JP2010059201A - Adjuvant for transcutaneous immunization - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a formulation for transcutaneous immunization containing an antigen and an adjuvant. <P>SOLUTION: The transcutaneous immunization system delivers the antigen to immune cells without perforation of the skin, and induces an immune response in an animal or human. This system uses the adjuvant (preferably ADP-ribosylating exotoxin) to induce an antigen-specific immune response (e.g. humoral and/or cellular effectors) after transcutaneous application of the formulation containing the antigen and the adjuvant to the intact skin of the animal or human. The efficiency of immunization is 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. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

The present invention relates to transcutaneous immunity and adjuvants useful therefor for inducing antigen-specific immune responses.

  Transcutaneous immunity requires both passage of the antigen through the outer barrier of the skin (which normally does not pass the antigen) and an immune response to the antigen. US patent application Ser. No. 08 / 749,164 has demonstrated that the use of cholera toxin as an antigen induces a reproducible and strong antibody response. Antigen was administered to the skin in saline with or without liposomes. In this application we demonstrate transcutaneous immunization using adjuvants such as bacterial exotoxin, its subunits and related toxins.

  There is a report of transcutaneous immunization using transferosomes by Paul et al. (1995). In this document, transferosomes are used as carriers for proteins (bovine serum albumin and gap junction proteins) to which complement-mediated lysis of antigen-sensitized liposomes is directed. Placing a solution containing this protein on the skin did not induce an immune response, and only the transferosome could transport the antigen through the skin and achieve immunity. As described in US patent application Ser. No. 08 / 749,164, transferosomes are not liposomes.

  FIG. 1 of Paul et al. (1995) showed that only antigen and transferosome formulations induce an immune response (measured by lysis of antigen-sensitized liposomes). Formulations of antigens in solution applied to the skin, antigens and mixed micelles, and antigens and liposomes (ie, smectic mesophases) did not induce an immune response equal to that induced by subcutaneous injection. . Thus, there was a positive regulation (ie antigen and transferosome) that justified the negative conclusion that the antigen and liposome formulation did not cause transcutaneous immunity.

  Paul et al. (1995), on page 3521, the skin is an effective protective barrier "impervious to substances with a molecular weight of up to 750 daltons", non-invasive immunity through large skin of the whole immunogen. It is said that it is excluded. Therefore, this document denies the use of molecules such as cholera toxin (which is 85,000 daltons) because such molecules are not expected to pass through the skin and therefore cannot achieve immunity. ing. That is, the skin is a barrier that an adjuvant or antigen such as cholera toxin cannot be considered without the disclosure of the present invention.

Paul and Cevc (1995), page 145, “Large molecules do not normally pass through complete mammalian skin. Therefore, immunization from outside the skin with simple peptide or protein solutions. Is impossible. " They concluded that "liposomes or mixed micelle immunogens applied to the skin, whether combined with immunoadjuvant lipid A, are not as bioactive as simple protein solutions".
Wang et al. (1996) placed an aqueous solution of ovalbumin (OVA) on the skin of a shaved mouse to induce an allergic type response as a model of atopic dermatitis. Mice were anesthetized and covered with a closed patch containing up to 10 mg of OVA, which was placed on the skin for 4 consecutive days. This operation was repeated after 2 weeks.

  In FIG. 2 of Wang et al. (1996), the ELISA assay performed to measure the IgG2a antibody response did not show an IgG2a antibody response to OVA. However, IgE antibodies associated with allergic responses were detected. In further experiments, mice were more extensively patched with OVA solution for 4 days every 2 weeks. This was repeated 5 times, i.e. the mice were patched for a total of 20 days. Again, high doses of OVA did not produce significant IgG2a antibodies. Significant levels of IgE antibody were produced.

  The authors at page 4079, “We applied a protein antigen on the skin in the absence of adjuvant can sensitize animals and induce a predominantly Th2-like response with high levels of IgE. To prove this, we have established an animal model. " Extensive skin exposure to high doses of protein antigen failed to produce significant IgG antigens, but induced IgE antibodies that were evidence of an allergic type response. That is, Wang et al. (1996) teach that the described OVA exposure is a model of atopic dermatitis and not a mode of immunity. Thus, according to the teachings of this document, transcutaneous immunization with an antigen could be expected to induce high levels of IgE antibodies if it crosses the skin and induces an immune response. Instead, we have unexpectedly found that an antigen placed on the skin in saline with an adjuvant induces high levels of IgG and small amounts of IgA, but not IgE.

  In contrast to the cited references, we provide that the application of the antigen and adjuvant to the skin provides a transdermal delivery system of the antigen that can induce an antigen-specific immune response of IgG or IgA. I found. The adjuvant is preferably ADP-ribosylated exotoxin. From time to time, hydration, passage-enhancing substances or occlusive dressings may be used in the transdermal delivery system.

Summary of the Invention It is an object of the present invention to provide a system of transcutaneous immunity that induces an immune response (eg, humoral and / or cellular effectors) in an animal or human.

  This system provides a simple application of an antigen and adjuvant formulation to the intact skin of an organism to induce a specific immune response to the antigen.

  In particular, adjuvants activate antigen presenting cells of the immune system (eg, Langerhans cells of the epidermis, skin dendritic cells, dendritic cells, macrophages, B lymphocytes) and / or induce antigen presenting cells, Phagocytosis of antigen. The antigen presenting cell then presents the antigen to T and B cells. In Langerhans cells, antigen-presenting cells migrate from the skin to the lymph nodes and present the antigen to lymphocytes (eg, B cells and / or T cells), thus inducing an antigen-specific immune response.

  In addition to inducing an immune response to generate antigen-specific B lymphocytes and / or T lymphocytes (including cytotoxic T lymphocytes (CTL)), another object of the present invention is a transdermal immune system. Is used to positively or negatively control components of the immune system to affect antigen-specific helper T lymphocytes (Th1, Th2 or both).

  In a first embodiment of the invention, a formulation containing an antigen and an adjuvant is applied to the intact skin of an organism, the antigen is presented to immune cells, and an antigen-specific immune response is induced without puncturing the skin Is done. The formulation may contain additional antigens such that transdermal application of the formulation induces an immune response against multiple antigens. In such cases, the antigens may or may not be obtained from the same source, but the antigens may have different chemical structures so as to induce specific immune responses against different antigens. I will. Antigen-specific lymphocytes may participate in the immune response (by B cells if participating) and antigen-specific antibodies may be part of the immune response.

  In a second embodiment of the invention, the method is used to treat an organism. If the antigen is obtained from a pathogen, the treatment causes the organism to be vaccinated against pathogenicity as caused by infection by the pathogen or toxin secretion. Formulations containing tumor antigens provide cancer treatment, formulations containing self antigens provide treatment for diseases caused by the organism's own immune system (eg, autoimmune diseases), and formulations containing allergens treat allergic diseases May be used in immunotherapy to do.

  In a third embodiment of the invention, a patch for use in the above method is provided. The patch consists of a bandage and an effective amount of antigen and adjuvant. The bandage may be occlusive or non-occlusive. The patch may contain additional antigens such that its application induces an immune response against multiple antigens. In such cases, the antigen may or may not be obtained from the same source, but the antigen will have a different chemical structure so as to induce a specific immune response against different antigens. Let's go. For effective treatment, multiple patches are applied frequently or constantly over a long period of time.

  Further in a fourth embodiment of the invention, the formulation is applied to intact skin so as to cover two or more draining lymph node fields using single or multiple applications. Applied. The formulation may contain additional antigens such that application to intact skin induces an immune response against multiple antigens. In such cases, the antigen may or may not be obtained from the same source, but the antigen will have a different chemical structure so as to induce a specific immune response against different antigens. Let's go.

  The products of the methods of the invention can be used to treat an existing disease, prevent a disease, or reduce the severity and / or duration of a disease. However, induction of allergy, atopic disease, dermatitis, or contact hypersensitivity is not preferred.

  In addition to the antigen and adjuvant, the formulation may further contain hydration agents (eg, liposomes), penetration enhancers, or both. For example, antigen-adjuvant formulations include emulsions, emulsions (eg, aqueous creams) made with AQUAPHOR (petrolatum, mineral oil, mineral wax, wool wax, panthenol, bisabol, and glycerin). ), Oil-in-water emulsions (eg oil cream), anhydrous lipids and oil-in-water emulsions, anhydrous lipids and water-in-oil emulsions, fats, waxes, oils, silicones, wetting agents (eg glycerol), jelly (eg SURGILUBE, KY jelly), or combinations thereof. The formulation may be provided as an aqueous solution.

  This formulation preferably does not contain an organic solvent. The formulation may be applied after wiping the skin with alcohol. However, it is not preferred to remove the keratinocyte layer prior to application of the formulation to the extent achieved with a depilatory agent.

  Antigens may be derived from pathogens that can infect organisms (eg, bacteria, viruses, fungi, or parasites), or cells (eg, tumor cells or normal cells). The antigen may be a tumor antigen or a self antigen. Chemically, the antigen may be a carbohydrate, glycolipid, glycoprotein, lipid, lipoprotein, phospholipid, polypeptide, or a chemical or recombinant conjugate thereof. The molecular weight of the antigen is greater than 500 daltons, preferably greater than 800 daltons, and more preferably greater than 1000 daltons.

  Antigens are obtained by recombinant means, chemical synthesis, or purification from natural sources. Proteinaceous antigens or conjugates with polysaccharides are preferred. The antigen is at least partially purified and cell-free. Alternatively, the antigen is provided in the form of a live virus, a live attenuated virus, or an inactivated virus.

Inclusion of an adjuvant allows enhancement or modulation of the immune response. Further selection of suitable antigens or adjuvants include humoral or cellular immune responses, specific antibody isotypes (eg, IgM, IgD, IgA1, IgA2, IgE, IgG1, IgG2, IgG3, IgG4, or combinations thereof), and Preferential induction of a specific T cell subset (eg, CTL, Th1, Th2, T _DTR , or combinations thereof) is possible.

  Preferably the adjuvant is ADP-ribosylated exotoxin or a subunit thereof. A Langerhans cell activator can be used at any time.

  Optionally, the antigen, adjuvant, or both may be provided in the formulation by a nucleic acid (eg, DNA, RNA, cDNA, cRNA) encoding the antigen or adjuvant, as appropriate. This technique is called genetic immunity.

  As used herein, the term “antigen” means a substance that induces a specific immune response when presented to immune cells of an organism. The antigen may comprise a single immunogenic epitope, or multiple immunogenic epitopes recognized by a B cell receptor (ie, an antibody on the membrane of a B cell) or a T cell receptor. The molecule may be both an antigen and an adjuvant (eg, cholera toxin) and thus the formulation may contain only one component.

  As used herein, the term “adjuvant” means a substance added to a formulation that helps induce an immune response to an antigen. Certain substances may act as both adjuvants and antigens by inducing immune stimulation and specific antibody or T cell responses.

  As used herein, the term “effective amount” means the amount of antigen that induces an antigen-specific immune response. Induction of such an immune response may provide therapy such as immune defense, desensitization, immunosuppression, modulation of autoimmune disease, enhanced cancer immune surveillance, or therapeutic vaccination against established infections .

  As used herein, the term “draining lymph node” refers to the lymph fluid collected on a defined set of lymph nodes (eg, cervical, axilla, buttocks, pulleys, popliteal, abdominal and thoracic). Means the anatomical region filtered through the lymph nodes).

FIG. 1 shows that cholera toxin (CT) induces major histocompatibility complex (MHC) class II expression, LC morphology changes, and LC loss (possibly by migration) on Langerhans cells (LC) It shows that. BALB / c mice (H-2 ^d ) were immunized percutaneously in the ear with 250 g of cholera toxin CT or its B subunit (CTB) in saline. Previous experiments have established that mice can be easily immunized using ear skin (7000 anti-CT ELISA units after one immunization). After 16 hours, an epidermis sheet was prepared and stained for MHC class II molecules (scale bar is 50 μm). Panels are (A) saline alone as negative control, (B) transcutaneous immunization with CT in saline, (C) transcutaneous immunization with CTB in saline, (D) tumor necrosis factor-α as positive control. (10 μg) represents intradermal injection.

DETAILED DESCRIPTION OF THE INVENTION The transcutaneous immune system delivers substances to specialized cells that produce an immune response (eg, antigen presenting cells, lymphocytes) (Bos, 1997). These substances are called antigens as a class. An antigen consists of, for example, carbohydrates, glycolipids, glycoproteins, lipids, lipoproteins, phospholipids, polypeptides, proteins, conjugates thereof, or any other substance known to induce an immune response. Good. The antigen may be provided as a whole organism (eg, a bacterium or viral particle) and the antigen is obtained as an extract or lysate from whole cells or membranes only, or is the antigen synthesized chemically Or produced by recombinant means or obtained by viral inactivation.

  Methods for preparing drugs are known in the art, and the antigen and adjuvant are combined with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and preparations thereof are described, for example, in Remington's Pharmaceutical Sciences, E.W. Martin. Such formulations contain the antigen and adjuvant together with an appropriate amount of vehicle to prepare a pharmaceutically acceptable composition suitable for administration to humans or animals. The formulations are applied in creams, emulsions, gels, lotions, ointments, pastes, solvents, suspensions, or other dosage forms in the art. In particular, formulations that enhance skin hydration, penetration, or both are preferred. Other pharmaceutically acceptable additives (eg, diluents, binders, stabilizers, preservatives, and colorants) are also incorporated.

  Increased hydration of the stratum corneum increases the percutaneous absorption of certain solutes (Roberts and Walker, 1993). As used herein, the term “penetration enhancer” does not include substances such as water, physiological buffers, saline solutions, and alcohols that do not puncture the skin.

  The object of the present invention is to provide a novel means of immunizing through intact skin without the need to puncture the skin. The transcutaneous immune system provides a method by which antigens and adjuvants can be delivered to the immune system, particularly special antigen presenting cells (eg, Langerhans cells) under the skin.

  Without being bound by theory, to explain our observations, it is believed that the transcutaneous immune delivery system carries the antigen to cells of the immune system where an immune response is induced. The antigen passes through the normal protective outer layer of the skin (eg, the stratum corneum) and presents the antigen presented directly (or macrophages, dendritic cells, skin dendritic cells) to the T lymphocytes, The immune response is induced via B lymphocytes, or Kupfer cells). Optionally, the antigen may pass through the stratum corneum via hair follicles or skin organelles (eg, sweat glands, sebaceous glands).

  Transcutaneous immunization with bacterial ADP-ribosylated exotoxin (bARE) is known to be the most efficient antigen presenting cell (APC) (Udey, 1997). Target. We have found that bARE activates Langerhans cells when applied to the skin in saline solution. Langerhans cells direct the phagocytic immune response and migration of the antigen to the lymph nodes where it acts as an APC and presents the antigen to lymphocytes (Udey, 1997), thus producing a strong antibody response. Induce. Skin is generally considered a barrier to invading organisms, but imperfections in this barrier are innumerable throughout the epidermis to build an immune response against invading organisms. Of Langerhans cells are distributed (Udey, 1997).

According to Udey, 1997:
“Langerhans cells are bone marrow-derived cells that are present in the stratified squamous epithelium of all mammals. These contain all the accessory cell activities present in the non-inflammatory epidermis and are now on the skin. Essential for the initiation and propagation of an immune response to an applied antigen, Langerhans cells are members of a family of potent accessory cells (“dendritic cells”) that are widely distributed in epithelial and solid organs and lymphoid tissues. It appears only rarely ...

  “It is now recognized that Langerhans cells (and possibly other dendritic cells) have a life cycle with at least two distinct stages. Langerhans cells in the epidermis are normal for antigen capture“ sentinel ”cells. Configure your network. Epidermal Langerhans cells ingest granules containing microorganisms and are efficient processors of complex antigens. However, they have low expression levels of MHC class I and II antigens and costimulatory molecules (ICAM-1, B7-1, and B7-2) and do not stimulate much unprimed T cells. After contact with the antigen, some of the Langerhans cells are activated, exit the epidermis and migrate to the T cell-dependent region of the local lymph node, where they become mature dendritic cells. In the process of leaving the epidermis and migrating to lymph nodes, antigen-bearing epidermal Langerhans cells (now “messengers”) show dramatic changes in morphology, surface phenotype and function. In contrast to epidermal Langerhans cells, lymphoid dendritic cells are essentially non-phagocytic and have low processing efficiency of protein antigens, but express high levels of MHC class I and II antigens and various costimulatory molecules. It is the most cooperative among the native T cells that have been identified to date. "

  We believe that the strong antigen-presenting ability of epidermal Langerhans cells can be used for transdermally delivered vaccines. The transcutaneous immune response using the cutaneous immune system consists of passive diffusion to deliver vaccine antigen only to Langerhans cells in the stratum corneum (outermost layer of skin consisting of keratinocytes and lipids), ingest the antigen, The subsequent activation of Langerhans cells that migrate to cell follicles and / or T cell dependent regions and present antigens to T cells and / or T cells is required (Stingl et al., 1989). If antigens other than bARE (eg, BSA) are phagocytosed by Langerhans cells, these antigens are also transported to lymph nodes and presented to T cells, and then immunity specific for that antigen (eg, BSA). A response could be induced. That is, transdermal immunity is characterized by Langerhans cells, possibly by bacterial ADP-ribosylated exotoxins, ADP-ribosylated exotoxin binding subunits (eg, cholera toxin B subunit), or other Langerhans cell activators Activation.

  The mechanism of transcutaneous immunity by Langerhans cell activation, migration and antigen presentation is clearly demonstrated by upregulation of MHC class II expression in epidermal Langerhans cells from epidermal sheets immunized with CT or CTB . In addition, the extent of antibody response induced by transcutaneous immunity and isotype switching mainly to IgG is generally determined by antigen presentation such as Langerhans cells or dendritic cells (Janeway and Travers, 1996). Cell-stimulated T cell help and activation of both Th1 and Th2 pathways as suggested by the production of IgG1 and IgG2a (Paul and Seder, 1994; Seder and Paul) (Paul), 1994)). Furthermore, T cell proliferation against the antigen OVA is demonstrated in mice immunized with CT + OVA. Alternatively, a large antibody response is induced by thymus-independent antigen type 1 (TI-1) (Janeway and Travers, 1996), which directly activates B cells.

  The more commonly known range of skin immune responses is indicated by contact dermatitis and atopy. Contact dermatitis with pathological manifestations of LC activation engulfs antigen, migrates to lymph nodes, presents antigen, sensitizes T cells, and causes a strong destructive cellular response that occurs in affected areas of the skin Directed by Langerhans cells (Dahl, 1996; Leung, 1997). Atopic dermatitis utilizes Langerhans cells in a similar manner, but is the same as Th2 cells and is generally associated with high levels of IgE antibodies (Dahl, 1996; Leung) 1997).

  Transcutaneous immunization with cholera toxin and related bARE, on the other hand, is a superficial and microscopic post-immunization skin finding evidenced by lack of lymphocyte infiltration 24, 48, and 120 hours after immunization with cholera toxin ( That is, it is a novel immune response lacking non-inflammatory skin). This is because Langerhans cells "include all the accessory cell activity present in the non-inflammatory epidermis and are now essential for the initiation and propagation of an immune response to an antigen applied on the skin" (Uday ( Udey), 1997). Here the uniqueness of the transcutaneous immune response is also the high level of antigen-specific IgG antibody and the type of antibody produced (eg IgM, IgG1, ig2a, ig2b, IgG3 and IgA), and the anti-CT IgE antibody It is indicated by both lack.

  That is, we have found that bacterial-derived toxins applied to the surface of the skin can activate Langerhans cells or other antigen-presenting cells and are potent, as demonstrated by high levels of antigen-specific circulating IgG antibodies. We believe that an immune response can be induced. Such adjuvants can be used in transcutaneous immunization to enhance the IgG antibody response to proteins that are not themselves immunogenic when placed on the skin.

  Transdermal targeting of Langerhans cells can also be used to deactivate its antigen-presenting ability, thus preventing immunity or sensitization. Techniques for deactivating Langerhans cells include the use of, for example, interleukin-10 (Peguet-Navarro et al., 1995), interleukin-1β monoclonal antibody (Enk et al., 1993), Or there is a superantigen deficiency, such as through staphylococcal enterotoxin-A (SEA) induced epidermal Langerhans cell depletion (Shankar et al., 1996).

  Transcutaneous immunity is induced by the ganglioside GM1 binding activity of subunits such as CT, LT, or CTB (Craig and Cuatrecasas, 1975). Ganglioside GM1 is a ubiquitous cell membrane that is present in all mammalian cells (Plotkin and Mortimer, 1994). When pentameric CT B subunit binds to the cell surface, a hydrophilic hole is formed, allowing the A subunit to pass through the lipid bilayer (Ribi et al., 1988).

  We have shown that transcutaneous immunization with CT or CTB may require ganglioside GM1 binding activity. When mice were immunized transcutaneously with CT, CTA, and CTB, only CT and CTB caused an immune response. CTA has ADP-ribosylating exotoxin activity, but only CT and CTB with binding activity can induce an immune response, indicating that the B subunit is necessary and sufficient to immunize through the skin. ing. We conclude that Langerhans cells or other antigen presenting cells are activated by the binding of CTB to their cell surface.

Antigens of the present invention are expressed by recombinant means (preferably as fusions with affinity for epitope tags (Summers and Smith, 1987; Goeddle, 1990; Ausubel). ) Et al. (1996)), the chemical synthesis of oligopeptides (free or as a conjugate with a carrier protein) can be used to obtain the antigens of the present invention (Bodanzsky, 1993; Wisdom ( Wisdom), 1994). Oligopeptides are considered to be a type of polypeptide.

  Oligopeptides with a length of 6 to 20 residues are preferred. Polypeptides may also be synthesized as branched structures as disclosed in US Pat. Nos. 5,229,490 and 5,390,111. Antigenic polypeptides are, for example, synthetic or recombinant B cell and T cell epitopes from one organism or disease, universal T cell epitopes, and mixed T cell epitopes, and B cells from another organism or disease. It may contain an epitope.

  Antigens obtained by recombinant means or peptide synthesis, as well as antigens of the invention obtained from natural sources or extracts, are purified (preferably fractionated or chromatographed) using the physical and chemical characteristics of the antigen. (Janson and Ryden, 1989; Deutscher, 1990; Scopes, 1993)).

  Multivalent antigen formulations may be used to induce an immune response against two or more antigens simultaneously. Conjugates may be used to induce an immune response against multiple antigens, enhance an immune response, or both. In addition, toxoids, or toxoids enhanced using toxins may be used to enhance the toxins. First, transcutaneous immunization may be used to enhance responses induced by other immunization routes (eg, injection, or oral or nasal).

  Antigens include, for example, toxins, toxoids, subunits thereof, or combinations thereof (eg, cholera toxin, tetanus toxoid).

The antigen may be solubilized in water, a solvent (eg, methanol) or a buffer. Suitable buffers include, but are not limited to, phosphate buffered saline (PBS) without Ca ⁺⁺ / Mg ⁺⁺ , normal saline (150 mM NaCl aqueous solution), and Tris buffer. Antigens that do not dissolve in neutral buffer are solubilized with 10 mM acetic acid and then diluted to the desired volume with neutral buffer (eg, PBS). In the case of an antigen that dissolves only at acidic pH, it may be solubilized with dilute acetic acid, and then acetic acid-PBS may be used as a diluent at acidic pH. Glycerol can be a suitable non-aqueous buffer for use in the present invention.

  If the antigen, for example hepatitis A virus, is itself insoluble, the antigen may be present in the suspension or even in the preparation of the aggregate.

  Hydrophobic antigens (eg, polypeptides containing domains across the membrane) are solubilized in detergents. For formulations containing liposomes, the antigen in a surfactant solution (eg, cell membrane extract) is mixed with the lipid, then the surfactant is removed by dilution, dialysis, or column chromatography to remove the liposomes. It may be formed. Some antigens, such as from viruses (eg, hepatitis A virus), need not be soluble per se, but in the form of virosomes (Morein and Simons, 1985). Can be directly incorporated into liposomes.

  Plotkin and Mortimer (1994), which can vaccinate animals or humans to induce specific immune responses against specific pathogens, as well as methods for preparing antigens, determining the appropriate amount of antigen Methods of measuring induction of immune responses and methods of treating infection by pathogens (eg, bacteria, viruses, fungi, or parasites) are provided.

  Bacteria include, for example, Bacillus anthracis, Campylobacter, Cholera, Diphtheria, Toxigenic E. coli, Giardia, Neisseria, Helicobacter pylori (Lee and Chen, 1994), Hemophilus influenza B (Hemophilus influenza B), non-typeable hemophilus influenza (Henophilus influenzae non-typeable), Neisseria meningitidis, Bordetella pertussis, Streptococcus pneumoniae, Salmonella, Shigella, Streptococcus B, Group A There are cocci, tetanus, Vibrio cholerae, Yersinia, Staphylococcus, Pseudomonas species and Clostridia species.

  Viruses include, for example, adenoviruses, dengue serotypes 1-4 (Delenda et al., 1994; Fonseca et al., 1994; Smucny et al., 1995), Ebola (Yarling) (Jahring et al., 1996), enterovirus, hepatitis serotypes A to R (Blum, 1995; Katkov, 1996; Lieberman and Greenberg, 1996; mast (Mast), 1996; Shafara et al., 1995; Smedila et al., 1994; US Pat. Nos. 5,314,808 and 5,436,126), herpes simplex virus 1 or 2, Human Infectious disease virus (Deprez et al., 1996), influenza, Japanese equine encephalitis, measles, Norwalk, papillomavirus, parvovirus B19, polio, rabies, rotavirus, rubella, measles, vaccinia, malaria antigen There are vaccinia constructs, chickenpox, and yellow fever containing genes encoding other antigens such as:

  Parasites include, for example, Entamoeba histolytica (Zhang et al., 1995), Plasmodium (Bathhurst et al., 1993; Chang et al., 1989, 1992, 1994; Fries et al., 1992a, 1992b; Herrington et al., 1991; Khusmith et al., 1991; Malik et al., 1991; Migliolini et al., 1993; Pessi et al., 1991; Tam, 1988; Vreden et al., 1991; White et al., 1993; Wiesmueller , 1991), Leishmania (Leishmania) (Franken Burg (Frankenburg) et al., 1996), Toxoplasma gondii, and there is a roundworm.

  Antigens may also contain those used in biological battles, such as ricin, that are protected against by antibodies.

Adjuvant formulations also contain adjuvants, but one molecule may contain both adjuvants and antigenicity (eg, cholera toxin) (Elson and Dertzbaugh, 1994). An adjuvant is a substance used to specifically or non-specifically enhance an antigen-specific immune response. Usually, the adjuvant and the preparation are mixed before providing the antigen, or may be provided separately in a short time.

  Adjuvants are, for example, oil emulsions (eg, complete or incomplete Freund's adjuvant), chemokines (eg, defensin 1 or 2, RANTES, MIP1-α, MIP-2, interleukin-8), or cytokines (eg, Interleukin-1β, -2, -6, -10, or -12; γ-interferon; tumor necrosis factor-α; or granulocyte-monocyte-colony stimulating factor (Nohria and Rubin) Review, 1994), muramyl dipeptide derivatives (eg, murabutide, threonyl-MDP or muramyl tripeptide), heat shock proteins or derivatives, derivatives of Leishmania major LeIF (Skeiky et al., 1995), cholera toxin or cholera toxin B, lipopolysaccharide (LPS) derivatives (eg, lipid A or monophosphoryl lipid A), or superantigens (Saloga et al., 1996). . See also Richards et al. (1995) for adjuvants useful for immunization.

Adjuvants are antibodies or cell effectors, specific antibody isotypes (eg, IgM, IgD, IgA1, IgA2, secretory IgA, IgE, IgG1, IgG2, IgG3, and / or IgG4), or specific T cell subsets (eg, CTL, Th1, Th2, and / or T _DTR ) are selected to induce preferentially (Glenn et al., 1995).

  Cholera toxin is a bacterial exotoxin of the family of ADP-ribosylated exotoxins (referred to as bARE). Most bAREs are constructed as A: B dimers of the A subunit containing one binding B subunit and ADP-ribosyltransferase. Such toxins include diphtheria toxin, Pseudomonas exotoxin A, cholera toxin (CT), E. coli heat labile enterotoxin (LT), pertussis toxin, C. botulinum toxin C2, C. botulinum (C. cerevisiae). botulinum toxin C3, C. limosum exoenzyme, B. cereus exoenzyme, Pseudomonas exotoxin S, Staphylococcus aureus EDIN, B. sphaericus There is.

Cholera toxin is an example of bARE constructed by A subunit and B subunit. The B subunit is a binding subunit and consists of a B subunit pentamer that non-covalently binds to the A subunit. The B subunit pentamer is a symmetric donut-shaped structure that binds to GM ₁ -ganglioside on target cells. The A subunit ADP-ribosylates the alpha subunit of a subset of heterotrimeric GTP proteins (G proteins), including the Gs protein, thereby increasing intracellular levels of cyclic AMP. This stimulates the release of ions and fluids from small intestinal cells in the case of cholera.

Cholera toxin (CT) and its B subunit (CTB) have adjuvant properties when used as intramuscular or oral immunogens (Elson and Dertzbaugh, 1994; Trach et al., 1997). Another antigen, E. coli heat labile enterotoxin (LT), is 80% homologous to CT at the amino acid level and has similar binding properties. This also, GM 1 in the intestinal tract _- appears to bind to ganglioside receptor, having similar ADP- ribosylating exotoxin activity. Another bARE, Pseudomonas exotoxin A (ETA), binds to α ₂ -macroglobulin receptor-low density lipoprotein receptor related protein (Koonnas et al., 1992). bARE is reviewed by Krueger and Barbieri (1995).

  The toxicity of CT by the oral, nasal and intramuscular routes limits the doses used as adjuvants. In a comparative study of intramuscularly injected CT, extensive swelling was induced at the injection site. In contrast, equal or higher amounts of CT placed on the skin showed no toxicity.

  The following example shows that cholera toxin (CT), its B subunit (CTB), E. coli heat labile enterotoxin (LT) and pertussis toxin are powerful adjuvants for transcutaneous immunity and induce high levels of IgG antibodies However, IgE antibody is not induced. CTB without CT is also shown to induce high levels of IgG antibodies. That is, both bARE and its derivatives can be effectively immunized when applied on the skin in a simple solution. Furthermore, these examples show that CT, CTB and bARE can act as both adjuvants and antigens.

  When an adjuvant such as CT is mixed with BSA, a protein that is not normally immunogenic when applied to the skin, anti-BSA antibodies are induced. Using pertussis toxin as an adjuvant, an immune response to diphtheria toxoid was induced, but not diphtheria toxoid alone. That is, bARE can act as an adjuvant for non-immunogenic proteins in the transdermal immune system.

  Other proteins also act as adjuvants and antigens. For example, isolated virions influenza A and B, FLUZONE (Lederle) contain highly immunogenic neuraminidase and hemagglutinin, provide protection and act as their own adjuvants and antigens And effectively immunize through the skin. Of diphtheria toxoid denatured using formalin, pertussis toxoid denatured using hydrogen peroxide, or cholera or E. coli denatured using genetic techniques to destroy ribosyltransferase Toxoids, such as mutant toxins, such as thermolabile enterotoxins, continue to retain adjuvanticity and can act as antigens and adjuvants.

  Protection against life-threatening infections, diphtheria, pertussis and tetanus (DPT) can be achieved by inducing high levels of circulating antitoxin antibodies. Pertussis may be an exception because some researchers feel that antibodies against other parts of the invading organism are necessary for protection, but many new generations of cell-free pertussis vaccines have PT as a component of the vaccine. (Krueger and Barbieri, 1995). The pathology of the disease caused by DPT is directly related to the action of these toxins, and anti-toxin antibodies certainly play a role in defense (Schneerson et al., 1996).

  In general, toxins can be chemically inactivated to produce toxoids that are less toxic but retain immunogenicity. We believe that the transcutaneous immune system using toxin-based immunogens and adjuvants can achieve antitoxin levels sufficient to protect against these diseases. Anti-toxin antibodies can be induced by immunization with a toxin, or a genetically detoxified toxoid, or a toxoid and an adjuvant (eg, CT), or toxoid alone. Genetically modified toxin toxins with modified ADP-ribosylating exotoxin activity but no binding activity are particularly useful as non-toxic activators of antigen presenting cells used in transcutaneous immunization it is conceivable that.

  We also believe that CT can also act as an adjuvant to induce antigen-specific CTLs by transcutaneous immunization (for use of CT as an adjuvant in oral immunization, see Bowen et al. 1994; see Porgador et al. 1997).

  bARE adjuvants may be chemically conjugated to other antigens including, for example, carbohydrate, polypeptide, glycolipid, and glycoprotein antigens. Chemical binding of these antigens with toxins, their subunits, or toxoids is expected to enhance the immune response to these antigens when applied on the skin.

  Difficulties with toxin toxicity (eg diphtheria toxin is very toxic and one molecule can kill one cell) and difficult to handle powerful toxins like tetanus To overcome this, some researchers have created genetically produced toxoids by a recombinant approach. This is based on inactivation of the catalytic activity of ADP-ribosylated transferase by genetic deletion. These toxins retain binding ability but lack the toxicity of natural toxins. This approach is described by Brunette et al. (1994), Rappuoli et al. (1995), and Rappuoli et al. (1996). Such genetically modified exotoxins are useful for the transcutaneous immune system because they are free of toxins and have no safety concerns. They act as both antigens and adjuvants and enhance the immune response against themselves or added antigens. In addition, there are several techniques for chemically modified toxins that are related to the same problem (Schneerson et al., 1996). Alternatively, toxin or toxoid fragments may be used, for example, as a tetanus C fragment. These techniques may be important in some applications, especially in children where ingested toxins (eg, diphtheria toxin) can cause side effects.

  Optionally, a Langerhans cell activator may be used as an adjuvant. Examples of such activators include: heat shock protein inducers; contact sensitizers (eg, trinitrochlorobenzene, dinitrofluorobenzene, nitrogen mustard, pentadecyl catechol); toxins (eg, Shiga toxin) , Staph enterotoxin B); lipopolysaccharide; lipid A, or derivatives thereof; bacterial DNA (Stacey et al., 1996); cytokines (eg, tumor necrosis factor-α, interleukin-1β, − 10, -12); and chemokines (eg, defensin 1 or 2, RANTES, MIP-1α, MIP-2, interleukin-8).

  Different adjuvant combinations can be used in the present invention. For example, a combination of bacterial DNA containing a DpG nucleotide sequence and ADP-ribosylated exotoxin could be used to direct a T-helper response to a transdermally administered antigen. That is, Th1 or Th2-like responses to CT-adjuvanted antigens could be switched by the use of unmethylated CpG bacterial DNA, or other proteins (eg, LeIF) or calcium channel blockers.

  CpG is one of a class of structures with a pattern that allows the immune system to recognize its pathogenic origin and stimulate an underlying immune response that leads to an adaptive immune response. (Medzitov and Janeway, Curr. Opin. Immunol., 9: 4-9, 1997). These structures are called pathogen-associated molecular patterns (PAMPs) and include lipopolysaccharide, teichoic acid, unmethylated CpG motif, double stranded RNA and mannin.

  PAMP induces endogenous signals that can mediate inflammatory responses, acts as a costimulator of T cell function, and regulates effector function. The ability of PAMP to induce these responses plays a role in its potential as an adjuvant, and its target is APCs such as macrophages and dendritic cells. Skin antigen-presenting cells are similarly stimulated by PAMP transmitted in the skin. For example, Langerhans cells (a type of dendritic cell) are activated by a PAMP solution on the skin using molecules that are weakly immunogenic percutaneously, migrated, and these weakly immunogenic molecules Presents to T cells in the lymph nodes and induces an antibody response to less immunogenic molecules. PAMP is also used with other skin adjuvants, such as cholera toxin, to induce different costimulatory molecules and modulate different effector functions leading to immune responses (eg, Th2 to Th1 response).

  If the immunizing antigen has sufficient ability to activate Langerhans cells, another adjuvant would not be required as in the case of CT, which is the antigen and adjuvant. Whole cell preparations, live viruses, attenuated viruses, DNA plasmids, and bacterial DNA are considered sufficient to immunize transcutaneously. It may be possible to use low concentrations of contact sensitizers or other Langerhans cell activators to induce an immune response without causing skin lesions.

Liposomes and their preparation Liposomes are closed vesicles around an internal aqueous space. The internal compartment is separated from the external medium by a lipid bilayer consisting of discontinuous lipid molecules. In the present invention, antigen is delivered through intact skin to special cells of the immune system, where an antigen-specific immune response is induced. Transcutaneous immunity is achieved using liposomes, but as shown in the examples, liposomes are not required to induce an antigen-specific immune response.

  Liposomes are prepared using a variety of techniques and membrane lipids (reviewed in Gregoriadis, 1993). Liposomes may be prepared in advance and then mixed with the antigen. The antigen is dissolved or suspended and then used to make (a) pre-made liposomes in lyophilized state, (b) dry lipids as swelling solutions or suspensions, or (c) liposomes. Added to the lipid solution. They are also formed from lipids extracted from the stratum corneum, for example ceramides and cholesterol derivatives.

  Chloroform is a suitable solvent for lipids, but may be harmful during storage. Thus, at intervals of 1-3 months, chloroform is redistilled before use as a solvent to form liposomes. After distillation, 0.7% ethanol is added as a preservative. Ethanol and methanol are other suitable solvents.

  The lipid solution used to form the liposomes is placed in a round bottom flask. Pear-type boiling flasks are preferred, especially those sold by Lurex Scientific (Vineland, NJ, Catalog No. JM-5490). The volume of the flask is more than 10 times the volume of the expected aqueous suspension of liposomes to allow proper agitation during liposome formation.

  Using a rotary evaporator, the solvent is removed at 37 ° C. under negative pressure with a filtration aspirator attached to a water tap for 10 minutes. The flask is further dried in a desiccator for 1 hour under low vacuum (ie, less than 50 mm Hg).

  To encapsulate the antigen in the liposomes, the aqueous solution containing the antigen is added to the lyophilized liposomal lipid in a volume to a concentration of about 200 mM with respect to the liposomal lipid and shaken until all dry liposomal lipid is moistened. Or vortex mix. The liposome-antigen mixture is then incubated at 4 ° C. for 18 hours to 72 hours. Liposome-antigen preparations may be used immediately or stored for several years. It is preferred to use such formulations directly for transcutaneous immunization without removing unencapsulated antigen. To reduce the size of the liposomes (which can enhance transcutaneous immunity), methods such as bath sonication are used.

  Liposomes are formed as described above, but no antigen is added to the aqueous solution. The antigen is then added to the preformed liposome, so the antigen will be associated in solution and / or in the liposome (but not encapsulated in the liposome). This method for producing a liposome-containing preparation is preferred because it is simple. Techniques such as bath sonication may be used to modify liposome size and / or layer state to enhance immunity.

  Although not essential for practicing the present invention, stratum corneum hydration is enhanced by adding liposomes to the formulation. Liposomes, along with adjuvants, are used as carriers to enhance the immune response to antigens mixed, encapsulated, bound, or associated with liposomes.

Transdermal delivery of antigen Since transdermal delivery of antigen targets Langerhans cells, efficient immunization is achieved by the present invention. These cells are abundant in the skin and are efficient antigen-presenting cells, causing T cell memory and a strong immune response (Udey, 1997). Since there are numerous Langerhans cells in the skin, the efficiency of transdermal delivery is related to the surface area exposed to the antigen and adjuvant. Indeed, the reason that transcutaneous immunization is effective may be because it targets more of these efficient antigen-presenting cells than intramuscular immunity. However, even a small number of Langerhans cells or dendritic cells may be sufficient for immunity.

  We believe that the present invention improves immunity, while inducing a strong immune response. Transcutaneous immunity does not penetrate the skin and there are no complications and pains associated therewith, reducing the need for skilled personnel, aseptic techniques, and aseptic devices. Furthermore, the barrier to immunity or the barrier to multiple immunity is reduced at multiple sites. Immunization with a single application of the formulation is also contemplated.

  Immunization is performed using a simple solution of the antigen and adjuvant impregnated in gauze with a closed patch on the skin, or using other patch techniques. Creams, soaking, ointments and sprays are other possible applications. Immunization is possible even for non-skilled persons and is easy to self-apply. If immunization is easy, large-scale immunization will be possible. In addition, simple immunization techniques will also improve the immunity of pediatric patients, elderly people, and third world humans.

  Similarly, animals could be immunized using the present invention. Application to the ear, lower abdomen, toes, conjunctiva, interstitial area, or anal area, or application by subsidence or immersion could be used.

  Conventional vaccine formulations were injected into the skin using a needle. Injecting a vaccine using a needle can cause pain associated with the injection, the need for sterile needles and syringes, skilled medical personnel to administer the vaccine, injection discomfort, and puncture the skin with the needle There are several drawbacks, such as possible complications. The ability to immunize from the skin without the use of a needle (ie, transcutaneous immunization) is a major advance in vaccine delivery by avoiding the above disadvantages.

  The transdermal delivery system of the present invention also does not involve the penetration of intact skin by sound or electrical energy. A system that uses electrolysis to induce breakdown of the stratum corneum is disclosed in US Pat. No. 5,464,386.

  Furthermore, transcutaneous immunization is superior to immunization using a needle because it uses several locations that target a large surface area of the skin and more immune cells are targeted. A therapeutically effective amount of an antigen sufficient to induce an immune response is a site on the skin or multiple draining lymph nodes (eg, cervical, axilla, buttocks, on the pulley, popliteal, abdomen) And delivered across the area of intact skin covering the lymph nodes of the breast). When a small amount of antigen is injected into one location by intradermal, subcutaneous or intramuscular injection, such a location close to many different lymph nodes at a systemic location provides a broader stimulus to the immune system Will do.

  Antigens that pass through or enter the skin encounter antigen presenting cells, which process the antigen to induce an immune response. Multiple immune sites will collect a larger population of antigen-presenting cells, and this collected larger population will cause a greater induction of the immune response. Transcutaneous immunization can be applied in the immediate vicinity of the lymph node drainage site, thus improving the efficiency or efficacy of immunity. When absorbed through the skin, the antigen is delivered to phagocytic cells in the skin (eg, skin dendritic cells, macrophages, and other skin antigen presenting cells), and the antigen is also phagocytic in the liver, spleen, and bone marrow ( They are also delivered via the bloodstream or lymphatic system, which are known to function as antigen presenting cells). The result will be that the antigen is widely distributed in the antigen-presenting cells to the extent that current immunizations, if at all, are rarely achieved.

  The transcutaneous immune system is applied directly to the skin and air-dried; rubbed into the skin or escape; fixed with a bandage, patch, or absorbent material; stockings, slippers, gloves to maximize contact with the skin Or it may be fixed with a shirt or the like; or it may be sprayed onto the skin. The formulation may be applied with an absorbent bandage or gauze. Formulations include, for example, AQUAHOR (from Beiersdorf, petrolatum, mineral oil, mineral wax, wool wax, panthenol, bisabol, and glycerin), plastic films, impregnated polymers, COMFEEL (Coloplast), or an occlusive dressing such as petrolatum; or, for example, DUODERM (3M) or OPSITE (Smith & Napheu) Or a non-occlusive dressing such as An occlusive dressing completely eliminates the passage of water. Alternatively, a physically occlusive dressing such as TEGADERM can be applied to rehydrate and apply the patch for a longer period of time or prevent the skin from wiping.

  The formulation may be applied to one or more sites, or to one or more limbs, or a large surface area of the skin that is fully immersed. The formulation may be applied directly to the skin.

  Genetic immunity is described in US Pat. Nos. 5,589,466 and 5,593,972. The nucleic acid contained in the formulation may encode an antigen, an adjuvant, or both. The nucleic acid may be replicable or not. This may be non-integrative and non-infectious. The nucleic acid further comprises control regions operably linked to an antigen or adjuvant coding sequence (eg, promoter, enhancer, silencer, transcription start and stop sites, RNA splice acceptor and donor sites, polyadenylation signal, internal A ribosome binding site, a translation initiation site and a translation termination site). Nucleic acids can form complexes with agents that facilitate transfection (eg, cationic lipids, calcium phosphate, DEAE dextran, polybrene-DMSO, or combinations thereof). The nucleic acid may comprise a region derived from the viral genome. Such materials and techniques are described in Kriegler (1990) and Murray (1991).

The immune response can be humoral (ie antigen specific antibodies) and / or cellular (ie antigen specific lymphocytes such as B cells, CD4 ⁺ T cells, CD8 ⁺ T cells, CTL, Th1 cells, Th2 cells, And / or _TDTH cells) effector arms. Furthermore, the immune response may include NK cells that mediate antibody-dependent cellular cytotoxicity (ADCC).

  The immune response induced by the formulations of the present invention includes the induction of antigen-specific antibodies and / or cytotoxic lymphocytes (CTL, Alving and Wassef, reviewed in 1994). The antibody is detected by immunoassay and is expected to detect various isotypes (eg, IgM, IgD, IgA1, IgA2, secretory IgA, IgE, IgG1, IgG2, IgG3, or IgG4). The immune response can also be detected by neutralization assays.

  Antibodies are protective proteins produced by B lymphocytes. This is highly specific and generally targets one epitope of the antigen. Antibodies often react specifically with antigens from pathogenic pathogens and play an important role in protecting against the disease. Immunity induces antibodies specific for immune antigens (eg, cholera toxin). These antigen-specific antibodies are induced when the antigen is delivered to the skin by liposomes.

  CTLs are specific protective immune cells that are produced to protect against infection by pathogens. These are highly specific. Immunity induces antigen-specific CTLs, such as synthetic oligopeptides based on malaria proteins, along with self major histocompatibility complex antigens. CTL induced by immunization using a transdermal delivery system kills pathogen-infected cells. Immunity also produces a memory response as shown by enhanced antibody and CTL responses, lymphocyte proliferation by culture of antigen-stimulated lymphocytes, and delayed hypersensitivity response to intradermal antigen administration of antigen alone. .

  The T helper response induced by transcutaneous immunity can be manipulated by using calcium channel blockers that inhibit contact hypersensitivity reactions by inhibiting antigen catabolism and subsequent presentation by epidermal Langerhans cells. Transdermal application of calcium channel blockers would be expected to affect the surface expression of costimulatory molecules (eg, B7-related family) and the generation of subsequent T helper responses. It is also expected that the addition of calcium channel blockers can be used to inhibit delayed hypersensitivity responses and select immune responses that are primarily cellular or humoral responses.

  In the virus neutralization assay, serial dilutions of serum are added to the host cells, which are then observed for infection after challenge with infectious virus. Alternatively, serial dilutions of serum are incubated with infectious titer virus and then inoculated into animals, which are then observed for signs of infection.

  The transcutaneous immune system of the present invention is evaluated using an antigen challenge model in animals or humans, which assesses the ability of immunization with an antigen to protect a subject from disease. Such protection proves an antigen-specific immune response. It is presumed that achieving an anti-diphtheria antibody titer of 5 IU / ml or higher instead of antigen administration generally represents optimal protection and acts as a surrogate marker of protection (Plotkin and Mortimer, (1994).

  In addition, the Plasmodium faciparum challenge model is used to induce an antigen-specific immune response in humans. Human volunteers are immunized using a transcutaneous immune system containing oligopeptides or proteins (polypeptides) from malaria parasites and then exposed to malaria experimentally or in a natural setting. A mouse Plasmodium yoelii mouse malaria challenge model is used to assess the protection of mice against malaria (Wang et al., 1995).

  Alving et al. (1986) found lipid A as an adjuvant to induce an immune response against cholera toxin (CT) in rabbits and four tetrapeptides (Asn-Ala-Asn-Pro) conjugated to BSA. A liposome containing a synthetic protein consisting of a malaria oligopeptide containing The authors found that the immune response to cholera toxin or to a synthetic malaria protein is markedly increased by encapsulating the antigen in liposomes containing lipid A compared to similar liposomes lacking lipid A. It was. Several antigens derived from Plasmodium faciparum circumsporozoite protein (CSP) or merozoite surface protein are encapsulated in liposomes containing lipid A. All malaria antigens encapsulated in liposomes containing lipid A have been shown to induce humoral effectors (ie, antigen-specific antibodies) and some also induce cellular responses. The development of immune responses and immune protection in animals vaccinated with malaria antigens is measured by immunofluorescence against whole CSP-transfected target cells, fixed malaria sporozoites or CTL killing.

  Mice transcutaneously immunized with cholera toxin are protected against intranasal challenge with 20 μg of cholera toxin. Mallet et al. (Personal communication) found that C57B1 / 6 mice develop fatal hemorrhagic pneumonia in response to intranasal challenge with CT. Alternatively, mice are challenged with intraperitoneal administration of CT (Dragunsky et al., 1992). Cholera toxin-specific IgG or IgA antibodies protect against cholera toxin challenge (Pierce, 1978; Pierce and Reynolds et al., 1974).

  Similar protective effects are expected in humans immunized with LT or CT and challenged with LT-secreting E. coli or CT-secreting Vibrio cholerae, respectively. Furthermore, cross-protection has been demonstrated between CT and LT immunized subjects and CT and LT mediated diseases.

  As shown in the examples below, mucosal immunity is achieved by the transdermal route. Mucosal IgG and IgA can be detected in mice immunized transcutaneously with CT. This is important for defense in diseases where diseases such as LT or CT mediated diseases occur at mucosal sites, pathogen organisms enter at mucosal sites, or mucosal infection is critical to pathology.

  Transcutaneous immunity against diseases such as influenza is expected to be effective by inducing mucosal or systemic immunity, or by a combination of immunity such as humoral, cellular or mucosal.

  The vaccine may be effective against host actions such as red blood cell binding to malaria vascular endothelium by inducing anti-sequestrin antibodies.

  Protective antibodies such as anti-hepatitis A, B or hepatitis E antibodies are derived by the transdermal route using whole inactivated virus, virus derived subunits or recombinant products.

  Protection against tetanus, diphtheria and other toxin-mediated diseases is conferred by percutaneously derived antitoxin antibodies. Tetanus “boost” patches are contemplated that contain adjuvants such as CT, and toxoids such as tetanus and diphtheria, or fragments such as tetanus C fragment. Booster immunization is performed after the primary immunization by injection or transcutaneous immunization with the same or similar antigen. For injection immunization that induces immunity but may have side effects due to boosting, transcutaneous boosting is preferred. Oral or nasal immunization may also be boosted using the transdermal route. Simultaneous use of injectable and transcutaneous immunization could also be used.

  Vaccine administration is also used as a treatment for cancer and autoimmune diseases. For example, vaccination with tumor antigens (eg, prostate specific antigens) results in an immune response in the form of antibodies, CTLs and lymphocyte proliferation, which allows the body's immune system to recognize and kill tumor cells. To induce. Targeting dendritic cells, of which Langerhans cells are a specific subset, has proven to be an important strategy in cancer immunotherapy. Tumor antigens useful for vaccination are melanoma (US Pat. Nos. 5,102,663, 5,141,742 and 5,262,177), prostate cancer (US Pat. No. 5,538,866). And lymphomas (US Pat. Nos. 4,816,249, 5,068,177, and 5,227,159). Vaccination with T cell receptor oligopeptides can induce an immune response that stops the progression of autoimmune disease (US Pat. Nos. 5,612,035 and 5,614,192; Antel et al., 1996; Vandenberg et al., 1996). US Pat. No. 5,552,300 also describes antigens useful for the treatment of autoimmune diseases.

  The following are intended to illustrate the present invention. However, the practice of the invention is in no way limited or restricted to these examples.

EXAMPLE Hair of BALB / c mice 6-8 weeks old was shaved with a # 40 clipper. This shaving was done without damaging the skin. This was done from the middle of the chest to just below the neck. The mice were then allowed to rest for 24 hours. Prior to this, mice were labeled for recognition and blood was collected in advance to obtain a sample of preimmune serum. The mice were also immunized transcutaneously by applying 50 μl of immunization solution to each ear without shaving.

  Next, mice were immunized by the following method. Mice were anesthetized with 0.03-0.06 ml of a 20 mg / ml solution of xylazine and 0.5 ml of 100 mg / ml ketamine, and the mice were fixed with this dose of anesthesia for approximately 1 hour. The mouse was placed face down on a warm blanket.

The immune solution was then placed on the shaved skin on the dorsal side of the mouse as follows. A 1.2cm x 1.6cm stencil made of polystyrene is gently placed on the back and the skin is partially moistened using sterile gauze moistened with saline (this will apply the application of immune solution). The immune solution was then pipetted onto the area marked with a stencil to create a 2 cm ² patch of the immune solution. Alternatively, an aliquot of immune solution was applied evenly to the shaved area or ear. Care was taken not to scratch or rub the skin with the tip of the pipette. The immune solution was spread around this and covered with the smooth end of the pipette.

  The immunization solution (about 100 μl to about 200 μl) was left for 60-180 minutes on the left of the back of the mouse. At the end of 60 minutes, the mice were fixed with warm water at the neck and tail and washed for 10 seconds. The mouse was then gently patted with sterile gauze to dry and a second wash was performed for 10 seconds. The mice were then dried a second time and placed in cages. Mice did not show adverse effects from anesthesia, immunization, washing steps, or exotoxin-derived toxicity. After immunization, no skin irritation, swelling or redness was seen and the mice appeared to be growing. Immunization using the ear was performed as described above, except that it was not shaved prior to immunization.

Antigens The following antigens were used for immunization and ELISA and mixed using sterile PBS or normal saline. Cholera toxin or CT (List Biologicals, Catalog # 101B, Lot # 10149CB), CT B subunit (List Biologicals, Catalog # BT01, Lot # CVXG-14E), CT A subunit (List Biologicals, Catalog # 102A, Lot # CVXA-17B), CT A subunit (Calbiochem, Catalog # 608562); pertussis toxin, unsalted (List Bio Logistics (List Biologicals, Lot # 181120a); Tetanus Toxoid (List Biologicals, Lot # 913a and # 1915a); Pseudomonas exotoxin A (List Biologicals, Lot # ETA25a); diphtheria toxoid (List Biologicals, Lot # 15151); E. coli (E Heat labile enterotoxin (Sigma, lot $ 9640625); bovine serum albumin or BSA (Sigma, catalog # 3A-4503, lot # 31F-0116); and Hemophilus influenza ) B conjugate (Connaught, lot # 6J81401).

ELISA-IgG (H + L)
Antibodies specific for CT, LT, ETA, pertussis toxin, diphtheria toxoid, tetanus toxoid, Haemophilus influenzae B conjugate, influenza, sequestrin, and BSA are similar to Glenn et al. (1995). The measurement was performed using an ELISA. All antigens were dissolved in sterile saline at a concentration of 2 μg / ml. 50 μl of this solution (0.1 μg) per well was placed on an IMMULON-2 polystyrene plate (Dynatech Laboratories, Chantilly, VA) overnight at room temperature. Incubated. The plate was then blocked for 1 hour with 0.5% casein / 0.05% Tween 20 blocking buffer. Serum was diluted with 0.5% casein / 0.05% Tween 20 dilution; serial dilutions were made in rows on the plate. Incubation was for 2 hours at room temperature.

  The plates are then washed 4 times with PBS-0.05% Tween 20 wash and goat anti-mouse IgG (H + L) horseradish peroxidase (HRP) binding (Bio-Rad Laboratories, Richmond, Calif.). State, catalog # 170-6516) Secondary antibody was diluted in casein diluent at a dilution of 1/500 and left on the plate for 1 hour at room temperature. The plate was then washed 4 times with PBS-Tween wash. 100 μl of 2,2′-azino-di (3-ethyl-benzothiazolone) sulfonic acid substrate (Kirkegaard and Perry) was added to each well and the plate was developed at 405 nm for 20-40 minutes. read. Results are reported as the geometric mean and standard error of the average of individual sera in ELISA units (serum dilution with absorbance equal to 1.0) or as individual antibody responses in ELISA units.

ELISA-IgG (γ), IgM (μ) and IgA (α)
IgG (γ), IgM (μ) and IgA (α) anti-CT antibody levels were measured using an ELISA in a manner similar to Glenn et al. (1995). CT was dissolved in sterile saline at a concentration of 2 μg / ml. 50 μl of this solution (0.1 μg) per well was placed on an IMMULON-2 polystyrene plate (Dynatech Laboratories, Chantilly, VA) overnight at room temperature. Incubated. The plate was then blocked with 0.5% casein-Tween 20 blocking buffer for 1 hour. Serum was diluted with casein diluent and serial dilutions were made on the plates. This was incubated at room temperature for 2 hours.

  The plates are then washed 4 times with PBS-Tween wash and goat anti-mouse IgG (γ) HRP conjugated (Bio-Rad Laboratories, Richmond, Calif., Catalog # 172-1038), goat Anti-mouse IgM (μ) HRP binding (Bio-Rad Laboratories, Richmond, Calif., Catalog # 172-1030), or goat anti-mouse IgA HRP binding (Sigma, St. Louis, MO) Catalog # 1158985) The secondary antibody was diluted with casein diluent at a dilution ratio of 1/1000 and left on the plate for 1 hour at room temperature. The plate was then washed 4 times with PBS-Tween wash. 100 μl of 2,2′-azino-di (3-ethyl-benzothiazolone) sulfonic acid substrate (Kirkegaard and Perry, Gaithersburg, Md.) Was added to the wells and the plate at 405 nm. read. Results are reported as the geometric mean and standard error of the mean of individual sera in ELISA units (serum dilution with absorbance equal to 1.0).

ELISA-IgG subclass Antigen-specific IgG (IgG1, IgG2a, IgG2b, and IgG3) subclass antibodies to CT, LT, ETA, and BSA were performed as described by Glenn et al. (1995). Solid phase ELISA was performed on IMMULON-2 polystyrene plates (Dynatech Laboratories, Chantilly, VA). Wells containing each antigen (0.1 μg / 50 μl) in saline were incubated overnight and blocked with 0.5% casein-Tween 20. Each mouse serum diluted in 0.5% casein was serially diluted and incubated at room temperature for 4 hours. Secondary antibodies consisted of horseradish peroxidase-conjugated goat anti-mouse isotype specific antibodies (IgG1, IgG2a, IgG2b, IgG3, The Binding Site, San Diego, Calif.). Standard curves for each subclass were determined using mouse myeloma IgG1, IgG2a, IgG2b, and IgG3 (The Binding Site, San Diego, Calif.). Standard wells are used to capture myeloma IgG subclass standards added to serial dilutions, goat anti-mouse IgG (H + L) (Bio-Rad Laboratories, Richmond, Calif., Catalog # 170- 1054). The myeloma IgG subclass was also detected using a peroxidase-conjugated goat anti-mouse subclass specific antibody. Both test sera and myeloma standards were based on 2,2'-azino-di (3-ethyl-benzothiazolone) sulfonic acid (Kirkegaard and Perry, Gaithersburg, MD) as a substrate. Detected using. Absorbance was read at 405 nm. Individual antigen-specific subclasses were quantified using values from the linear titer curve calculated against the myeloma standard curve and reported as μg / ml.

ELISA-IgE
Quantification of antigen-specific IgE antibody was performed using the protocol from Pharmingen Technical Protocol (Pharmingen Technical Protocols), Research Product Catalog (Research Products Catalog), page 541 of 1996-1997. 50 μl of 2 μg / ml purified anti-mouse IgE-captured mAb (Pharmingen, catalog # 021111D) in 0.1 M NaHCO ₃ (pH 8.2) in an immuno (IMMUNO) plate (Nunc, catalog # 12- 565-136). Plates were incubated overnight at room temperature, washed 3 times with PBS-Tween 20, blocked with 3% BSA in PBS for 2 hours, and washed 3 times with PBS-Tween. Serum was diluted in 1% BSA in PBS, added at 1/100 dilution, and serially diluted along the line (eg 1/100, 1/200, etc.). Purified mouse IgE standard (Pharmingen, catalog # 0312D) was added at a starting dilution of 0.25 μg / ml and serially diluted along the row. Plates were incubated for 2 hours and washed 5 times with PBS-Tween.

  Biotinylated anti-mouse IgE mAB (Pharmingen, catalog # 02122D) was made up to 2 μg / ml with 1% BSA in PBS, incubated for 45 minutes and washed 5 times with PBS-Tween. Avidin-peroxidase (Sigma A3151, 1: 400 in 1 mg / ml solution) was added over 30 minutes and the plate was washed 6 times with PBS-Tween. Both test sera and IgE standards were based on 2,2′-azino-di (3-ethyl-benzothiazolone) sulfonic acid (Kirkegaard and Perry, Gaithersburg, MD) as a substrate. Detected using. Absorbance was read at 405 nm. Individual antigen-specific subclasses were quantified using values from the linear titer curve calculated against the IgE standard curve and reported as μg / ml.

Liposome Preparation When liposomes are included in formulations for transdermal immunization, multilamellar liposomes consisting of dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, and cholesterol were prepared by Alving et al. (1993). Dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, and cholesterol were purchased from Avanti Polar Lips Inc. (Alabaster, Alabama). The lipid stock solution in chloroform was removed from storage in a -20 ° C freezer.

  Lipids were mixed with dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, and cholesterol in a molar ratio of 0.9: 0.1: 0.75 in a pear-shaped flask. The solvent was removed using a rotary evaporator at 37 ° C. under negative pressure for 10 minutes. The flask was further dried in a desiccator for 2 hours under low vacuum to remove the remaining solvent. Liposomes were expanded to 37 mM phospholipids using sterile water, lyophilized and stored at -20 ° C. These liposomes were mixed with physiological saline (pH 7.0) in their lyophilized state to achieve the specified phospholipid concentration in saline. Alternatively, the dried lipids were increased in saline (pH 7.0) to make liposomes and not lyophilized.

Example 1
A group of 5 BALB / c mice aged 6-8 weeks were immunized transcutaneously as described above in the “immunization process”. Mice were immunized using 100 μl of immunization solution prepared as follows: Liposomes prepared as described above in “Liposome Preparation” were mixed with saline to form liposomes. The preformed liposomes are then diluted in saline (liposome alone group) or with CT in saline, and immunized containing liposomes with 10-150 mM phospholipid with 100 μg CT per 100 μl immune solution. A solution was obtained. CT was mixed in saline to make an immune solution containing 100 μg CT per 100 μg solution in the CT alone administration group. The solution was vortex mixed for 10 seconds prior to immunization.

Mice 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 boost and compared to preimmune serum. As shown in Table 1, the levels of anti-CT antibodies induced by CT without liposomes differed from the levels of anti-CT antibodies produced using liposomes, except for mice when using 150 mM liposomes. CT alone in saline was able to immunize mice against CT and produce high titer antibodies.

Example 2
A group of 5 BALB / c mice aged 6-8 weeks were immunized transcutaneously as described above in the “immunization process”. Mice were immunized at 0 and 3 weeks using 100 μl of immunization solution prepared as follows: For the BSA alone administration group, BSA was mixed in saline and 100 μl saline of the BSA alone administration group. An immunization solution containing 200 μg of BSA per unit was made. For the BSA and CT administration groups, BSA and CT were mixed in saline to make an immune solution containing 200 μg BSA and 100 μg CT per 100 μl saline. When using liposomes, the liposomes were prepared as described above in “Liposome Preparation” and first mixed with saline to form liposomes. These were then diluted in BSA or BSA and CT in saline to give an immune solution containing liposomes at 50 mM phospholipid with 200 μg BSA per 100 μl immune solution or 200 μg BSA + 100 μg CT per 100 μl immune solution. . The solution was vortex mixed for 10 seconds prior to immunization.

Antibodies were measured in sera 3 weeks after secondary immunization using ELISA as described above for “ELISA IgG (H + L)”. The results are shown in Table 2. BSA alone with or without liposomes failed 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 titer anti-BSA antibodies were produced.

Example 3
A group of 5 BALB / c mice aged 6-8 weeks were immunized transcutaneously as described above in the “immunization process”. Mice were immunized at 0 and 3 weeks using 100 μl immunization solution prepared as follows: LT was mixed in saline to give 100 μg LT per 100 μl saline in the LT alone group. Containing immunization solution was made. When using liposomes, the liposomes were prepared as described above in “Liposome Preparation” and first mixed with saline to form liposomes. The preformed liposomes were then diluted in LT in saline to obtain an immune solution containing liposomes at 50 mM phospholipid with 100 μg LT per 100 μl immune solution. The solution was vortex mixed for 10 seconds prior to immunization.

Anti-LT antibody was measured 3 weeks after secondary immunization using ELISA as described above for “ELISA IgG (H + L)”. The results are shown in Table 3. LT was clearly immunogenic both with and without liposomes and no significant difference could be detected between groups. LT and CT are members of the family of bacterial ADP-ribosylated exotoxins (bARE). These are constructed as A: B proenzymes with the ADP-ribosyltransferase activity contained in the A subunit and the target cell binding function of the B subunit. LT is 80% homologous to CT at the amino acid level, has a non-covalently linked subunit organization, stoichiometric ratio (A: B5), the same binding target, ganglioside GM1, and is the same size Degree (MW ~ 80,000). The similarity between LT and CT appears to affect its immunogenicity by the transdermal route, as reflected by the similar magnitude of the antibody response to both CT and LT (Tables 1 and 3). .

Example 4
Six to eight week old C57B1 / 6 mice were immunized transcutaneously in groups of 5 mice as described above in the “immunization step”. Mice were immunized once using 100 μl of immunization solution prepared as follows: LT was mixed in saline to make an immunization solution containing 100 μg LT per 100 μl saline. The solution was vortex mixed for 10 seconds prior to immunization.

Anti-LT antibody was measured 3 weeks after a single immunization using ELISA as described above for “ELISA IgG (H + L)”. The results are shown in Table 4. LT was clearly immunogenic with a single immunization and antibody was produced in 3 weeks. Rapid enhancement of antibody titer and response to a single immunization would be a useful aspect of transdermal immunization. Rapid single immunization may be useful in epidemics, for travelers and when it is difficult to access medical care.

Example 5
8-12 week old C57B1 / 6 mice were immunized transcutaneously in groups of 5 mice as described above in the “immunization step”. Mice were immunized once using 100 μl of immune solution prepared as follows: CT was mixed in saline to make an immune solution containing 100 μg CT per 100 μl saline. The solution was vortex mixed for 10 seconds prior to immunization.

Anti-CT antibodies were measured 3 weeks after a single immunization using ELISA as described above for “ELISA IgG (H + L)”. The results are shown in Table 5. CT was highly immunogenic with a single immunization. Rapid enhancement of antibody titer and response to a single immunization would be a useful aspect of transdermal immunization. Rapid single immunization may be useful in epidemics, for travelers and when it is difficult to access medical care.

Example 6
A group of 5 BALB / c mice aged 6-8 weeks were immunized transcutaneously as described above in the “immunization process”. Mice were immunized at 0 and 3 weeks using 100 μl immunization solution prepared as follows: ETA was mixed in saline to give 100 μg ETA per 100 μl saline in the ETA alone group. Containing immunization solution was made. When using liposomes, the liposomes were prepared as described above in “Liposome Preparation” and first mixed with saline to form liposomes. The preformed liposomes were then diluted with ETA in saline to obtain an immune solution containing liposomes at 50 mM phospholipid with 100 μg of ETA per 100 μl of immune solution. The solution was vortex mixed for 10 seconds prior to immunization.

Antibodies were measured in sera 3 weeks after secondary immunization using ELISA as described above for “ELISA IgG (H + L)”. The results are shown in Table 6. ETA was clearly immunogenic both with and without liposomes and no significant difference could be detected between groups. ETA is a single 613 amino acid peptide with A and B domains on the same peptide, and an entirely different receptor, α2-macroglobulin receptor / low density lipoprotein receptor related protein (Koonnas et al., 1992) and is different from CT and LT. Although not similar in ETA and CT in size, structure, and binding target, ETA also induced a transdermal antibody response.

Example 7
A group of 5 BALB / c mice aged 6-8 weeks were immunized transcutaneously as described above in the “immunization process”. Mice were immunized using 100 μl immunization solution prepared as follows: CT mixed in saline to make 100 μg CT per 100 μl immunized solution, LT mixed in saline. Make 100 μg LT per 100 μl immunization solution, mix ETA in saline, make 100 μg ETA per 100 μl immunization solution, and mix CT and BSA in 100 μl 100 μg CT per immunization solution and 200 μg BSA per 100 μl immunization solution were made. The solution was vortex mixed for 10 seconds prior to immunization.

Mice were immunized transcutaneously at 0 and 3 weeks, and antibody levels were determined using ELISA as described above in “ELISA IgG subclass” after 3 weeks of booster, and against preimmune serum. And compared. IgG subclass responses to CT, BSA and LT are at similar levels as IgG1 and IgG2a, reflecting the activation of T help from both Th1 and Th2 lymphocytes (Seder and Paul). ), 1994), on the other hand, IgG subclass responses to ETA consisted almost exclusively of IgG1 and IgG3, consistent with a Th2-like response (Table 7). That is, it is believed that all IgG subclasses can be produced using transcutaneous immunization.

Example 8
A group of 5 BALB / c mice aged 6-8 weeks were immunized transcutaneously as described above in the “immunization process”. Mice were immunized using 100 μl immunization solution prepared as follows: LT mixed in saline to make an immunization solution containing 100 μg LT per 100 μl saline for the LT alone administration group. CT was mixed in saline to make an immune solution containing 100 μg CT per 100 μl saline for the CT alone group, and ETA was mixed in saline and 100 μl for the ETA alone group. An immunization solution containing 100 μg of ETA per saline solution was made and BSA and CT were mixed in saline and contained 100 μg BSA and 100 μg CT per 100 μl saline for the BSA and CT administration groups An immunization solution was made. The solution was vortex mixed for 10 seconds prior to immunization.

Mice were immunized transcutaneously at 0 and 3 weeks, and antibody levels were determined using ELISA as described above for “ELISA IgE” one week after boost and against preimmune serum. Compared. As shown in Table 8, the sensitivity of detection was 0.003 μg / ml, but no IgE antibody was found. IgG antibodies were measured in the same mice using “ELISA IgG (H + L)” in serum 3 weeks after secondary immunization. IgG antibody responses to LT, ETA, CT and BSA demonstrate that mice were successfully immunized and responded with high titers of antibodies against each antigen.

Example 9
A group of 5 BALB / c mice aged 6-8 weeks were immunized transcutaneously as described above in the “immunization process”. Mice were immunized at 0 and 3 weeks using 100 ml immunization solution prepared as follows: CT was mixed in saline to give an immunization solution containing 100 mg CT per 100 ml immunization solution. Created. The immunization solution was vortex mixed for 10 seconds before immunization.

Mice were immunized transcutaneously at 0 and 3 weeks, and antibody levels were determined using ELISA as described above for “ELISA IgG (H + L)” and “ELISA IgG (γ)”. Measurements were taken 1 and 4 weeks after the first immunization and compared to pre-immune serum. As shown in Table 9, high levels of anti-CT IgG (γ) antibody were induced by CT in saline. A small amount of IgM could be detected by using an IgM (μ) specific secondary antibody. Up to 4 weeks, the antibody response was mainly IgG. Data is reported in ELISA units.

Example 10
A group of 5 BALB / c mice aged 6-8 weeks were immunized transcutaneously as described above in the “immunization process”. Mice were immunized once using 100 μl of immune solution prepared as follows: CT was mixed in saline to make an immune solution containing 100 μg CT per 100 μl saline. The solution was vortex mixed for 10 seconds prior to immunization. Mice were immunized transcutaneously at 0 and 3 weeks. Antibody levels were determined using ELISA as described above for “ELISA IgG (H + L)” 5 weeks after boost and compared to preimmune serum. As shown in Table 10, serum anti-CT IgA antibodies were detected.

Example 11
A group of 5 BALB / c mice aged 6-8 weeks were immunized transcutaneously as described above in the “immunization process”. Mice were immunized using 100 μl of immunization solution prepared as follows: CT was mixed in saline to make an immunization solution containing 100 μg CT per 100 μl immunization solution. The immunization solution was vortex mixed for 10 seconds before immunization.

  Mice are immunized transcutaneously at 0 and 3 weeks with 100 μl of immunization solution, and antibody levels are determined using ELISA as described above for “ELISA IgG (H + L)” and “ELISA IgG (γ)”. Asked. Antibody measurements were taken 8 weeks after the first immunization and compared to pre-immune serum. As shown in Table 11, high levels of serum anti-CT antibodies were induced by CT in saline. Lung lavage IgG could be detected by ELISA using IgG (H + L) or IgG (γ) specific antibodies. Antibodies found on the pulmonary mucosal surface are diluted with the lavage method used to recover the mucosal antibodies, so the exact amount of antibody detected is more significant than simply the presence of detectable antibody. Not something.

  Lung lavage was obtained after the mice were sacrificed. The trachea and lungs were exposed by gentle incision, and the trachea was incised laterally above the bifurcation. A 22 gauge polypropylene tube was inserted and tied over the trachea to form a tight seal at the rim. 0.5 ml PBS was injected using a 1 ml syringe attached to the tube and the lungs were gently inflated with fluid. The fluid was collected and reinjected for a total of 3 rounds of wash solution. The lung lavage fluid was then frozen at -20 ° C.

Table 11 shows IgG (H + L) and IgG (γ) antibody responses to cholera toxin in week 8 serum and lung lavage fluid. Data is expressed in ELISA units. The antibody was clearly detectable in the lung lavage fluid of all mice. The presence of antibodies in the mucosa is important for protection against diseases active in the mucosa.

Example 12
BALB / c mice were immunized transcutaneously in groups of 4 mice at 0 and 3 weeks as described above in the “immunization process”. Liposomes were prepared as described above in “Preparation of liposomes” and were first mixed with saline to form liposomes. Pre-formed liposomes are then diluted with either CT, CTA or CTB in saline to contain the liposomes at 50 mM phospholipid with 50 μg of antigen (CT, CTA or CTb) per 100 μl of immune solution. A solution was obtained. The solution was vortex mixed for 10 seconds prior to immunization.

Antibodies were determined using ELISA as described above for “ELISA IgG (H + L)” one week after boost and compared to preimmune serum. The results are shown in Table 12. CT and CTB were clearly immunogenic, while CTA was not. That is, the B subunit of CT is necessary and sufficient to induce a strong antibody response.

Example 13
One group of 5 BALB / c mice were immunized transcutaneously as described above in the “Immunization Step”. Mice were immunized at 0 and 3 weeks with 100 μg diphtheria toxoid and 10 μg pertussis toxin per 100 μl saline. The solution was vortex mixed for 10 seconds prior to immunization.

Antibodies were quantified using ELISA as described above for “ELISA IgG (H + L)”. Anti-diphtheria toxoid antibody was detected only in animals immunized with both pertussis toxin and diphtheria toxoid. The best responding mice were 1,038 anti-diphtheria toxoid antibody ELISA units. That is, a small amount of pertussis toxin acts as an adjuvant for the diphtheria toxoid antigen. Toxoid alone did not induce an immune response, suggesting that the toxoid process affected the part of the molecule responsible for the adjuvant action found in ADP-ribosylated exotoxins.

Example 14
One group of 5 BALB / c mice were immunized transcutaneously as described above in the “Immunization Step”. Mice were immunized at 0, 8 and 20 weeks with 50 μg pertussis toxin (List, catalog # 181, lot # 181-20a) per 100 μl saline.

Antibodies were quantified using ELISA as described above for “ELISA IgG (H + L)”. Anti-pertussis toxin antibody was detected one week after the last boost in mice immunized with pertussis. All five mice had elevated levels of anti-pertussis toxin antibody after the last immunization. That is, pertussis toxin acts as its own adjuvant to induce PT-specific IgG antibodies. The adjuvant action of PT would be useful in enhancing antibody responses against co-administered antigens as well as against PT itself in combination vaccines such as diphtheria / pertussis / tetanus / Hib.

Example 15
One group of 5 BALB / c mice were immunized transcutaneously as described above in the “Immunization Step”. Mice were immunized once at week 0 with 50 μg tetanus toxoid and 100 μg cholera toxin per 100 μl saline.

Antibodies were quantified using ELISA as described above for “ELISA IgG (H + L)”. Anti-tetanus toxoid antibody was detected in mouse 5173 at 443 ELISA units at 8 weeks.
Example 16

The likelihood that oral immunization would occur due to hair patching following epidermal application and subsequent cleaning of the application site was assessed using ¹²⁵ I-labeled CT to track antigen / adjuvant fate. Mice were anesthetized and immunized transcutaneously with 100 μg of ¹²⁵ I-labeled CT (150,000 cpm / μg CT) as described above in the “immunization step”. Control mice were left anesthetized for 6 hours without hair repair, and experimental mice were anesthetized for 1 hour and then allowed to groom after washing. Mice were sacrificed at 6 hours, organs were weighed, and ¹²⁵ I was counted on a Packard gamma counter. A total of 2-3 μg CT was detected at the immune site of the shaved skin (14,600 cpm / μg tissue), while the largest 0.5 μg CT was in the stomach (661 cpm / μg tissue) and intestine (9 cpm / (μg tissue).

Oral immunization with 10 μg CT in saline at 0 and 3 weeks (n = 5) induced a 6-week average IgG antibody response of <1,000 ELISA units, whereas less than 5 μg CT in the washed skin Transcutaneous immunization with 100 μg CT, which was demonstrated above, resulted in an anti-CT response of 42,178 ELISA units at 6 weeks. Induction of an immune response against orally taken CT requires the addition of NaHCO ₃ to the immune solution (Piece, 1978; Lycke and Holmglen, 1986) . That is, oral immunization does not contribute significantly to the antibodies detected when CT is applied over the skin.

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

BALB / c mouse ears were either dorsally coated with 100 μg CT in saline, 100 μg CTB in saline, saline alone, or 100 pg of positive control for 1 hour while the mice were anesthetized. Intradermal injections of LPS or 10 μg TNFα were performed. The ears were then washed thoroughly and 24 hours later, the ears were removed and epidermal sheets were collected and stained for MHC class II expression as described in Caughman et al. (1986). Epidermal sheets were stained with MKD6 ^{(anti-I-A d)} or negative control Y3P (anti ^{I-A k),} were used goat anti-mouse FITC F _{(ab) 2} as the second step reagent. It was previously found that mice immunized percutaneously in the ear (not shaved as described above) had 7,000 ELISA units of anti-CTab 3 weeks after a single immunization.

  Enhanced expression of MHC class II molecules as detected by staining intensity, decreased number of Langerhans cells (especially with cholera toxin), and changes in Langerhans cell morphology were immunized with CT and CTB comparable to controls Found in the mouse epidermal sheet (FIG. 1), this suggests that Langerhans cells were activated by cholera toxin applied on the skin (Aiba and Katz). ), 1990; Enk et al., 1993).

Example 18
Langerhans cells are the epidermis part of a family of powerful accessory cells called “dendritic cells”. Langerhans cells (and possibly related cells in the dermis) are thought to be necessary for an immune response directed against foreign antigens encountered in the skin. The “life cycle” of Langerhans cells is characterized by at least two distinct stages. Epidermal Langerhans cells (“Sentinel”) are able to take up particles and efficiently process antigens, but are weak stimulators of unprimed T cells. In contrast, Langerhans cells ("messengers") that induce lymph node migration following contact with the epidermal antigen have less phagocytic action and limited antigen processing capacity, but untreated T It is a powerful stimulator of cells. If Langerhans cells fulfill both their “sentinel” and “messenger” roles, they must remain in the epidermis and exit the epidermis in a controlled manner after exposure to antigen. Need to be able to. That is, control of Langerhans cell-keratin cell adhesion is an important regulatory point in Langerhans cell trafficking and function.

  Langerhans cells express E-cadherin (Blauvelt et al., 1995), a homophilic adhesion molecule that appears prominently in the epithelium. Keratinocytes also express this adhesion molecule, and E-cadherin clearly mediates adhesion of mouse Langerhans cells to keratinocytes in vitro. It is known that E-cadherin is involved in the localization of Langerhans cells in the epidermis. For a characterization of Langerhans cells and keratinocytes and a review of their properties, see Stingl et al. (1989).

  Migration of epidermal Langerhans cells (LC) from the skin to the draining lymph nodes and transport of antigens are known to be important in the induction of cutaneous immune responses (eg, contact sensitization). In migration to the lymph nodes, Langerhans cells undergo many phenotypic changes necessary for gaining the ability to migrate from the skin and present antigen. Besides up-regulation of MHC class II molecules, there is a change in the expression of adhesion molecules that control the interaction with the surrounding tissue matrix and with T lymphocytes. Langerhans cell migration is known to be associated with a marked decrease in E-cadherin expression (Schwarzenberger and Udey, 1996, and parallel upregulation of ICAM-1). (Uday, 1997)).

  Transcutaneous immunization with bacterial ADP-ribosylated exotoxin (bARE) targets Langerhans cells in the epidermis. bARE activates Langerhans cells and converts it from its role as a sentinel to its role as a messenger. Ingested antigen is then taken to the lymph nodes where it is presented to B and T cells (Streilin and Grammer, 1989; Kripke et al., 1990; (Tew et al., 1997). In this process, epidermal Langerhans cells mature in lymph nodes to become antigen-presenting dendritic cells (Shuler and Steinman, 1985). Lymphocytes entering the lymph nodes separate into B cell follicles and T cell regions. The activation of Langerhans cells to become migratory Langerhans cells is associated with not only a significant increase in MHC class II molecules, but also a significant decrease in E-cadherin expression and ICAM-1 upregulation. It has been known.

  We show that cholera toxin (CT) and its B subunit (CTB) up-regulate ICAM-1 expression, down-regulate E-cadherin expression on Langerhans cells, and MHC class II molecules on Langerhans cells. It is considered that the expression of is up-regulated. CT or CTB releases the sentinel Langerhans cells, like BSA or diphtheria toxoids phagocytosed by Langerhans cells at the same location and time of encounter with CT or CTB (when they are acting as an adjuvant) To present the correct antigen. Activating Langerhans cells to upregulate ICAM-1 expression and downregulate E-cadherin expression is mediated by release of cytokines (including TNFα and IL-1β) from epidermal cells or Langerhans cells themselves Is done.

  This method of assisting transcutaneous immunization is believed to work with any compound that activates Langerhans cells. Activation occurs to down-regulate E-cadherin and up-regulate ICAM-1. The Langerhans cells then carry antigens made from a mixture of such Langerhans cell activating compounds and antigens (eg, diphtheria toxoid or BSA) to the lymph nodes, where the antigens are presented to T cells and immune Induce a response. That is, an activator such as bARE activates Langerhans cells, phagocytoses antigens such as diphtheria toxoid, migrates to lymph nodes, matures into dendritic cells, and presents antigens to T cells By doing so, it can be used as an adjuvant for antigens (eg, diphtheria toxoid) that are originally non-immunogenic percutaneously.

  T cell helper responses to antigens used in transcutaneous immunization are affected by the application of cytokines and / or chemokines. For example, interleukin-10 (IL-10) directs the antibody response to a Th2 IgG1 / IgE response, and anti-IL-10 can enhance the production of IgG2a (Bellinghausen et al., 1996).

Example 19
Sequestrin is a molecule expressed on the surface of malaria-infected erythrocytes that functions to fix erythrocytes infested with malaria to the vascular endothelium. This is essential for the survival of the parasite and directly contributes to the pathogenicity of P. faciparum in children who die from brain malaria. In cerebral malaria, brain capillaries are clogged with countless parasitized red blood cells because of the specific interaction between the sequestrin molecule and the host endothelial receptor CD36. Okkenhouse et al. Identified both the host receptor CD36 and a parasite molecule (sequestrin) that mediate this receptor-ligand interaction. Okkenhause et al. Cloned and expressed the domain of the sequestrin molecule that interacts with the CD36 receptor as a recombinant protein produced in E. coli. The sequestrin product, which is 79 amino acids truncated, was used in the following examples.

  Active immunization with recombinant sequestrin or DNA encoding the sequestrin gene blocks the adhesion of malaria parasitized erythrocytes to the host endothelial CD36 and therefore cannot bind to the endothelium, causing the parasite to die. To induce antibodies that interfere with the completion of the parasite's life cycle. The strategy is to develop an immunization that induces high-titer blocking antibodies. One such method is to deliver the vaccine transdermally. Measurement of total antibody titer as well as both blocking activity and opsonization is the basis of this approach using transcutaneous immunity. The recombinant sequestrin protein used in this experiment is 79 amino acids long (˜18 kDa) and contains the CD36 binding domain of the molecule. We also generated a naked DNA construct consisting of this domain and induced the antibody using epidermal gene gun delivery.

  BALB / c mice (n = 3) were immunized transcutaneously as described above under “Immunization”. Mice were immunized at 0 and 8 weeks using 120 μl of immunization solution prepared as follows. Plasmids encoded for P. faciparum sequestrin are mixed in saline to contain 80 μg plasmid in 100 μl saline, 80 μg CT (List Biologicals). An immunization solution was made. After gently washing the ear with an alcohol swab (Triad Alcohol pad, 780% isopropyl alcohol), 120 μl was applied to the unlabeled ear. The immunization solution was not removed by washing.

  Serum collected from the tail vein at 3, 4, 7, and 9 weeks after primary immunization was measured for antibodies to sequestrin using ELISA as described above under “ELISA IgG (H + L)”. The results are shown in Table 15.

  Sequestrin DNA with CT induced a detectable antibody response against the expressed protein after the second boost. In order for immunity to occur, proteins need to be expressed and processed by the immune system. That is, CT acted as an adjuvant for the immune response against the sequestrin protein expressed by the plasmid encoding sequestrin.

DNA vaccines, such as malaria (Gramzinski, Vaccine 15: 913-915, 1997) and HIV (Shriver et al., Vaccine 15: 884-887, 1997) in non-human primates It has been demonstrated to induce neutralizing antibodies and CTL against various diseases, and several models (McCllements et al., Vaccine 15: 857-60, 1997) showed different degrees of protection. DNA immunization through the skin is expected to induce a response similar to that of a gene gun that targets the skin's immune system (Playaga et al., Vaccine 15: 1349-1352, 1997).

Example 20
As described above in “Immunization”, 5 groups of BALB / c mice were immunized transcutaneously using sequestrin. Mice were immunized at weeks 0, 2 and 8 using 100 μl of immunization solution prepared as follows: For the group receiving sequestrin and CT at 0 weeks, mice were 59 μg CT in 410 μl. And 192 μg sequestrin, 192 μg in 410 μl for sequestrin alone, and 120 μg CTB and 250 μg sequestrin in 520 μl for the group receiving sequestrin and CTB. Two weeks later, the mice were 345 μl saline containing 163 μg sequestrin for the sequestrin alone group and 345 μl saline containing 163 μg sequestrin and 60 μg CT for the CT + sequestrin group. For the Sequestrin + CTB group, immunization was performed with 345 μl saline containing 163 μg sequestrin and 120 μg CTB. For the second booster, mice, 120 μg sequestrin for the sequestrin alone group, 120 μg sequestrin and 120 μg CT for the CT + sequestrin group, and 120 μg for the sequestrin + CTB group. Sequestrin and 120 μg CTB were administered.

Antibodies were measured on sera at 3, 5, 7, 8, 10, 11 and 15 weeks after the first immunization using ELISA as described above for “ELISA IgG (H + L)”. 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 against sequestrin, and CTB induced a stronger immune response than sequestrin alone. CT and CTB acted as adjuvants for the immune response to the recombinant protein sequestrin.

Example 21
As described above in the “immunization method”, BALB / c mice were immunized transcutaneously in 5 mice per group. Mice were immunized at week 0 using 100 μl of immunization solution prepared as follows: FLUSHIELD (Wyeth-Ayerst, purified subviral particles, 1997-98 formulation, lot # U0980 -35-1) was freeze-dried, mixed with saline, and for the influenza virus single administration group, an immune solution containing 90 μg of FLUSHIELD subviral particles in 100 μl of saline was used. For the CT administration group, influenza and CT were mixed with saline to prepare an immune solution containing 90 μg FLUSHIELD antigen and 100 μg CT in 100 μl saline.

Antibodies were measured on sera 3 weeks after the first immunization using ELISA as described above for “ELISA IgG (H + L)”. 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 than that observed with influenza alone. That is, CT acted as an adjuvant for the immune response to FLUSHIELD (subviral particle influenza vaccine) (a mixture of virus-derived antigens).

Example 22
LT is a family of ADP-ribosylated exotoxins, similar in molecular weight to CT, binds to ganglioside GM1, is 80% homologous to CT, and has a similar A: B5 stoichiometry. That is, LT was also used as an adjuvant for DT in transcutaneous immunization. BALB / c mice (n = 5) at 0, 8, and 18 weeks as described above, 100 μg LT (Sigma, catalog # E-8015, lot 17hH12000) and 100 μg CT at 100 μl saline. Immunization with saline solution containing (List Biologicals, Catalog # 101b). LT induced a moderate response to DT as shown in Table 18.

ETA (List Biologicals, Lot #ETA 25A) is a family of ADP-ribosylating exotoxins, but a single polypeptide that binds to different receptors. 100 μg of ETA was delivered to the back of BALB / c mice at 0, 8, and 18 weeks as described above in 100 μl saline solution containing 100 μg CT. Boosting ETA enhanced the response to DT at 20 weeks. That is, other ADP-ribosylated exotoxins could act as adjuvants for co-administered proteins (Table 18).

Example 23
As described above in the “immunization method”, BALB / c mice were immunized transcutaneously in 5 mice per group. At 0, 8 and 18 weeks, 100 μg of cholera toxin (List Biologicals, Catalog # 101B), 50 μg of tetanus toxoid (List Biologicals, Catalog # 191B, lot # 1913a and 1915b) and immunized with 100 μl saline containing 83 μg diphtheria toxoid (List Biologicals, catalog # 151, lot # 15151).

Antibodies against CT, DT, and TT were quantified using ELISA as described above for “ELISA IgG (H + L)”. Anti-CT, DT, or TT antibody was detected 23 weeks after primary immunization. Anti-diphtheria toxoid and cholera toxin antibodies were elevated in all immunized mice. Shows the highest response, anti-tetanus toxoid antibody ELISA units 342 ^- a, was about 80 times the antibody levels detected in non-immunized animals. That is, the unrelated antigen combination (CT / TT / DT) can be used to immunize against individual antigens. This demonstrates that cholera toxin can be used as an adjuvant for multivalent vaccines.

Example 25
Transcutaneous immunization using CT induces a strong immune response. The immune response to 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 was orally administered to BALB / c mice (n = 5) using a 200 μl pipette tip. Mice swallowed the immune solution easily. 25 μl of 1 mg / ml CT in saline was administered to the percutaneous group as described above. 25 μl CT in saline was injected intramuscularly in the front thigh of the intramuscular group.

Mice injected intramuscularly with CT showed significant swelling and tenderness at the injection site and showed high levels of anti-CT antibodies. Mice immunized transcutaneously showed no redness or swelling at the site of immunization and showed high levels of anti-CT antibodies. Mice immunized orally showed much lower levels of antibody compared to mice immunized transcutaneously. This indicates that oral immunization by hair repair in transcutaneous immunized mice is not responsible for the high level of antibodies induced by transcutaneous immunization. Overall, transcutaneous route immunity is superior to oral or intramuscular immunization because high levels of antibodies are achieved without side effects to immunity.

Example 26
As described above in the “immunization method”, BALB / c mice were immunized transcutaneously in 5 mice per group. At 0, 8 and 20 weeks, mice were immunized with 100 μl of immunization solution prepared as follows: Hib conjugate (Connaught, lot # 6J81401, 86 μg / ml) for antibody enrichment. Lyophilized. The lyophilized product is mixed with saline, and for the Hib conjugate alone administration group, an immune solution containing 50 μg Hib conjugate in 100 μl saline, and for the Hib conjugate and CT administration group, Hib binding. The body and CT were mixed with saline to make an immune solution containing 50 μg Hib conjugate and 100 μg CT in 100 μl saline.

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

Example 27
Emulsions, creams and gels will provide practical advantages for conveniently spreading immune compounds on the skin surface in the hair or body wrinkles. Furthermore, such formulations may provide benefits such as closure or hydration that can enhance the efficiency of immunization.

  E. coli heat-labile enterotoxin (LT) (Sigma, catalog # E-8015, lot 17hH1200), using simple saline and commonly available petroleum-based ointments AQUAHOR (which can be used alone or in combination with virtually any ointment with an aqueous solution or in combination with other oily substances and all common topical medicines) ( 507, PDR (PDR), non-prescription drugs (1994, 15th edition)) was used to compare the efficiency of transcutaneous immunization. Mice were treated with a range of doses to assess relative antibody responses to decreasing doses in the comparison vehicle.

  BALB / c mice were immunized as described above except that the immunization solution was applied to the back for 3 hours. The saline solution of LT uses a 2 mg / ml, 1 mg / ml, 0.5 mg / ml or 0.2 mg / ml solution, respectively, for a 50 μl dose of solution, and 100 μg, 50 μg, 25 μg or 10 μg of antigen in the solution. Was prepared to deliver. After 3 hours, the immune solution was removed by gently wiping the back with a damp gauze.

  The water-in-oil preparation was made as follows: an equal volume of AQUAHOR and saline antigen in a 15-gauge glass tuberculin syringe with 1 ml luer tablets connecting two syringes. An emulsifying needle was used to mix until the mixture was homogeneous. Each was mixed with an equal volume of Aquaphor (AQUAAPHOR) using 4 mg / ml, 2 mg / ml, 1 mg / ml or 0.5 mg / ml solutions of LT in saline. 50 μl of this mixture was applied to the shaved back for 3 hours and then gently removed by wiping with gauze. To deliver 50 μl, the antigen dose of the water-in-oil LT containing emulsion was weighed. The weight-to-volume ratio is determined by adding the specific gravity of saline (1.00 g / ml) and aquaphor (AQUAPOR) (0.867 g / ml) and dividing the total by 2 to obtain a final specific gravity of 0.9335 g / ml. Calculated by A water-in-oil emulsion containing about 47 mg LT was delivered to mice for immunization.

A dose-response relationship was evident for both saline and water-in-oil emulsions delivering LT (Table 22). The highest level of antibody was induced at 100 μg and a lower but stronger immune response was induced at 10 μg. LT emulsified in a water-in-oil form seems to provide a convenient delivery mechanism for transcutaneous immunity, inducing a response comparable to LT in saline. Similarly, more complex formulations such as gels, creams or oil-in-water in oil could be used to deliver antigen for transdermal immunization. Such compositions can be used with patches, closed bandages, or reservoirs, and allow for long-term or short-term application of immune antigens and adjuvants.

Example 28
Five mice per group were immunized transcutaneously as described above in the “immunization process”. Mice were treated at 0, 8 and 18 weeks with 50 μg tetanus toxoid (List Biologicals, Catalog # 191B, Lots # 1913a and # 1915b) and 83 μg diphtheria toxoid (List Biologicals). ), Catalog # 151, lot # 15151) alone or in combination with 100 μl saline containing 100 μg cholera toxin (List Biologicals, catalog # 101B, lot # 10149CB).

Anti-diphtheria toxoid antibody was quantified using ELISA as described above under “ELISA IgG (H + L)”. Increased anti-toxoid antibody levels were detected in certain mice immunized with either TT / DT or CT / TT / DT. However, antibody titers were far superior in animals that included CT as an adjuvant. This anti-toxoid titer was clearly elevated in both groups after each immunization (8 and 18 weeks). That is, although DT can induce a small but significant response to itself, the extent of the response is 1) including cholera toxin as an adjuvant, and 2) boosting with adjuvant cholera toxin and antigen (diphtheria toxoid) It can be increased by applying. The classical booster response is dependent on T cell memory, and boosting with anti-DT antibodies in this experiment indicates that T cells are involved by transcutaneous immunization.

Example 29
C57B1 / 6 mice were immunized transcutaneously with CT (without azide, Calbiochem) as described above, on the shaved back of the mice. Mice were challenged 6 weeks after immunization using a lethal challenge model (Mallet et al., Non-toxic variant of Vibrio cholerae toxin in mouse cholera toxin intranasal challenge model (CTK63) and the immunopreventive effect of Escherichia coli heat labile toxin (LTK63), under submission to Immunology Letters). For antigen administration, mice were anesthetized with 20 μl of ketamine-rompin, and 20 μg of CT (Calbiochem, azide-free) dissolved in saline was administered intranasally with a plastic pipette tip. In test # 1, 12/15 immunized mice survived after 14 days of challenge and 1/9 non-immunized control mice survived. Five control mice died prior to challenge due to anesthesia. Antigen # 1 mice had 15,000 ELISA units (geometric mean) of anti-CT serum antibodies, and 5 immunized mice sacrificed at the time of antigen challenge were 5/5 mice with lung lavage fluid. IgG was detected. Lung lavage fluid was collected as described above.

Immunization and challenge were repeated for untreated C57B1 / 6 mice, and 7/16 immunized mice survived after challenge, while non-immunized mice survived only 2/17 after challenge. Antigen # 2 mice had 41,947 ELISA units (geometric mean) of anti-CT IgG antibody. Both anti-CT IgG and IgA were demonstrated in lung lavage fluid from 5 mice sacrificed at the time of challenge (Table 24). Fecal samples from 8/9 mice demonstrated both anti-CT IgG and IgA (Table 25). Fecal samples were freshly collected from animals that defecate spontaneously at the time of antigen administration. Feces were frozen at -20 ° C. At the time of ELISA, stool was thawed, homogenized in 100 μl of PBS, centrifuged and the supernatant was subjected to ELISA. The combined survival of immunized mice was 19/31, or 61%, while the combined survival of non-immunized mice was 3/26, or 12%.

Example 30
C57B1 / 6 female mice were obtained from Charles River Laboratories. Mice were treated with 200 μg ovalbumin (OVA) (Sigma, lot # 14H7035, stock concentration of 2 mg / ml in PBS) and 50 μg cholera toxin (List Biologicals, lot # 101481B, 5 mg / ml). immunization with a stock concentration of ml). The amount of ⁵¹ Cr released was measured using a Packard Cobra Gamma Counter (sequential number 102389).

C57B1 / 6 mice were anesthetized with 0.03 ml of ketamine-rompin, shaved back with clippers without damaging the skin, and rested for 24 hours. Mice were anaesthetized and then immunized on days 0 and 28 by placing 150 μl of immunization solution over a 2 cm ² area on the shaved skin for 2 hours. The mice were then wiped twice with moistened gauze. Mice did not show deleterious effects from either anesthesia, immunization, or washing steps. This operation was repeated every week for 3 weeks.

Spleen lymphocytes were collected one week after boost. Cells with or without antigen, 5% rat concanavalin A supernatant as a source of IL-2, RPMI-1640 and 10% FBS (penicillin-streptomycin, glutamine, non-essential amino acids, sodium pyruvate And 2-mercaptoethanol) for 6 days in vitro. Target cells consist of syngeneic gene (H-2 ^b ) EL4 cells alone, EL4 cells to which the CTL peptide SINFEKKL is applied, heterologous gene (H-2k) L929 cells, and EG7 cells. Target cells (1 × 10 ⁶ cells per well) are labeled with 0.1 mCi ⁵¹ Cr (Na ₂ CrO ₄ source, Amersham) per well for 1 hour and 3: 1 to 100: 1. Were added to effector cells in a range of ratios. The cell mixture was cultured in 96-well round bottom tissue culture plates (Costar, catalog # 3524) in 0.2 ml complete RPMI-1640, 10% FBS medium at 37 ° C. with 5% CO ₂ humidified atmosphere. Incubated for hours. At the end of the 5 hour culture, the supernatant was absorbed with cotton gauze and processed for measurement of ⁵¹ Cr release. Specific dissolution was determined as follows:
% Specific dissolution = 100 × [(experimental release−spontaneous release) / (maximum release−spontaneous release)].

As shown in Part 1 of Table 26, CTLs were detected on cells pulsed with EL4 peptide in the group immunized with 100 + E: T ratio of CT + OVA. CTL measurements are not positive if the percent specific lysis is 10%, ie, the media does not clearly exceed the stimulated percent effector background lysis. Similarly, as shown in Part 2 of Table 26, CTL were detected against EG7 (OVA transfected cells) in a group immunized with 100: 1 E: T ratio of CT + OVA. That is, CT acted as an adjuvant to CTL production by the transdermal route.

Example 31
C57B1 / 6 mice (n = 6) were immunized transcutaneously as described above in the “immunization step”. Mice were treated with 100 μg of cholera toxin (List Biologicals, catalog # 101B, lot # 10149CB) and 250 μg of ovalbumin protein (Sigma, albumin chicken egg, grade V, catalog # A5503, lot #. Immunization at 0 and 4 weeks with 100 μl saline containing 14H7035).

Single cell suspensions were prepared from spleens removed from mice 8 weeks after the first immunization. Spleen cells were incorporated into the culture at 8 × 10 ⁵ cells per well in a volume of 200 μl containing OVA protein or irrelevant protein conalbumin at a given concentration. The culture was incubated for 72 hours at 37 ° C. in a CO ₂ incubator, at which time 0.5 μCi / well of ³ H thymidine was added to each well. After 12 hours, the plates were collected and proliferation was assessed by quantifying the incorporated radiolabeled thymidine by liquid scintillation counting. The raw value of ³ H uptake is shown in cpm, and the fold increase (experimental cpm / medium cpm) is shown to the left of each sample. An increase factor greater than 3 is considered significant.

Significant proliferation was detected only when spleen cells were stimulated with the protein, ovalbumin, and animals were immunized in vivo against this protein, but not with the irrelevant protein conalbumin. That is, transcutaneous immunization with cholera toxin and ovalbumin protein induces antigen-specific proliferation of spleen cells in vitro, indicating that a cellular immune response is triggered by this method.

  The disclosures of all patents and all other printed materials cited herein are hereby incorporated by reference in their entirety. Such references are cited as indicating the state of the art.

  Although the present invention has been described in connection with what is presently considered practical and preferred embodiments, it is not to be understood that the invention is limited or limited to the disclosed embodiments, On the contrary, it is intended to cover combinations equivalent to the various modifications falling within the spirit and scope of the appended claims.

  That is, variations in the described invention will be apparent to those skilled in the art without departing from the novel aspects of the invention, and such variations are intended to be encompassed by the following claims It should be understood that

References

Claims (36)

  A preparation for transcutaneous immunization comprising an antigen and an adjuvant, wherein the application of the preparation to intact skin induces an immune response specific to the antigen without puncturing the skin .   The formulation of claim 1 further comprising a bandage for forming a patch for transcutaneous immunity.   The formulation according to claim 2, wherein the bandage is an occlusive dressing.   The formulation according to claim 2, wherein the bandage covers two or more draining lymph node fields.   The formulation of claim 1, wherein the adjuvant enhances presentation of the antigen to lymphocytes.   The preparation according to claim 1, wherein the adjuvant activates antigen-presenting cells.   The preparation according to claim 6, wherein the antigen-presenting cell is a Langerhans cell or a skin dendritic cell.   The formulation of claim 1, wherein the adjuvant increases major histocompatibility complex class II expression on antigen presenting cells.   The preparation according to claim 8, wherein the antigen-presenting cell is a Langerhans cell or a skin dendritic cell.   The preparation according to claim 1, wherein the adjuvant causes the antigen-presenting cells under the application site to migrate to the draining lymph node.   The preparation according to claim 10, wherein the antigen-presenting cell is a Langerhans cell or a skin dendritic cell.   The preparation according to claim 1, wherein the adjuvant gives a signal to Langerhans cells to mature into dendritic cells.   2. A formulation according to claim 1 consisting essentially of an antigen and an adjuvant.   2. A formulation according to claim 1 wherein the components of the formulation are both an antigen and an adjuvant.   The preparation according to claim 1, which is an aqueous solution.   The preparation according to claim 1, which does not contain an organic solvent.   The formulation according to claim 1, which does not contain a penetration enhancer.   2. A formulation according to claim 1 formed as an emulsion.   2. The formulation of claim 1 wherein the antigen is obtained from a pathogen selected from the group consisting of bacteria, viruses, fungi, and parasites.   The preparation according to claim 1, wherein the antigen is a tumor antigen.   The preparation according to claim 1, wherein the antigen is an autoantigen.   The preparation according to claim 1, wherein the antigen is an allergen.   The formulation of claim 1, wherein the antigen has a molecular weight greater than 500 Daltons.   2. The formulation of claim 1 comprising at least two different additional antigens.   The preparation according to claim 1, wherein the antigen is provided as a nucleic acid encoding the antigen.   26. The formulation of claim 25, wherein the nucleic acid is non-integrating and non-replicating.   26. The formulation of claim 25, wherein the nucleic acid further comprises a control region operably linked to a sequence encoding the antigen.   26. The formulation of claim 25, which does not contain a penetration enhancer, viral particles, liposomes, or charged lipids.   The preparation according to any one of claims 1 to 28, wherein the adjuvant is an ADP-ribosylated exotoxin.   30. The formulation of claim 29, wherein the ADP-ribosylated exotoxin is cholera toxin or a functional derivative thereof.   30. The formulation of claim 29, wherein the ADP-ribosylated exotoxin is E. coli heat labile enterotoxin, pertussis toxin, or a functional derivative thereof.   30. The formulation of claim 29, wherein the adjuvant is provided as a nucleic acid encoding an ADP-ribosylated exotoxin.   29. A formulation according to any one of claims 1 to 28, wherein the formulation is applied without using physical, electrical or sonic energy that pierces the intact skin.   The preparation according to any one of claims 1 to 28, wherein the immune response is not an allergic reaction, dermatitis, or atopic reaction.   The manufacturing method of the formulation of any one of the said Claims.   Use of a formulation according to any one of the preceding claims for inducing an immune response in a non-human animal.