Media geo 76535 5176 CT / TT / DT DT 64707 5177 CT / TT / DT DT 17941 5179 CT / TT / DT DT 114503 5216 CT / TT / DT DT 290964 5219 CT / TT / DT DT 125412 Combined < 4 Media geo 86528 5176 CC / TT / DT TT 21 5177 CC / TT / DT TT 30 5179 CT / TT / DT TT 342 5216 CT / TT / DT TT 36 5219 CT / TT / DT TT 30 Combined < 2 Geo media 47 Example 25 Transcutaneous immunization using CT induces potent immune responses. The immune response to an intramuscular injection and oral immunization was compared to transcutaneous immunization using CT as an adjuvant and antigen. 25 μg of CT (List Biologicals, Catalog # 101b) dissolved in saline were orally administered in 25 μl to BALB / c mice (n = 5) using the tip of a 200 μl pipette. The mice swallowed the immunization solution easily. Twenty-five μl of 1 mg / ml CT in saline was administered to the ear as described for the transcutaneous labeled group. Twenty-five μg of CT in saline were injected intramuscularly into the anterior thigh in the intramuscular labeled group. Mice injected intramuscularly with CT developed marked swelling and sensitivity at the site of injection, and developed high levels of anti-CT antibodies. Transcutaneously immunized mice had no redness or swelling at the site of immunization and developed high levels of anti-CT antibodies. Orally immunized mice developed very low levels of antibodies compared to mice immunized transcutaneously. This indicates that oral immunization through grooming in transcutaneously immunized mice did not explain the high levels of antibodies induced by transcutaneous immunization. In general, the transcutaneous route of immunization is superior to either oral immunization or IM, since high levels of antibodies are achieved without adverse reactions to immunization.

Table 20. Kinetics of antibody titers against cholera toxin in animals immunized by the transcutaneous, oral, or intramuscular route Route of Units of ELISA IgG (H + L) Animal # Immunization Bleeding Week 6 8962 Transcutaneous 23489 8963 Transcutaneous 30132 8964 Transcutaneous 6918 8965 Transcutaneous 20070 8825 Transcutaneous 492045 Combined 16 Media geo 34426 8951 Oral 743 8952 Oral 4549 8953 Oral 11329 8954 Oral 1672 Combined 14 Media geo 2829 955 intramuscular 35261 958 Intramuscular 607061 8959 Intramuscular 452966 8850 Intramusc lar 468838 8777 Intramuscular 171648 Combined 12 Media geo 239029 Example 26 BALB / c mice were immunized transcutaneously as described above for the "Immunization Procedure", in groups of five mice. Mice were immunized at 0, 8 and 20 weeks using 100 μl of immunization solution prepared as follows: Hib conjugate (Connaught, lot # 6J81401, 86 μg / ml) was lyophilized in order to concentrate the antigen. The lyophilized product was mixed in saline to make an immunization solution containing 50 μg of Hib conjugate per 10 μl of saline for the group that received the Hib-conjugate alone; the Hib conjugate and CT were mixed in saline to make an immunization solution containing 50 μg of Hib conjugate and 100 μg of CT per 100 μl of saline for the group that received Hib and CT conjugate.

The antibodies were determined using ELISA as described above for "ELISA IgG (H + L)" in sera 3 weeks after the second immunization. The results are shown in Table 21. The Hib conjugate alone induced a small but detectable antibody response. However, the addition of CT stimulated a much stronger immune response for the Hib conjugate. CT acted as an adjuvant for the immune response to the Hib conjugate. This indicates that an antigen conjugated to polysaccharide can be used as a transcutaneous antigen by the method described.

Table 21. Antibody for Haemophilus infl uenzae b (Hib) Animal # Group Inm. ELISA units. IgG (H + L) 5211 Hib 57 5212 Hib 29 5213 Hib 28 521-4 Hib 63 5215 Hib 31 Medium Geo 39 5201 CT / Hib 1962 5202 CT / Hib 3065 5203 CT / Hib 250 5204 CT / Hib 12 5205 CT / Hib 610 Geo 406 Medium combined bleed Example 27 Emulsions, creams and angels may provide practical advantages for the convenient diffusion of the immunization compound on the surface of the skin, on the hair or on the body folds. In addition, such preparations can provide advantages such as occlusion or hydration which can improve the efficiency of immunization. Heat-labile enterotoxin (LT) from E. col i (Sigma, Catalog # E-8015, lot 17hH1200) was used to compare the efficiency of transcutaneous immunization using a simple saline solution and a commonly available petrolatum-based ointment, AQUAPHOR, which "can be used alone or in composition with virtually any ointment using aqueous solutions or in combination with other oil-based substances and all common topical medications "(page 507 PDR, for Non-Prescription Drugs, 1994, 15th Edition). The mice were treated with a range of doses to evaluate the relative response of the antibody for dose reduction in comparative vehicles. BALB / c mice were immunized as described above, except that the immunization solution was applied for 3 hours on the back. The saline solutions of LT were prepared to distribute a dose of 50 μl of solution and 100 μg, 50 μg, 25 μg or 10 μg of antigen in the solution, using a solution of 2 mg / ml, 1 mg / ml, 0.5 mg / ml or 0.2 mg / ml, respectively. After 3 hours the back gently rubbed using a wetted gauze to remove the immunization solution.

The water-in-oil preparation was performed as follows: equal volumes of AQUAPHOR and antigen in saline were mixed in 1 ml tuberculin glass syringes with luer latches using a 15 gauge emulsification needle connecting it to two syringes and mixing until the mixture was homogeneous. A solution of 4 mg / ml, 2 mg / ml, 1 mg / ml or 0.5 mg / ml of LT in saline, respectively, was used to mix with an equal volume of AQUAPHOR. 50 μl of this mixture were applied to the shaved back for three hours and then removed only by rubbing with gauze. The antigen doses for water-in-oil emulsions containing LT were weighed in order to distribute 50 μl. The weight-for-volume ratio was calculated by adding the specific gravity of the saline solution (1.00 g / ml) and AQUAPHOR, 0.867 g / ml, and dividing the sum by 2 for a final specific density of 0.9335 g / ml. Approximately 47 mg of water in oil emulsion containing LT, were distributed to the mouse for immunization. A dose-response relationship was evident for the saline solution and the water-in-oil emulsion for distributing LT (Table 22). One hundred μg induced the highest level of antibodies and 10 μg induced a lower but potent immune response. LT emulsified as water in oil induced a similar response to LT in saline, and seems to offer a convenient distribution mechanism for transcutaneous immunization. Similarly, more complex gels, creams or formulations such as oil-in water-in oil could be used to distribute antigen for transcutaneous immunization. Such compositions could be used in conjunction with patches, occlusion bandages, or reservoirs, and may allow long-term application or short-term application of the immunization antigen and adjuvant.

Table 22. Serum antibody against heat-labile enterotoxin (LT) of E. coli in animals immunized with varying doses of LT in saline or emulsion of AQUAPHOR ELISA IgG Units ELISA Units IgG (H + L) (H + L) Inm Group. Emulsion Animal Id "Bleeding Week .1 emulsion Animal Id # Bleeding Week 3 LT lOO μg saline 8741 184.14 Aquaphor 8717 6487 LT 100 μg Saline 8742 16320 Aquaphor 8719 4698 LT 100 μg Saline 874.1 19580 Aquaphor 8774 18843 LT 1 0 μg Saline 8744 19313 Aquaphor 8775 18217 or LT 100 μg saline 8745 22875 aquaphor 8861 16230 Combined .12 Combined 54 19190 11117 Medium Gco saline 8736 19129 Aquaphor 8721 4160 LT 50 μg Saline 8737 3975 Aquaphor 8722 12256 LT 50 μg Saline 8738 6502 Aquaphor 8725 12262 LT 50 μg Saline 8739 6224 Aquaphor 8771 12982 LT 50 μg Saline 8740 18449 Aquaphor 8772 15246 LT 50 μg Combined 54 Combined 57 8929 10435 Media Gco Salina 8768 3274 Aquaphor 8727 3585 Saline 87.11 3622 Aquaphor 8728 3 LT25μg Saline 8732 557 Aquaphor 8729 4206 LT 25 μg Saline 87.13 626 Aquaphor 8862 7353 LT 25 μg Saline 87.14 1725 Aquaphor 8769 5148 LT25μg Combined 56 Combined 53 LT25μg 1481 1114 Salina 8848 621 Aquaphor 8748 1968 Salty Media Gco 8849 475 Aquaphor 8749 1935 Saline 8757 858 Aquaphor 8750 646 LTl μg Saline 8759 552 Aquaphor 8747 1569 i LT 1 μg Saline 8760 489 Aquaphor 8764 1 LTOμg Combined 4.1 Combined 39 LT 10 μg 585 329 LT 10 μg MediaGeo Example 28 Mice were transcutaneously immunized as described above for the "Immunization Procedure", in groups of five mice. The groups were immunized at 0, 8 and 18 weeks with 100 μl of saline containing 100 μg of tetanus toxoid (List Biologicals, Catalog # 191B, lots # 1913a and # 1915b) and 83 μg of diphtheria toxoid (List Biologicals, Catalog # 151, lot # 15151) alone or in combination with 100 μg of cholera toxin (List Biologicals, Catalog # 101B, lot # 10149CB). Antibodies with anti-diphtheria toxoid were quantified using ELISA as described for "ELISA IgG (H + L)". High levels of anti-toxoid antibodies were detected in animals immunized with either TT / DT or CT / TT / DT. However, the antibody titers were much higher in animals in which CT was included as an adjuvant. This anti-toxoid titre was obviously increased in both groups after each subsequent immunization (8 and 18 weeks). Thus, while DT can induce a small but significant response against itself, the magnitude of the response can be increased by 1) the inclusion of cholera toxin as an adjuvant and 2) the reinforcement with adjuvant cholera toxin and antigen (diphtheria toxoid). The responses of classical reinforcers are dependent on the memory of the T cells, and the reinforcement of the anti-DT antibodies in this experiment indicates that the T cells are compromised by the transcutaneous immunization.

Table 23. Kinetics of titers of anti-diphtheria toxoid (DT) antibody in animals immunized with tetanus toxoid (TT) and DT or cholera toxin (CT), TT and DT Detection Cluster I're- Units of ELISA IgG (H + L) - Nimató De Inni. from?? no Bleeding Sem 2 Sem 4 Sem.6 Sem.8 Sem. 10 Sem. 14 Sem. 17 San. 18 Sem.20 San. 5196 TI / DI '1) 1' 7 12 18 23 49 56 33 18 219 166 5197 TT / DT DT 5 11 11 10 15 17 16 17 125 75 5198 'ITDT DG 13 20 16 - 28 25 27 7 48 172 5199 TJ.7DT DT 13 8 10 10 11 22 12 217 178 263 5200 IT / DT DI '4 10 4 7 120 149 127 - 17309 1453 MeansGeo 12 10 11 31 38 29 26 332 382 5176 CT /? VDT DT 26 21 14 3416 5892 1930 1826 63087 647C 5177 CT / TT / DT DT 6 7 8 424 189 149 175 16416 1794 5179 CT / TT / DT DT 3 4 8 4349 1984 2236 1921 124239 11451 5216 CT / TT / DT DT 12 5 9 11 3238 2896 2596 1514 278281 2909? 5219 CT / TT / DT DT 15 13 12 5626 4355 2036 1941 343161 1254 MediaGeo 10 2582 1945 1277 1125 104205 8652 Garttttb Example 29 C57B1 / 6 mice were transcutaneously immunized with CT (azide-free, Calbiochem) as described above on the shaved back of the mouse. The . mice were challenged using a lethal challenge model 6 weeks after immunization (Mallet et al., "Immunoprophylactic efficacy of non-toxic mutants of Vijbrio chol toxin was e (CTK63) and heat labile toxin from Esch eri chi to col i (LTK63) in an intranasal challenge model of cholera toxin in mouse ", sent to Immunol ogy Let t ers).

In the challenge, the mice were administered with 20 μg of CT (Calbiochem, free of azide) dissolved in saline intranasally by means of the tip of a plastic pipette, while being anesthetized with 20 μl of ketamine-rompin. In test # 1, 12715 immunized mice survived the challenge after 14 days, and 1/9 unimmunized control mice survived. Five control mice were lost before the challenge due to anesthesia. Mice in challenge # 1 had serum anti-CT antibodies of 15,000 units of ELISA (geometric mean), and five immunized mice, killed at challenge time had pulmonary lavage IgG detected in 5/5 mice. Lung washes were collected as described above. Immunization and challenge were repeated with intact C57B1 / 6 mice, and 7/16 immunized mice survived the challenge, while only 2/17 non-immunized mice survived the challenge. Mice immunized in challenge # 2 had anti-CT IgG antibodies from 41,947 units of ELISA (geometric mean) . Pulmonary washings from five mice sacrificed at the time of the challenge demonstrated anti-CT IgG and IgA (Table 24).

Excrement samples of 8/9 mice demonstrated anti-CT IgG and IgA (Table 25). The samples of excrement were collected in fresh from the animals that defecated spontaneously at the time of the challenge. Stools were frozen at -20 ° C. At the time of ELISA, the faeces were thawed, homogenized in 100 μl of PBS, centrifuged and the ELISA was run on the supernatant. The combined survival rate among the immunized mice was 19/31 or 61%, while the combined survival rate among the non-immunized mice was 3/26 or 12%.

Table 24. Pulmonary Wash IgG and IgA Cholera Antitoxin Antibody Titers Animal Identification Number Dilution From the 8969 8970 8971 8972 8995 Sample IgG (H + L) anti -CT (D < Optical sine) 1: 10 3,613 3,368 3,477 3,443 3,350 1: 20 3,302 3,132 3,190 3,164 3,166 1: 40 3.090 2.772 2.825 2 .899 2.692 1: 80 2,786 2,287 2,303 2,264 2,086 1: 160 2,041 1,570 1,613 1,624 1,441 1: 320 1.325 0.971 1.037 1 .041 0.965 1: 640 0.703 0.638 0.601 0 .644 0.583 1: 1280 0.434 0.382 0.350 0.365365 Anti-CT IgG (Optical Density) 1: 2 1,235 2,071 2,005 2,115 1,984 1: 4 1,994 1,791 1,836 1.85 1,801 1: 8 1,919 1,681 2,349 1,796 1,742 1: 16 1.8 1,457 1,577 1,614 1,536 1: 32 1,503 1,217 1.36 1,523 1.23 1: 64 1,189 0.863 1,044 1.101 0.88 1: 128 0.814 0.57 0.726 0.74 0.595 1: 356 0.48 0.334 0.436 0.501 0.365 Table 25. IgG and IgA anti-cholera toxin antibody titers, in feces Mouse identification number (immunization group) Dilution of 8985 8997 8987 9090 8977 8976 8975 8988 8994 8979 9000 8983 sample (CT) (CT) (CT) (CT) (CT) (CT) (CT) (CT) (CT) (CT) (NONE) (NONE) IgG (H + L)? Ti-CT (optical density) 1: 10 1.01 1.91 2.33 0.03 0.74 1.98 1.20 1.45 0.09 0.05 0.02 0.18 1: 20 0.42 0.94 1.26 0.31 1.19 0.50 0.91 0.04 0.08? 1: 40 0.20 0.46 0.68 0.12 • 0.58 0.24 0.49 - 0.02 00 1: 80 0.10 0.21 0.34 0.05 0.31 0.09 0.25 - 1: 160 0.03 0.09 0.18 0.02 0.14 0.05 0.12 _ IgA? Nti-CT (optical density) 1: 4 0.32 1.14 0:43 0.00 0.19 1.00 0.58 1.21 0.02 0.07 1: 8 0.16 0.67 0:24 0.08 0.56 0.36 0.77 1: 16 0.08 0.33 0: 1 1 0.03 0.27 0.17 0.40 1:32 0.06 0.16 0:05 0.03 0.12 0.08 0.20 1: 64 0.01 0.07 0:03 _ 0.05 0.03 0.10 Example 30 Female C57B1 / 6 mice were obtained from Charles River Laboratories. Mice were immunized with 200 μg of ovalbumin (OVA) (Sigma, lot # 14H7035, stock concentration of 2 mg / ml in PBS) and 50 μg of cholera toxin (List Biologicals, lot # 101481B, stock concentration of 5 mg / ml). A Packard Cobra Gamma Counter (serial # 102389) was used to measure the amount of 51Cr released. C57B1 / 6 mice were anesthetized with 0.03 ml of ketamine-rompin and shaved on the back with a razor, without traumatizing the skin, and left to rest for 24 hours. The mice were anesthetized and then immunized at 0 and 28 days with 150 μl of immunization solution placed on the shaved skin over an area of 2 cm2 for 2 hours. The mice were then cleaned twice with wet gauze. The mice showed no adverse effects by anesthesia, immunization or by the washing procedure. This procedure was repeated weekly for three weeks. Splenic lymphocytes were harvested one week after the booster immunization. The cells were cultured in vi tro in RPMI-1640 and 10%. of FBS (with penicillin-streptomycin, glutamine, non-essential amino acids, sodium pyruvate and 2-mercaptoethanol) for 6 days with the addition of 5% supernatant of rat concanavalin A as a source of IL-2, with or without antigen . The target cells consist of syngeneic EL4 cells (H-2b) alone, and EL4 cells pulsed with the CTL peptide, SINFEKKL, allogeneic L929 cells (H-2k) and EG7 cells. The target cells (1 x 106 cells per well) were labeled for 1 hour with 0.1 mCi per well of 51 Cr (source, Na? CrO Amersham) and were added to the effector cells at ratios in the range of 3: 1 to 100: 1. Cell mixtures were incubated in 96-well round-bottom tissue culture plates (Costar, Catalog # 3524) in 0.2 ml of complete RPMI-1640, 10% FBS medium for 5 hours at 37 ° C in an atmosphere humidified of 5% C02. At the end of the 5-hour culture, the supernatants were absorbed with cotton pieces and processed for the determination of 51 Cr release. The specific lysis was determined as:% specific lysis = 100 x [(experimental release-spontaneous release) / (maximum release-spontaneous release)]. As shown in Table 26, the CTLs of part 1 were detected against the cells pulsed with the EL4 peptide at an E: T ratio of 100: 1 for the group immunized with CT + OVA. The CTL assays are not positive if the percent specific lysis is not above 10% or clearly above the preceding percentage lysis of the effectors stimulated with the medium. Similarly, as shown in Table 26, the CTLs of part 2 were detected against EG7 (cells transfected with OVA) at an E: T ratio of 100: 1 for the group immunized with CT + OVA. In this way, CT contributed to the production of CTLs via the transcutaneous route.

Table 26. Translucently Induced OVA-Specific CTL Part 1 - Target Cells: EL4 + Peptide Group Inm. CT + OVA CT + OVA CT CT OVA OVA Stimulated with Average Proportion; Ova Half Ova Half Ova E: T 100: 1 9.5 13.1 1 1.1 12.5 23.1 21.5 : 1 6.9 6.8 5.9 8.9 14.2 10.7 : 1 4.9 3.5 3.5 8.5 7.7 5.2 Part 2 - Target Cells: EG7 (Transfected with OVA) Inm Group CT + OVA CT + OVA CT CT OVA OVA Stimulated with Half Oval Half Oval Half Ova E.T 100: 1 10.6 17.6 14.5 16.8 23.8 26 : 1 4 9 9.5 8.2 10.1 13.6 10.7 : 1 6.4 4.4 4 5 7.3 4.2 E xemployment 31 C57B1 / 6 mice (n = 6) were transcutaneously immunized as described above for the "Immunization Procedure". Mice were immunized at 0 and 4 weeks with 100 μl of saline containing 100 μg of cholera toxin (List Biologicals, Catalog # 101B, lot # 10149CB) and 250 μg of ovalbumin protein (Sigma, chicken egg albumin , Grade V Catalog # A5503, lot # 14H7035). Single cell suspensions were prepared from the spleens harvested from animals at eight weeks after the first immunization. Splenocytes were established in culture at 8 x 10 5 cells per well in a volume of 200 μl containing OVA protein or protein irrelevant with albumin at the indicated concentrations. The cultures were incubated for 72 hours at 37 ° C in a C02 incubator at which time 0.5 μci / well of tritiated thymidine was added. (3H-thymidine) to each well. Twelve hours later, proliferation was evaluated by harvesting the plates and quantifying the radiolabeled thymidine, incorporated, by liquid scintillation counting. The crude values of the incorporation of 3H are indicated in cpm and the rate of increase (experimental cpm / cpm of the medium) is indicated to the left of each sample. Proportional increases greater than three are considered significant.

Significant proliferation was only detected when the splenocytes were stimulated with the protein, ovalbumin, to which the animals had been immunized in vi with and without the irrelevant protein conalbumin. Thus, transcutaneous immunization with cholera toxin and ovalbumin protein induces antigen-specific proliferation of splenocytes in vi tro, indicating that a cellular immune response is evoked by this method.

Table 27. Specific antigen proliferation of spleen cells from animals immunized with cholera toxin (CT) and Ovalbumin (OVA).

Concentration Group of Increase Incorporation of 3-H in the 3-H incorporation immunization of proportional OVA protein Conalbumin stimuli in vitro ine d i a Increase cpm cpm proportional CT / OVA 10 μg / ml l427 13450 9.4 3353 2.3 1 μg / ml 4161 2.9 2638 1 .8 0.1 μg / ml 2 198 1 .5 2394 1 .7 0.01 μg / ml 3419 2.4 2572 1 .8 The descriptions of all the Patents, as well as all other printed documents, cited in this specification are incorporated herein by reference in their entirety. Such references are cited as indicative of experience in the art. While the present invention has been described in relation to what is currently considered to be the practical and preferred embodiments, it is understood that the present invention does not have to be limited or restricted to the described modalities but on the contrary, it is intended to cover the various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Thus, it is understood that the variations in the invention described will be obvious to those of skill in the art, without departing from the novel aspects of the present invention, and such variations are intended to fall within the scope of the following claims.

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It is noted that in relation to this date, the best method known to the applicant to carry out the aforementi invention, is that which is clear from the present description of the invention.

Claims (36)

RE I VIND I CAC I ONE S Having described the invention as above, the content of the following claims is claimed as property:

1. A formulation for the transcutaneous immunization, comprising an antigen and an adjuvant, characterized in that the application of the formulation to the intact skin induces a specific immune response for the antigen, without piercing the skin.

2. The formulation according to claim 1, further characterized in that it comprises a bandage to form a patch for transcutaneous immunization.

3. The formulation according to claim 2, characterized in that the bandage is an occluder bandage.

4. the formulation of, according to claim 2, characterized in that the bandage covers more than one field of draining lymph nodes.

5. The formulation according to claim 1, characterized in that the adjuvant improves the presentation of the antigen to a lymphocyte.

6. The formulation according to claim 1, characterized in that the adjuvant activates an antigen presenting cell.

7. The formulation according to claim 6, characterized in that the antigen-presenting cell is a Langerhans cell or a dermal dendritic cell.

8. The formulation according to claim 1, characterized in that the adjuvant increases the expression of the major histocompatibility complex of class II, or an antigen presenting cell.

9. The formulation according to claim 8, characterized in that the antigen-presenting cell is a Langerhans cell or a dermal dendritic cell.

10. The formulation according to claim 1, characterized in that the adjuvant causes an antigen-presenting cell underlying an application site to migrate to a draining lymph node.

11. The formulation according to claim 10, characterized in that the antigen-presenting cell is a Langerhans cell or a dermal dendritic cell.

12. The formulation according to claim 1, characterized in that the adjuvant signals a Langerhans cell to mature into a dendritic cell.

13. The formulation according to claim 1, characterized in that it consists essentially of antigen and adjuvant.

14. The formulation according to claim 1, characterized in that the component of the formulation is antigen and adjuvant.

15. The formulation according to claim 1, characterized in that it is an aqueous solution.

16. The formulation according to claim 1, characterized in that it does not include an organic solvent.

17. - The formulation according to claim 1, characterized in that it does not include a penetration enhancer.

18. The formulation according to claim 1, characterized in that it is formed as an emulsion.

19. The formulation according to claim 1, characterized in that the antigen is derived from a pathogen selected from the g consisting of bacteria, viruses, fungi and parasites.

20. The formulation according to claim 1, characterized in that the antigen is a tumor antigen.

21. The formulation according to claim 1, characterized in that the antigen is a self antigen.

22. The formulation according to claim 1, characterized in that the antigen is an allergen.

23. The formulation according to claim 1, characterized in that the antigen is of molecular weight greater than 500 daltons.

24. The formulation according to claim 1, characterized in that it includes at least two different separated antigens.

25. The formulation according to claim 1, characterized in that the antigen is provided as a nucleic acid encoding the antigen.

26. The formulation according to claim 25, characterized in that the nucleic acid is non-integrative and non-replicating.

27. The formulation according to claim 25, characterized in that the nucleic acid further comprises a regulatory region operably linked to the sequence encoding the antigen.

28. The formulation according to claim 25, characterized in that it does not include a penetration enhancer, viral particle, liposome or charged lipid.

29. The formulation according to any of claims 1-28, characterized in that the adjuvant is an ADP ribosylating exotoxin.

30. The formulation according to claim 29, characterized in that the ADP ribosylating exotoxin is the cholera toxin or a functional derivative thereof.

31. The formulation according to claim 29, characterized in that the ADP ribosylating exotoxin is a heat-labile enterotoxin, from E. coli, pertussis toxin or a functional derivative thereof.

32. The formulation according to claim 29, characterized in that the adjuvant is provided as a nucleic acid encoding an ADP ribosylating exotoxin.

33. The formulation according to any of claims 1-28, characterized in that the application of the formulation does not involve the physical, electrical or sonic energy that pierces the intact skin.

34. The formulation according to any of claims 1-28, characterized in that the immune response is not an allergic reaction, dermatitis or atopic reaction.

35. The preparation of the formulation according to any of the preceding claims.

36. The use of the formulation according to any of the preceding claims, to induce an immune response in a non-human animal.