EP2701740A1

EP2701740A1 - Microparticle and/or nanoparticle vaccine for prophylactic and/or therapeutic application

Info

Publication number: EP2701740A1
Application number: EP12721408.8A
Authority: EP
Inventors: Peter Walden; Jürgen LADEMANN; Barbara Baude; Lars DÄHNE; Karl-Heinz Wiesmüller
Original assignee: Charite Universitaetsmedizin Berlin
Current assignee: Charite Universitaetsmedizin Berlin
Priority date: 2011-04-23
Filing date: 2012-04-23
Publication date: 2014-03-05
Also published as: DE102011018499A1; WO2012146364A1

Abstract

The invention relates to novel topical vaccines that, with the aid of a particle-based support system, specifically address the dendritic cells of the skin and thereby induce effective prophylactic and/or therapeutic T‑cell‑mediated immunity. The invention further relates to the composition of such a vaccine of antigen-determinants for cytotoxic effector-T cells and helper-T cells and also the use of synthetic analogues of bacterial lipopeptides as TLR-based adjuvants for reinforcing the specific immune response. A combination of the vaccine components is immobilized on micro- and/or nano-scale support particles, after application to the skin are transported by these in an active transport process into the hair follicles and are there temporarily deposited. The active ingredients liberated in the hair follicle under the physiological conditions diffuse to the target structures in the skin, stimulate the cell surface receptors of the dendritic cells and induce a targeted T-cellular immune response. The novel topical micro- and/or nanoparticle vaccines and formulations thereof are suitable, firstly, for prevention, and secondly for treatment, of diseases such as infections, cancer, autoimmune disease, chronic infections and allergies in humans and animals.

Description

Micro- and / or nanoparticle vaccine for prophylactic and / or therapeutic

application

The present application claims the priority of a notification on Topical Nanoparticle Vaccines for Immune Stimulation of Dendritic Cells in the Skin "filed on 21 Apr. 2011 with the German Patent and Trademark Office, where it was assigned the reference DE 102011 018499.6. The content of this application, filed on Apr. 21, 2011, is hereby incorporated by reference for all purposes, including inclusion of any part or element or part of the specification, claims or drawings not incorporated herein by reference 20.5 (a) of the PCT, in accordance with Rule 4.18 of the PCT.

The present invention relates to a micro- and / or nanoparticle vaccine for prophylactic and / or therapeutic use, in particular for topical application to the skin surface. Moreover, the invention relates to a pharmaceutical preparation containing the micro- and / or nanoparticle vaccine, as well as the use of the micro- or nanoparticle vaccine or such a pharmaceutical preparation for the treatment or prophylaxis of a disease.

Background of the invention

The search for alternatives to the usual vaccination methods via the parenteral and oral routes of administration is receiving increasing attention (O'Hagan et al.2004).

In the systemic uptake of the active ingredients, which are distributed throughout the body via the blood and lymphatic system, only a fraction of the pharmacologically active constituents reach the target structures. In addition, many active ingredients have low bioavailability due to their physicochemical properties. Less stable compounds are metabolized by enzymatic degradation processes, especially in the gastrointestinal tract, before they reach the site of action. Thus, with the usual vaccination procedures very high dosages are often needed to achieve an adequate immunization success, including the corresponding side effects.

For this reason, increasing efforts have been made in recent years to use controlled drug delivery systems to control the active ingredients in a stabilized form and in a sufficiently high dose at a defined site of action and release them there with a defined release profile.

Further limiting conditions are the risks and side effects associated with systemic, mostly invasive forms of administration, such as secondary infections. allergic reactions, irritation, intoxication, hematomas and the like. Last but not least, the injection needles cause a large amount of potentially infectious hazardous waste.

Advantages of a topical vaccination, in contrast to the simple, flexible and painless handling also the targeted addressing of relevant structures of the immune system in the skin, which allow an effective immunization without burdening the entire organism with the often poorly tolerated substances, and the Avoidance of any risk of infection. Trained medical staff is often expendable.

The topical application of medicaments to the skin is based on the direct application of the active ingredient formulation to the surface of the skin, the passage of the optionally carrier-bound active ingredients through the skin and the penetration / diffusion of the active components to the site of action.

For this purpose, some application strategies have already been developed. However, few concepts have been put into clinical use, i. converted into clinical trials.

Already in 1998, the company lomai Corporation established the development of the patch technology with a patent application, the transcutaneous immunization on the skin Langerhans cells by application of antigenic determinants in combination with ADP-ribosylierenden exotoxins or other adjuvants directly to the skin surface , has the subject (W0 1999/43350). However, it is also the subject of this application that the penetration process through the skin is supported by the application of physical or chemical methods of barrier damage.

In a further application (WO 2002/074244), the topical application of only the adjuvants is described in order to intensify the immune reaction triggered by parenterally administered vaccines. The skin penetration mediation technology used conforms to the above principle involving separation of the upper epidermal layer. Thereafter, the patch is adhered and the exiting wound fluid wets the vaccine, which is then to diffuse into the skin. For the mechanism of this penetration intercellular penetration within the lipid layers and, primarily for polar substances, the transfer through liquid intercellular channels between the keratinocytes are discussed. The hair follicles and sweat glands are expressly granted a "minimal role" in transcutaneous drug delivery, particles are proposed only for better stabilization of suspensions and as depots for the delayed release of the active ingredient components. On the basis of these inventions, the company Intercell AG has now developed a vaccine patch against travel diarrhea (WO 2005/103073). However, the approval procedure was halted because no adequate efficacy could be demonstrated in Phase III clinical trials. A system for vaccination against influenza in which the immunizing effect of low doses of the conventionally administered antigens is enhanced by a patch containing a toxic adjuvant and a vaccine patch against hospital germs are under development (WO 2003/047602, Glenn et al., 2007) ,

Other approaches to topical vaccination achieve the transdermal / intradermal application of antigens by needle-free injection using air, liquids and / or pressure (W01999 / 31262, Chen et al., 2000, 2002, Mitragotri 2006) or micro-needles (Choi et al., 2007) , Sanofi Pasteur's Intanza ^® and IDflu ^® approved the first intradermal microinjection vaccine system for seasonal influenza vaccination in the EU in December 2008. With only 1, 5 mm long micro-needles, the antigen is injected minimally invasive directly into the skin. However, this does not reach the epidermis, which is particularly dense with the immunocompetent cells, as the ideal target region. Sullivan et al. (2010) propose the use of plasters with self-dissolving micro-needles of polyvinylpyrrolidone in which the vaccines are included.

Experiments with cell-penetrating peptides were less successful. By coupling TAT peptides to lipid nanoparticles, they could reach the epidermis to a depth of 120 μm. Also in the hair follicles particles were detectable at this depth. However, this is not sufficient for addressing the DCs required for immunization (Patlolla et al. 2010). In other studies, the transport effect is limited to a few proteins (Huang et al.2010).

For some years, liposomes have also been used as transport systems for topically applied drugs in and through the barrier of human skin (Barry 2002). Investigations have shown that these vesicular structures do not penetrate the skin barrier as intact units, but release the active ingredients, which then further penetrate on their own.

However, there are studies that nanoparticles below a certain size are able to penetrate the skin barrier after pre-damage. Vogt et al. For example, nanoparticles with a diameter of 40-1500 nm were applied to excised skin, the barrier of which had previously been damaged by a cyanoacrylate tear. In this case, it could be shown that especially the smallest investigated particles could penetrate into the tissue and were taken up there by the Langerhans cells (Vogt et al., 2006). A subsequent vaccination strategy with classical influenza vaccine suspensions is in clinical phase 1a (Combadiere et al., 2010, WO 2006 / 136959). The same principle applies to the DNA-based HIV vaccine DermaVir from Genetic Immunity (clinical phase II), in which the nucleic acids are bound to mannosylated polyethyleneimine nanoparticles. A release of the active ingredients is not provided in these concepts. Rather, one starts from the particulate structure of the antigens, for example in the form of viral particles, nanoparticles or DNA.

Numerous nanoscale carrier systems have been developed, the main purpose of which is to transport the carrier-bound drugs directly to and into the immunocompetent cells. In WO 2010/006753, Si0 ₂ nanoparticles having a size of 25 nm are used as the antigen carrier, the effectiveness of the uptake in the antigen-presenting cells of the lymph nodes and thus of the immunization via the size of the particles being controllable. Also in WO 2008/118861 the addressing of the immunocompetent cells is achieved by size and geometry of the carrier particles, largely independent of their composition. The application is parenteral in both cases by intradermal or subcutaneous injection. The carrier particles, together with the covalently bound active ingredients, are taken up directly by the antigen-presenting cells of the lymph nodes. Similar approaches are based on biodegradable carrier particles (WO 2008/048298, WO 2007/067744, WO 1998/28357), wherein the active ingredient components can be integrated into the matrix or bound to the surface by adsorptive interactions or complex formation. Carrier particles with an intrinsic adjuvant effect are also described (Chen et al., 2010, Florindo et al., 2010).

By non-covalent coupling of the active ingredients to the carrier particles on the basis of physicochemical interactions, there is the possibility of controlled release of active ingredient after reaching the site of action (WO 2009/046498, WO 2007/067744).

So far, however, it has not been possible in any application to realize the transdermal drug delivery for any and any number of vaccine components without pre-damage of the skin or injection systems and without entry of the carrier into the living body tissue (Prausnitzetal, 2008). While the penetration of nanoparticles into the hair follicles has become well-researched, the use of larger and thus non-cell carrier particles, the effective binding and targeted release of the active ingredients as well as the independent diffusion of the active ingredients into the skin's immunocompetent structures are still the subject of current research ,

The development of vaccines away from the complete organisms towards high purity synthetic vaccine components increases the requirement of adjuvants to enhance the immune response. Many classical adjuvants simulate the immunologically effective ones through certain molecular structures called Pathogen-Associated Molecular Patterns (PAMPs) Components of the pathogens and are recognized by the immunocompetent cells of the innate immune system, including the antigen-presenting cells, such as DCs, preferably via the toll-like receptors (TLR). These cells also play an important role in the expression of the antigen-specific adaptive immune response by the presentation of the antigens to the lymphocytes and the expression of cytokines.

Among others, bacterial lipopeptides, which primarily stimulate the T-cellular immune response, are of great importance (Brown et al., 2005). Because the TLRs for lipopeptides, unlike the TLRs for most other immunomodulators, are expressed on the surfaces of the cells, bacterial lipopeptides are particularly well suited for targeting. In addition, these can be replaced by synthetic analogues, which are characterized by the unusual amino acid S- [2,3-di (hydroxy) -propyl] - [f?] Cysteine at the N-terminus, bound to the two or three fatty acids as a characteristic feature , exhibit. Thus, the TLR 1 / TLR2 heterodimer also reacts to triacylated synthetic lipopeptides, such as the Pam ₃ Cys derivatives (EP 0210412). TLR6 / TLR 2 heterodimers are sensitive to diacylated synthetic lipopeptides, such as the Pam ₂ Cys derivatives (EP 0519327).

For example, is by Mühlradt etal. claims a diacylated dihydroxypropyl cysteine peptide having a Mycoplasma fermentans derived peptide (MALP-2) for various applications in immunological medicine (WO 1998/27110, WO 1999/59610, WO 2002/028887, WO 2003/084568, WO 2004 / 108145).

Based on these findings, various drugs and vaccines containing adjuncts of acylated lipopeptides as adjuvants have already been developed for use in immunomodulatory therapies. Typically, the lipopeptides, preferably Pam ₃ Cys and Pam ₂ Cys derivatives as TLR 2 targeting substance, this mixed with an antigenic component for B lymphocytes or for cytotoxic T cells and other excipients to induce synergistic effects (WO 2007 / 103322, WO 2006/104389, US Pat. No. 7,387,271). WO 2006/040076 also describes a topical application of such an active ingredient formulation.

Also, formulations as liposomes or virus like particles which, in addition to antigens and optionally cytostatics, also contain lipopeptides, preferably Pam ₃ Cys, are claimed for the treatment of various diseases and allergies (WO 2005/063201, WO 2005/063288).

Also described are conjugates of Pam ₂ Cys and Pam ₃ Cys derivatives and antigens as vaccines to specifically bind or infiltrate the antigens to the immunocompetent cells; in WO 2007/078879 a combination of a membrane protein of the influenza virus and a Pam ₃ Cys derivative, in WO 2009/046498 a DNA A vaccine which is coupled by polar amino acids to a Pam ₂ Cys derivative, in WO 2007/079448 a linear tandem molecule consisting of a B cell epitope, a T cell epitope and Pam ₃ Cys or in WO 2004/014956 and WO 2004/014957 a molecule of a B-cell or CTL epitope and a helper T-cell epitope, which are bridged by a Pam ₃ Cys lipopeptide. Already Borges etal. (1994) describe the immunoenhancing effect by the direct coupling of Pam ₂ Cys and / or Pam ₃ Cys derivatives with CTL and / or helper T cell epitopes.

Very few concrete applications are known in the context of particulate biotransporters for lipopeptides. The focus of related patent applications is the nature of the particles. As a rule, the active ingredient components are not specified. Lipopeptides are mentioned among a variety of possible compounds as immune cell-targeting substances. An application of lipid-like structures are nanoscale fat and fat-like droplets as drug carriers (EP 0605497). Heuking et al. In 2009, a system of Pam _{3 described} Cys-PEG coupled to chitosan (particles) as a potential drug delivery system, with the lipopeptide acting as the anchor molecule for targeting the chitosan biotransporters. Through the lipopeptide as TLR ligand, the drug carriers should be specifically bound to the immunocompetent cells to allow an intense interaction or uptake into the cell.

Task Based on the presented advantages and disadvantages of currently available vaccination methods, there is a need for further vaccination strategies for prophylaxis and / or therapy, which utilize the advantages of a topical vaccination in comparison to the previously dominant systemic and invasive application form.

It is therefore an object of the present invention to provide novel carrier-based and topically applicable prophylactic and / or therapeutic vaccines which allow effective and at the same time simple and painless immunization without side effects and risks.

The object is achieved by a topically applicable micro- and / or nanoparticle vaccine as defined in claim 1. This vaccine can be used for prophylactic and / or therapeutic use in infections, cancer, autoimmune diseases and inflammatory and allergic conditions. A vaccine according to the present invention is typically able to address with the DCs the sensor cells of the immune organ skin directly through the hair follicle as portal of entry and additionally to stimulate by a TLR-based adjuvant. Summary of the invention

The present disclosure relates to a micro- and / or nanoparticle vaccine, also referred to hereinafter as a micro- and / or nanoparticle vaccine, as well as uses and methods based on this vaccine.

The present invention relates to novel topical vaccines which, with the aid of a particle-based carrier system, specifically target the dendritic cells (DC) of the skin via the hair follicle as portal of entry and thus induce effective prophylactic and / or therapeutic T-cell-mediated immunity.

The present invention further relates to the composition of such a vaccine of antigenic determinants for cytotoxic effector T cells and helper T cells, and to the use of synthetic analogs of bacterial lipopeptides as TLR-based adjuvants for enhancing the specific immune response.

A combination of the vaccine components is immobilized on micro- and / or nanoscale carrier particles, transported to the skin by these in an active transport process in the hair follicles and temporarily deposited there. The active ingredients released under the physiological conditions in the hair follicle diffuse to the target structures in the skin, stimulate the cell surface receptors of the DCs and induce a targeted vaccine antigen-specific T cell immune response.

The new topical microparticles and / or nanoparticle vaccines and their formulations are / are suitable as medicines on the one hand for the prevention and on the other hand for the treatment of diseases such as infections, cancer, autoimmune diseases, chronic inflammations and allergies in humans and animals.

In a first aspect, the present invention provides a micro and / or nanoparticle vaccine. The vaccine has basic components as individual molecules or conjugates. The vaccine has a first basic component, which is one or more antigenic determinants of cytotoxic effector T cells. The vaccine has a second basic component, which is one or more antigenic determinants of helper T cells. Furthermore, the vaccine has a third basic component, which is one or more immunomodulators for activating the antigen-presenting dendritic cells. The vaccine components are coupled to micro- and / or nanoscale carrier particles. The micro- and / or nanoscale carrier particles are designed in such a way that after topical application to the skin surface they are actively transported into the hair follicles where they are temporarily deposited. As a result, the vaccine components induce T cell-mediated immunity via stimulation of the skin's dendritic cells. In some embodiments, the micro- and / or nanoparticle vaccine according to the first aspect is a vaccine for medical use. The micro- and / or nanoparticle vaccine is, in some embodiments, for therapeutic and / or prophylactic use. In some embodiments, the micro- and / or nanoparticle vaccine is a vaccine for use in a therapeutic and / or prophylactic process.

In a second aspect, the present invention provides a prophylactic and / or therapeutic method using a micro- and / or nanoparticle vaccine. The method comprises inducing T-cell mediated immunity. Inducing T-cell mediated immunity occurs by stimulating the dendritic cells of the skin by the micro- and / or nanoparticle vaccine. The vaccine has basic components as individual molecules or conjugates. The vaccine has a first basic component, which is one or more antigenic determinants of cytotoxic effector T cells. The vaccine has a second basic component, which is one or more antigenic determinants of helper T cells. Furthermore, the vaccine has a third basic component, which is one or more immunomodulators for activating the antigen-presenting dendritic cells. The vaccine components are coupled to the micro- and / or nanoscale carrier particles. Typically, the vaccine is a vaccine according to the first aspect. The method further typically includes topically applying the micro- and / or nanoparticle vaccine to the skin surface of a subject. The method further comprises allowing the micro- and / or nanoscale particles to be actively transported into the hair follicles. In addition, the procedure includes allowing temporary storage of the micro- or nanoscale particles in hair follicles. As a result, the vaccine components induce T cell-mediated immunity via stimulation of the skin's dendritic cells.

The method according to the second aspect is typically a method for the prophylactic and / or therapeutic treatment of a disease of a subject, ie a disease in humans and / or animals. In some embodiments, the method according to the second aspect is a method for the prophylactic and / or therapeutic treatment of a disease in humans and / or animals associated with infections, cancer, an autoimmune disease, or an inflammatory and / or allergic condition. In some embodiments, the method according to the second aspect is a method for prophylactically specific immunization against and / or for therapeutic vaccination in a disease. In some embodiments, the method according to the second aspect is a method of supporting a conventional vaccine. In some embodiments, the method according to the second aspect is one A method of enhancing the immune response following vaccination performed by conventional vaccination techniques.

In a related third aspect, the present invention provides a method of vaccinating a subject. The method comprises administering to a subject a pharmaceutically acceptable quality of a micro and / or nanoparticle composition. The method typically involves topically applying the micro- and / or nanoparticle composition to the skin surface of a subject. Typically, administering the micro and / or nanoparticle composition is sufficient to trigger an immune response of the subject. The micro / nanoparticle composition comprises micro- and / or nanoparticles. Basic components of the micro- and / or nanoparticle composition are coupled to the micro- and / or nanoparticles. The basic components are present as single molecules or as conjugates. The micro / nanoparticle composition has a first base component that is one or more antigenic determinants of cytotoxic effector T cells. The micro and / or nanoparticle composition has a second base component which is one or more antigenic determinants of helper T cells. Furthermore, the micro / nanoparticle composition has a third basic component, which is one or more immunomodulators for activating the antigen-presenting dendritic cells. Typically, the micro / nanoparticle composition is a vaccine according to the first aspect. The method further comprises allowing the micro- and / or nanoparticles to be actively transported into the hair follicles. In addition, the method involves allowing the micro- and / or nanoparticles to be temporarily deposited in the hair follicles. This allows the basic components of the micro / nanoparticle composition to induce T-cell mediated immunity by stimulating the dendritic cells of the skin.

In a fourth aspect, the present invention provides a pharmaceutical preparation. The pharmaceutical preparation contains a micro- and / or nanoparticle vaccine according to the first aspect, alone or in combination. The preparation is in a formulation suitable for topical application. Typically, the pharmaceutical preparation contains a therapeutically effective amount of the micro- and / or nanoparticle vaccine.

In some embodiments, the pharmaceutical preparation according to the fourth aspect is present as a paste, ointment, cream, lotion, emulsion, gel, hydrogel, suspension, solution, spray or patch. In some embodiments, such a preparation contains further constituents, such as otherwise therapeutically relevant pharmaceutical active ingredients and adjuvants, as well as further pharmaceutically suitable ingredients. Examples of other pharmaceutically acceptable ingredients include, but are not limited to, excipients, adjuvants and / or adjuvants. Substances such as detergents, stabilizers and preservatives. Such other ingredients which may be included in the preparation may be mixed, dissolved or associated with a physical or biological carrier.

In a fifth aspect, the present invention relates to the use of a micro- and / or nanoparticle vaccine according to the first aspect or a pharmaceutical preparation according to the fourth aspect for a medical indication.

Such a medical indication can cause a disease such as an infection, cancer, an autoimmune disease, or an inflammatory and / or allergic condition. In some embodiments, the use of a microparticle nanoparticle vaccine or pharmaceutical preparation according to the fifth aspect relates to prophylactic specific immunization against disease and / or therapeutic vaccination in a disease. In some embodiments, the use relates to enhancement of the immune response following vaccination performed by conventional vaccination techniques.

In some embodiments, the use relates to the preparation of a medicament for human and / or animal for immunization. Such immunization may be a prophylactic specific immunization against a disease. Such immunization may also be a therapeutic vaccination in a disease. Furthermore, such immunization may be to enhance the immune response following vaccination performed by a conventional vaccination procedure.

Brief description of the illustrations

Fig. 1 Principle of pH triggered adsorption and desorption of the polarized by a charge tag vaccine components on a ^® in the LBL method with charged polymers coated carrier particles. The release of the vaccine components takes place by recharging the top polymer layer as a function of the pH, as a result of which the electrostatic attraction is abolished.

FIG. 2 Dependence of the loading of the carrier particles on the order of addition of the vaccine components and the residence time in the loading solution with sequential addition (GILG = GILGFVFTL-KKKKK fluorescein, SEQ ID NO: 11 coupled to the fluorescent dye fluoerszein, P2C = Pam ₂ Cys-GDPKHPKSF-KKKKK-Tamra, SEQ ID NO: 9 coupled to the fluorescent dye Tamra, Cha = aK-L-Cha-VAAWTLKAAa-Aca-C-KKKKK-Cy5, SEQ ID NO: 10 coupled to the fluorescent dye Cy5). FIG. 3 Release kinetics of the three vaccine components GILGFVFTL-KKKKK-fluorescein (GILG), Pam ₂ Cys-GDPKHPKSF-KKKKK-Tamra (P2C) and aK-L-Cha-VAAWTLKAAa-Aca-C-KKKKK-Cy5 (Cha) after conversion into a 10 mM acetate buffer solution pH 5 with the addition of 0.1 M sodium chloride (1% suspension).

Fig. 4 Schematic drawing of a loaded with the individual vaccine components carrier particle. The number of components, the loading of the particles and the real stoichiometric ratios are variably adjustable according to the spectrum of action of the vaccine (the epitopes are shown by way of example for the antigenic determinants).

Fig. 5 Schematic drawing of a carrier particle loaded with a conjugate of the vaccine components. The loading of the particles is variably adjustable according to the spectrum of action of the vaccine (the epitopes are shown by way of example for the antigenic determinants).

FIG. 6 Penetration of the micro- / nanoparticle vaccine into the hair follicle of the pig ear model (laser scan microscopic image of the cryosections) 60 min after application to the skin. The rhodamine-labeled epitope GILGFVFTL-K4 penetrates further into the hair follicle after detachment from a silica carrier particle (labeled with Cy5).

Fig. 7 penetration depth of the silica carrier particles as a function of the particle size compared to the length of the terminal hair / Vellushaar follicles. The target region Infundibulum is reached at about 600 μητι depth.

FIG. 8 Accumulation and residence time of the silica carrier particles in the hair follicles and in the stratum corneum.

FIG. 9 Effect of the oligoamino acid tags on the immunological activity of the CTL epitope GILGFVFTL in the cytotoxicity assay with the human CD8 T cell line 10-21A and the B lymphoblastoid target cell line JY (titration in the radiochrome release assay). The insert shows the values transformed to Clark. The functional avidity values in the table indicate the peptide concentration for half-maximal cytolysis. Factors for activity loss are related to the functional avidity of the untethered peptide.

FIG. 10 Comparison of the immunological activity of the particle-bound and the free individual components in the ELISpot assay on IFN-γ with human CD8 T cells. 11 Titration of the immunological activity of mixtures of the particle-bound and the free vaccine components in the ELISpot assay on IFN-γ with human CD8 T cells (ordinate: optical units of the densitometric measurement).

Fig. 12 Differentiation and maturation of DCs using mixtures of differently loaded carrier particles based on the expression of the costimulatory

Cell surface molecules CD80 (A) and CD86 (C) and the DC activation marker CD83 (B). Components in parentheses were coupled together to a silica carrier particle.

Figure 13 Activation of DCs using mixtures of differently loaded carrier particles by expression of costimulatory cell surface molecules

CD80, CD86, CD40 and HLA-DR and the DC activation marker CD83. Components in brackets were coupled together to a carrier particle. TNF-α, IL-1-β and IL-6 is an inflammatory cytokine cocktail as a positive control.

Figure 14 Differential biological effects in induction of T cells and co-action of the helper T cell epitope PADRE and the immunomodulator P2C in the proliferation and differentiation of naive T cells by micro / nanoparticle vaccine activated DCs. TNF, IL-4 and IL-6 is an inflammatory cytokine cocktail as a positive control (above: proportion of memory T cells formed, below: proliferation rate).

Fig. 15 Differential biological effects in the induction of T cells and co-action of the helper T cell epitope PADRE and the immunomodulator P2C in the proliferation of memory T cells by micro / nanoparticle vaccine activated DCs. TNF, IL-4 and IL-6 is an inflammatory cytokine cocktail as a positive control (proliferation rate).

FIG. 16 Induction of antigen-specific T cell reactions by various combinations of the vaccine components on silica particles using the example of the CMV epitope

NLVPMVATV (SEQ ID NO: 6), detected by recombinant tetramerized MHC-peptide complexes.

FIG. 17 Induction of antigen-specific T-cell reactions by various combinations of the vaccine components on silica particles using the example of the influenza epitope GILGFVFTL, detected on the basis of the recombinant tetramerized MHC-peptide complexes. Detailed description

The present invention relates to a micro- and / or nanoparticle vaccine and its use. This vaccine comprises a micro- or nanoparticle to which immunologically active components are coupled. The vaccine may also include both a microparticle and a nanoparticle to which immunologically active components are coupled. In some embodiments, the vaccine is a nanoparticle to which are coupled three basic components as defined below. Typically, the vaccine has a plurality of micro- and / or nanoparticles to which immunologically active components are coupled. The term "microparticle" as used herein refers to any particle that has an extent of about 5000 nm or less in one or more dimensions, such as an extent, eg, a diameter, in the range of about 500 nm to about 3000 nm, in the range from about 500 nm to about 2000 nm, in the range of about 500 nm to about 1000 nm, in the range of about 700 nm to about 1500 nm, in the range of about 500 nm to about 800 nm, or in the range of about 600 nm to about 900 nm. The term "nanoparticle" as used herein refers to any particle having an extent in one or more dimensions, eg a diameter of about 500 nm or less, such as about 400 nm or less, about 300 nm or less, for example, in the range of about 1 nm to about 500 nm, about 5 nm to about 450 nm, about 1 nm to about 250 nm or from about 10 nm to about 150 nm. Such a micro- and such a nanoparticle is suitably dimensioned in order to be transported to hair follicles (see below).

A particle contained in a vaccine according to the present invention typically has an extent of about 5000 nm or less in one or more dimensions, such as an extent, eg, a diameter, of about 3000 nm or less, a diameter of about 1000 nm, a diameter of about 700 nm or less, about 500 nm or less, about 250 nm or less, about 100 nm or less, about 80 nm or less, about 60 nm or less, about 50 nm or less , about 35 nm or less, or about 35 nm or less. For example, a micro- or nanoparticle may have a maximum extension in the range of about 1 nm to about 1000 nm, such as about 1 nm to about 500 nm, or about 1 nm to about 100 nm. Examples of nanoparticles include, but are not limited to, a nanocrystal, a nanosphere, a nanorod, a nanoshell or a nanofilament such as a nanotube or a nanowire. Typically, a nanoparticle, as well as a microparticle according to the invention, is an inorganic particle. A micro- and / or nanoparticle may contain any suitable material - or consist thereof - such as carbon Semi-metal such as boron, germanium or silicon, or a metal such as gold, silver or copper, including an alloy thereof or a polymer.

A corresponding micro- and / or nanoparticle may be of any surface finish. For example, a microparticle and / or nanoparticle may have a polar or apolar surface and thus be hydrophilic or hydrophobic. Hydrophobic material is water repellent or tends to separate from water. Typically, hydrophobic material has a uniform electron density distribution. In contrast, hydrophobic material contains molecules or regions which can enter into dipole-dipole interactions with water molecules and thus have a high wettability for water.

A use or method according to the present invention based on the use of the micro- and / or nanoparticles may be applied to any subject, such as e.g. carried out on a mammal. Examples of a suitable mammal include, but are not limited to, a mouse, rabbit, rat, guinea pig, hamster, dog, cow, pig, goat, sheep, horse, rhesus monkey Schimpanze, a gorilla or a human. A use or method according to the invention directly addresses the sensor cells of one of the most important organs of the immune system, namely the DCs of the skin, including the Langerhans cells.

In their role as mediators of immunity, DCs have two key functions that can be clearly distinguished in time. As immature cells, they are responsible for the uptake and processing of antigens. Only a little later, after activation, they provide as mature cells for the stimulation mainly of T cells and indirectly via the T cells also of B cells, by giving them the processed antigen in the form of MHC-peptide complexes, together with costimulatory molecules, present on their surface while releasing cytokines that support and stabilize immune responses. As guardians and alerters of the immune system, they thus exercise a higher-level control function over the actual actors of the cellular immune response. Only one DC is needed to activate 100 to 3,000 antigen-specific T cells.

Because of their ability to induce, enhance, and build immune responses, DCs have become an important cellular tool for immunotherapy. New approaches to fully activate and differentiate these immunocompetent cells can significantly increase the efficacy of prophylactic and therapeutic vaccines, as only mature DCs have a specific T cell-based cytotoxic immune response and the expression of proinflammatory cytokines can trigger. At the same time, mature DCs are able to reverse the effect of tumors on immunosuppressive factors.

A special form of DCs are the Langerhans cells. These are still inactive antigen-presenting cells that form a continuous network in the epidermis of the skin. Upon contact with the antigen, their activation and differentiation into mature DCs transports the antigens into the regional lymph nodes where they are presented to the T lymphocytes.

Against this background, the DCs of the skin are ideal target structures for topical vaccination. As a portal of entry for the topical vaccine, the hair follicle is particularly well suited, since the epidermis is crossed in this area with a particularly dense network of immunocompetent cells, which serves to monitor and defend against by the non-closed hair barrier in the hair follicle penetrating foreign substances.

With the usual methods for topical vaccination, however, it has not yet been possible to use these structures for overcoming the skin barrier. All previously known methods are based on a penetration of the active ingredients directly through the horny layer of the skin or on the penetration of micro- or nanoscale particulate antigens directly into the DCs of the skin, in most cases after chemical or physical pre-damage or swelling of the skin. For solid particles a penetration into the hair follicles could be detected. However, there were no indications for the passage into the area of living cells (Lademann et al., 2009).

It is therefore all the more surprising that the system of the micro- and / or nanoparticle vaccines according to the invention has succeeded for the first time in rendering carrier-bound active substances into the hair follicles and thus into the deeper layers of the epidermis by targeted utilization of a natural transport mechanism. the stratum spinosum, to convey. In this regard, the micro- and / or nanoparticles of a vaccine of the present invention are typically configured so that, after being administered by application to the skin surface, it may be allowed for the nanoparticles to be transported into the hair follicles. Components of the vaccine coupled to the micro- and / or nanoparticles thus likewise reach the hair follicle. Furthermore, in embodiments of the invention, it may be allowed for the micro- and / or nanoparticles to remain in the hair follicle for a certain period of time (see below).

In this region, the highest density of immunocompetent cells of the skin is located, so that the vaccine components come in close proximity to the target structures and only have to diffuse a short distance after detachment from the carrier particle. The carrier particles typically have a quadruple function. First, as vectors for efficient targeting of the vaccine, they enable active transport into the hair follicles using a biomimetic principle. Second, as multifunctional carriers, they organize the molecular components of the vaccine in highly efficient spatial proximity. Third, they act as modulators of targeted release of the vaccine components after successful transport. Fourth, they enhance their effect through the multivalent organization of the vaccine components. Without being bound to a particular theory, it could surprisingly be found in the examinations that the movement of the hair in the hair follicle sets in motion a type of pumping mechanism which causes the transport of particles into the hair follicle. It is assumed that for this purely mechanical effect, the particulate structure of the support materials and the massaging are essential. The transport mechanism is all the more effective, the better the particle size and the shed density of the hair are coordinated. It is believed that the structure and surface finish of the particles have less influence.

Without wishing to be bound by theory, it is believed that in order to achieve an immunologically relevant depth in the hair follicle, in particular the dimension of the micro- / nanoparticles is / are significant. It has been shown that one or more particles, in particular one or more spherical particles, in a size range of 100 nm to 3,000 nm, for example 300 nm to 1,500 nm (supra), are / are particularly well suited to the various vaccine components to be transported by the described pumping mechanism to the optimal position in the hair follicle, wherein the penetration depth can be adjusted by the particle size (Fig. 7). After only 1 h, the infundibulum is reached at a depth of 600 μm, where the highest density of DC of the skin is found in human terminal hair. Further penetration within the next 24 h is insignificant for vaccination success.

In addition, it was surprisingly found that, in addition to the transport into the hair follicle, a long-term storage of the carrier particles takes place (FIG. 8). Only after a time interval of about 5 to 10 days, the carrier particles are typically transported out of the hair follicle again by sebum secretion and hair growth, while the particles lying in the stratum corneum are repelled relatively quickly by the continuous Abschilferung the horny cells. This allows the development of retarding topical formulations. At the same time, natural processes guarantee that the carrier particles are transported out of the hair follicle.

The possibility of a transfollicular penetration of the particles through the hair follicle barrier into the area of the living cells can not be ruled out, but so far it has not been possible be detected. Such a transition was only observed after pre-damaging the skin structure, for example by cyanoacrylate tearing and for particle sizes below 50 nm. Only the released drugs diffuse into the body tissue, where they reach the DCs as target structures and trigger a specific immune response.

For the effective loading of the carrier particles with the vaccine components, their stable binding and safe transport into the hair follicle as well as the targeted release of the further fully effective active ingredients on the target structures, a reversible coupling mechanism based on physical or chemical interactions can be used.

In some embodiments, reversible adsorption may be based on electrostatic interactions, van der Waals bonds, hydrophobic interactions, and / or hydrogen bonds. For example, an interaction can occur between a polarized surface of carrier particles and charged regions of the vaccine components, which leads to adsorption. If necessary, negative or positive net charge regions may be incorporated into vaccine components in the form of one or more additional molecular tags, called tags.

As triggering for the release of the vaccine components at the destination, the pH shift within the hair follicle can advantageously be used. Particles were designed whose surface potential (ζ-potential) changes in dependence on the pH in such a way that the vaccine components stably bind to the carrier particles during loading and transport and desorb again under the physiological conditions in the hair follicles.

For this purpose, in exemplary embodiments, particles were coated in the layer-by-layer (LBL) process. The LBL ^® technology uses the electrostatic interaction between charged polymers (polyelectrolytes) and surfaces. By adsorption of one or more polymers on oppositely charged particle surfaces, in the present invention, for example, on silica particles with negative surface potential, a reloading of the particles takes place, whereby the deposited amount of polymer by itself to a thickness of about 2-7 nm regulated. For a second layer, the process can be repeated with an oppositely charged polymer. In this way, any number of layers can be built up on substrates.

Any biocompatible polymer can be used. For the given application, the cationic polymers used are poly (allylamine hydrochloride) (PAH), poly (diallyldimethylammonium chloride) (PDA) and poly (acryloyloxyethyltrimethylammonium chloride) (PAOET) and as anionic polymers, the sodium salts of poly (vinyl sulfate) (PVS), sulfoethyl cellulose and polystyrene sulfonate (PSS) have been found to be particularly suitable.

In some embodiments of the present invention, ^LBL® surfaces having 2 to 20 layers can be used, using a weak polyanion or polycation for at least the outermost layer which alters its charge as a function of pH, such as chitosan. Polyvinylpyridine, polyvinylcarbazole, polyacrylate (RAA) or polymethacrylate (PMAA). Natural proteins are also suitable as pH-switchable materials, depending on their isoelectric point.

For the polarization of the vaccine components, these may be mixed with one day, i. an appendage having charged molecular regions such as amino acids. Amino acids are known as organic molecules having a carboxylic acid group (carboxyl group) and an amino group, typically α- or β-amino acids. For example, an appendage may have about two to about 20, about three to about 15, or about 4-10 charged α-amino acids, or be composed of such amino acids. Any number of amino acids can be used, including naturally occurring amino acids and isomers thereof. An appendage that has a positive or negative charge under physiological conditions is also referred to below as a charge tag. In some embodiments, such an appendage is over a peptide segment of up to about 8 amino acids in length, e.g. 1,2,3,4,5,6 or 7 amino acids coupled to a vaccine component. A corresponding peptide segment can also be referred to as a spacer. Such a charge tag contains one or more amino acids which have a side chain which have a positive or negative total charge under physiological conditions or consists of such amino acids. A chemical group or an amino acid having a total positive charge under physiological conditions is also referred to as a basic group or amino acid. A chemical group or an amino acid with a total negative charge is also referred to as an acidic group or amino acid.

In some embodiments, the basic amino acids lysine, 5-hydroxy-lysine, pyrrolysine, hypusine (N6- (4-amino-2-hydroxybutyl) -lysine), histidine, ornithine, and / or arginine may be used as positively charged amino acids under physiological conditions become. The presence of such amino acid units can serve for positive polarization. Glutamic acid-aspartic acid or sulfotyrosine units are illustrative examples of amino acids that can be used for negative polarization. They can ensure that the vaccine components accumulate in analogy to the polymers on the oppositely charged particles and are desorbed when the particle surface is recharged by pH. The length and composition of the charge tags as well as an optionally insertable spacer must be individually tailored to the respective vaccine component, since these molecules may be sterically hindered or further interactions, such as hydrogen bonds or hydrophobic interactions, may occur with the polymer surfaces. In addition, the capacity and efficiency of the loading process as well as the separation kinetics can be individually adjusted by modifying the charge tags.

In the context of the present invention (PAH / PSS) ₂ PAH / PMAA coated silica particles have proven to be particularly suitable. While the loading of the ^LBL® particles in some embodiments takes place under basic conditions, the desorption of the vaccine components in such embodiments typically occurs after the transport process in the acidic environment of the hair follicle and vice versa. Due to the increased hydrogen ion concentration occurs below about pH 7 to protonate the PMAA ~ - groups, whereby the outer LBL discharges ^® layer or recharges cationic by said underlying PAH layer. This leads to repulsion of the positively polarized vaccine components from the particle surface (Figure 1). An additional positive effect on the release of the vaccine is the increased salt concentration in the hair follicle.

Typically, between 20 and 150 pg of the individual vaccine components modified with corresponding oligoamino acid tags per 1 mg of silica carrier particles were adsorbed by this method. With the added amount, the adsorption time, the day length and the tag structure, the loading for each vaccine component can be adjusted individually. As a result, the stoichiometric composition of the microparticle and / or nanoparticle vaccine according to the invention can be adjusted in accordance with the individual medical indication, the form of administration and the efficiency of the vaccine components (Example B).

In addition to the unmixed loading, the simultaneous loading of a carrier particle with different, differently interacting vaccine components in a defined molar ratio, as well as their simultaneous and uniform release at the site of action, could be realized for the micro- and / or nanoparticle vaccine according to the invention, using the example of some active ingredient combinations.

A system be found particles which enables stable, effective and uniform adsorption of the vaccine components and their uniform release in the hair follicle - could therefore with the example described in the embodiments LBL ^®. In particular, the yields of the released in the hair follicles active ingredients in the order of 30 - 90% of the adsorbed amount, are in a sufficient for vaccination order of magnitude. Unwanted aggregation effects could be low loading methods, the choice of coating materials and the order of the polymer layers can be prevented.

By directly addressing the immunocompetent cells of the skin with the ability to induce T-cell mediated immunity in addition to antibody-mediated immunity, the composition of the subject, typically topical, micro- and / or nanoparticle vaccine of the present invention may be minimized Components and lowest levels of drugs are limited.

For the exemplary investigations cited in the exemplary embodiments, a synthetic minimal vaccine was used which consists of the following essential basic components for addressing various synergistic targets:

(i) epitope for cytotoxic effector T cells (CTL),

(ii) Epitope for helper T cells, for example, a Pan-DR epitope and

(iii) bacterial lipopeptide as immunomodulator.

In general, a vaccine according to the present invention, these components are interchangeable according to the medical indication and combined in any ratio. Instead of single epitopes, any antigenic determinants for T cells, e.g. multiple peptides, polyepitopes, biopolymers, or other antigenic information carriers, such as a nucleic acid, e.g. DNA or RNA, a lipid and / or an oligo- or polysaccharide. The term "antigen determinant" refers to one or more regions of an antigen or hapten that can be bound by an immunoglobulin, a T cell receptor, or a binding reagent such as a lipocalin mutein An antigenic determinant has at least one epitope (see below) The antigenic determinants for CTL epitopes are selected according to the desired immunization The helper T-cell epitope Pan-DR-E is universal for all HLA-DR allotypes, but must be adapted to the specific species and may be advantageous when a vaccine contains a plurality of antigen determinants / epitopes to achieve adequate vaccine protection in an immunogenetically heterogeneous population.

As used herein, the term "nucleic acid" refers to any nucleic acid in any configuration, such as single-stranded, double-stranded, or a combination thereof, to nucleic acids include DNA molecules, eg, genomic DNA or cDNA, RNA molecules, such as mRNA, analogues of DNA or RNA prepared by nucleotide analogues or by chemical nucleic acid synthesis, such as protein nucleic acid (PNA) .A nucleic acid may, of course, contain non-native nucleotide analogs or be coupled to an affinity tag or label. Numerous nucleotide analogs that may be included are known to those skilled in the art. A nucleotide analog is a nucleotide having a modification to, for example, the base, the sugar and / or the phosphate group. By way of illustration, as an example, it is known that a substitution of 2'-OH residues of siRNA with 2'F, 2'O-Me or 2'H residues increases the wVO stability of the RNA in question. Modifications to the base group include natural and synthetic modifications of A, C, G and T / U, various purine or pyrimidine bases such as uracil-5-yl, hypoxanthine-9-yl, and 2-aminoadenine-9-yl, as well as non Purine or non-purine bases. Other nucleotide analogues serve as universal bases. For example, universal bases include 3-nitropyrrole and 5-nitroindole. Universal bases are able to form a base pair with each base. Often, base modifications can be combined with, for example, a sugar modification, eg, 2-O-methoxyethyl, for example, to achieve unique properties such as improved duplex stability.

The third basic component of a vaccine of the present invention is an immunomodulator, i. a compound that activates or slows down or inhibits a subject's immune response. An example of an immunomodulator is immunostimulator-lipopolysaccharide (LPS). Another example of an immunomodulator is cationic lipid and lipid protamine DNA, which are described in U.S. Pat. 7,303,881. Another example of an immunomodulator is a synthetic bacterial lipopeptide. Lipopeptides are part of the bacterial cell wall and are recognized by the Toll-like receptors TLR 1, 2, 6 and 10 of the dendritic and other immunocompetent cells. Via a biochemical signal cascade, a corresponding intracellular mechanism, usually the expression of specific, the antigen-specific defense of pathogens serving genes is triggered in the TLR carrier cells, which leads to the differentiation of pathogen-specific T cells. As a result, in the first stage of infection defense, the nonspecific immune response is initiated, which subsequently follows the response of the acquired immune system to the formation of specific antibodies. Examples of a synthetic analog of a bacterial lipopeptide having immunostimulatory effect are e.g. disclosed in the German patent application DE 102009034779 A1.

The use of the lipopeptide immunomodulator can be particularly advantageous, since with a micro- and / or nanoparticle vaccine according to the invention specifically the DCs of the skin are addressed, which have a high density of all TLR and in their guard function for Entrittspforte skin via particularly effective mechanisms as the first line of defense of the innate immune system. In addition, the TLRs for the lipopeptides are expressed on the surfaces of the cells so that the lipopeptides can act directly on the cells, if appropriate also particle-bound, without being recorded in advance.

Another example of an immunomodulator is murabutide (N-acetylmuramyl-L-alanyl-D-glutamine-alpha-n-butyl-ester), a derivative of the bacterial muramyl dipeptide. Two illustrative and well-known examples of immunomodulators that are immunosuppressants are cyclosporin and methotrexate. Two other immunomodulating modulators are 4- (3-chlorophenyl) -N- (4- (trifluoromethyl) phenyl) pyrimidin-2-amine (VAF347) and fingoli- mod (720, 2-amino-2- (2- (4- octylphenyl) ethyl) propane-1,3-diol).

The direct coupling of the antigen-specific vaccine components with the immunomodulatory effective adjuvant allows a highly focused abolition of the conditioned immunosuppressive state of the skin and thus a marked increase in the T cell induced by the micro- and / or nanoparticle vaccine according to the invention -based, specific immune response. This further minimizes the possibility of immunological side effects.

In some embodiments, a lipopeptide immunomodulator according to the present invention has the following general structure (I):

R1-0- CH ₂

In the structural formula (I)

R1 and R2, which may be the same or different, represent a saturated or unsaturated acyl radical containing from about 6 to 28 carbon atoms or from about 7 to about 25, e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16. 17, 18, 19, 20, 21, 22, 23 or 24 C atoms, for example for a saturated acyl radical having 16 C atoms,

R3 is a saturated or unsaturated acyl radical containing from about 6 to 28 carbon atoms or from about 7 to about 25, e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16. 17, 18, 19, 20, 21, 22, 23 or 24 C atoms, for example for a saturated acyl radical having 16 C atoms, or H and R4 is a from 1 to 25 amino acid residues, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16. 17, 18, 19, 20, 21, 22, 23 or 24 amino acid residues, physiologically acceptable and in the treated species not per se immunogenic peptide, for example with the amino acid sequence GDPKHPKSF (SEQ ID NO: 1). The term

"Treated species" refers to the species that belongs to the subject being treated or In this context, the use of other known adjuvants, such as squalene or alumina, is not excluded.

The induction of the desired immunological reaction is fundamentally dependent on the simultaneous administration of all three components, which however should be coupled to the same or different carrier particles depending on the situation and antigen (embodiment D). In the latter case, the vaccine of the invention consists of a plurality of micro / nanoparticles.

For simultaneous loading of a carrier particle with multiple vaccine components, each component can be bound to the polarized particle surface via its own charge tag (Figure 4).

In some embodiments, for example, where peptidic vaccine components are present, two or more vaccine components are linked by covalent linkage to conjugates. Examples of such conjugates are the following general structures (II), (III) or (IV).

Lipopeptide O R5 tag R6

(Ii)

T-cell epitope,

In these structural formulas, n is in each case a natural number from 0 to 8, for example 1, 2, 3, 4, 5, 6 or 7. R 1, R 2, R 3, R 4, R 5 and R 6 are molecular units which may be identical or different can. Typically, R1, R2, R3, R4, R5 and R6 are independently selected peptide regions having amino acid sequences that are from 0 to about 8 amino acids in length, such as 1, 2, 3, 4, 5, 6 or 7 amino acids. Any such peptide region can serve as a molecular spacer and accordingly can be referred to as an amino acid spacer. T cell epitope and T cell epitope ₂ represent an amino acid sequence of an epitope for cytotoxic effector T cells or an epitope for helper T cells. T cell epitope and T cell epitope ₂ may be the same or different.

An "epitope" is antigenic and thus may be understood to be defining as an "antigenic structure" or an "antigenic determinant." Immunoglobulins and T cell receptors can recognize and bind an epitope, and an epitope is also a region of an antigen to which An epitope is a region of a molecule that may be linear, ie, an amino acid sequence, for example, in a linearly contiguous form, that is derived from, for example, an immunoglobulin, a T cell receptor, or a binding reagent such as a lipocalin mutein Imunglobulin or T cell receptor recognized epitope. A linear epitope generally contains at least three, and typically at least about 5, eg, 8-10 amino acids. An epitope may also be a conformationally-dependent epitope in which, unlike a linear epitope, the primary amino acid sequence is not the only component of the recognized epitope, for example because it can not be recognized as such by an immunoglobulin or T cell receptor. An immunoglobulin, a T-cell receptor, or a binding reagent such as a lipocalin mutein recognize a three-dimensional structure of the antigen in a conformationally-dependent epitope. A conformationally-dependent epitope typically contains a greater number of amino acids than a linear epitope. A conformation-dependent epitope can be determined, for example, by X-ray crystallography, two-dimensional NMR spectroscopy, site-directed spin labeling, and electron paramagnetic resonance spectroscopy.

In the structural formulas (II) to (IV), lipopeptide stands for an immunomodulator according to formula (I), day stands for a sequence of 4 to 12, e.g. 4-10, including about 5, about 6, about 7, about 8, or about 9 basic (positive charge) or acid (negative charge) amino acids.

Such a conjugate is then immobilized on the polarized carrier particle over the charge tag, as shown in FIG.

For which vaccines such or similar conjugates are advantageous, depends on the medical indication as well as the contained vaccine components and antigen determinants. In the investigations it could be shown that the spatial proximity of certain epitopes in some cases has an advantageous effect on the immunization success, in other cases has no influence.

Like the components of the micro- and / or nanoparticle vaccine and its molar ratios, the form of binding to the carrier particle (charge tags, single / mixed loading) and the type of conjugation must also be specifically adjusted.

On the basis of these investigations, a surprisingly effective system for topical vaccination could be found and it could be shown that the vaccine components after application on the skin surface by means of micro / nanoscale carrier particles in an active transport process and using a biomimetic principle, in the hair follicle and Thus, they reach the immediate vicinity of the immunocompetent cells of the skin where, after diffusion to the target structures, the DCs of the skin, they trigger the desired specific T cell immune response.

This is all the more surprising given that all previous topical vaccination strategies assume, in part, a need for direct penetration of the active ingredients through the skin even explicitly exclude the transport through the hair follicles as a possible way to overcome the skin barrier.

Furthermore, it could be surprisingly found that the immediate presence of the lipopeptide immunomodulator in close proximity to the other vaccine components, a significant increase in the T cell-based specific immune response could be achieved.

The present invention therefore relates to a micro- and / or nanoparticle vaccine which is suitable for prophylactic and / or therapeutic use. The vaccine consists of at least the basic components

(i) one or more antigenic determinants of cytotoxic effector T cells

(CTL epitopes, polyepitopes, biopolymers, proteins, DNA, RNA, polysaccharides,

Glycolipids, glycoproteins or similar carriers of antigenic information),

(ii) one or more antigenic determinants of helper T cells

Pan-DR epitopes as degenerate peptides for all HLA-DR allotypes, and

(iii) one or more immunomodulators for activating the antigen-presenting

DCs, preferably synthetic analogs of bacterial lipopeptides,

as individual molecules or conjugates,

at them

(a) the vaccine components are reversibly coupled by a charge tag to micro / nano-scale carrier particles,

(b) after topical application to the skin surface active in the hair follicle

transported and temporarily deposited there, in which

(C) the vaccine components are selectively released under the physiological conditions prevailing there and then

(d) by diffusion to the target structures, the DCs in the epidermis of the skin, (e) where they trigger a T cell-based vaccine antigen-specific immune response, and their use for various medical indications, such as for prophylactic specific immunization against and / or for therapeutic vaccination in diseases such as infections, cancer, autoimmune diseases and / or inflammatory and / or allergic conditions, as well as for enhancing the immune response after vaccination in human and veterinary medicine carried out by conventional vaccination methods.

The immunological potential of the micro- and / or nanoparticle vaccines according to the invention was determined by means of in vitro experiments with model vaccines containing the CTL epitopes GILGFVFTL (SEQ ID NO: 5) from the influenza matrix protein M1 or NLVPMVATV (SEQ ID NO: 6 ) out the cytomegalovirus CMV early antigen pp65 containing helper T-cell epitope Pan-DR-E (aK-L-Cha-VAAWTLKAAa-Aca-C) and Pam ₂ Cys-GDPKHPKSF as immunomodulator (Examples C and D) ).

Specifically, it could be shown in the continuing experiments that

(i) a defined adjustable loading of the carrier particles and a loading, under loading,

Storage and transport conditions stable binding of the vaccine components to the polarized surface of the particles is possible;

(ii) reliable active transport of the vaccine components into the hair follicle bis

to about 600 pm depth takes place;

(iii) the carrier particles are deposited in the hair follicles for about 5 to 10 days and by a

natural purification process are excreted again;

(iv) a uniform and simultaneous release of the vaccine components occurs at the desired depth of the follicular structure;

(v) the released vaccine components to the DCs of the skin (Langerhans cells)

diffuse and be absorbed by them;

(vi) the released vaccine components remain fully immunological

are active;

(vii) the particle-bound vaccine components of the model vaccines in human effector T cells elicit a specific T cell mediated immune response that is qualitatively and quantitatively comparable to the response to the unbound vaccine components and also the charge tags have no deleterious effect on the have immunological activity of the vaccine components;

(viii) the lipopeptides lead to the activation of the antigen-presenting DCs, resulting in a

Increase in the T cell-based specific immune response and a marked increase in the vaccination effect;

(ix) the helper T cell epitope Pan-DR-E additionally stimulates the T cells and, via the T cells, also the DCs;

(x) the advantage of the proximity of the vaccine components, specifically their conjugation with the lipopeptide, is individually dependent on the antigenic determinants used for CTL cells;

(xi) the combination of helper T-cell epitope Pan-DR-E and lipopeptide immunomodulator enhances or even makes possible the proliferation and differentiation of naive T cells.

Toxic effects or incompatibilities of the micro- and / or nanoparticle vaccines according to the invention were not observed in the previous in vitro and in vivo tests in the relevant concentration ranges, as well as in cytotoxicity tests on RKO cells. Another object of the invention is the preparation of the vaccine components according to the invention and their modification with charge tags (see above), as illustrated in Example A.

The synthesis of the epitopes, as well as the charge tags, in some embodiments consisting of a series of 4-10 basic (positive charge) or acid (negative charge) amino acids, can be carried out in solution or solid phase synthesis according to methods of protecting group chemistry known to those skilled in the art , The charge tags attached N-terminal or C-terminal, in some embodiments C-terminal, are already considered in the construction of the amino acid sequence.

As already explained in the foregoing, for example, lysine, pyrrolysine, histidine and / or arginine can be used as basic amino acids. A payday may consist of a sequence of identical amino acid units or of a sequence of different amino acid units. Typically, a charge tag contains or consists of negatively charged but not positively charged amino acid units or positively charged but not negatively charged amino acid units. In addition to charged amino acid units, in some embodiments a charge tag may also have uncharged amino acid units. Usually, however, one charge day contains more charged than uncharged amino acid units. In some embodiments, a vaccine or a nanoparticle or microparticle of the invention contains a charge tag that is a series of four, five, six, or seven lysine units, a series of four, five, six, or seven pyrrolysine units, a series of four , has five, six or seven histidine units and / or a sequence of four, five, six or seven arginine units or consists thereof. In some embodiments, a vaccine or a nanoparticle or microparticle of the invention contains a payday that is a series of four, five, six or seven sulfotyrosine units, a series of four, five, six or seven aspartate units and / or a sequence of four, five, six or seven glutamate units or consists thereof.

For the preparation of the synthetic analogues of bacterial lipopeptides of formula (I), typically the unusual amino acid Na-fluorenylmethoxycarbonyl-S- [2,3-di (hydroxy) - (2RS) -propyl] - [f?] Cysteine ( Fmoc-RS-Dhc-OH) in solution or on solid phase with the side chain protected peptide synthesized on a synthetic resin. This precursor can subsequently be bisacylated on the hydroxy groups. In the synthesis of the Pam ₃ Cys derivative additionally takes place the acylation of the N-terminal amino group.

If the synthesis of the lipopeptides according to the invention with the building block Noc-fluorenylmeth- oxycarbonyl-S- [2,3-di (hydroxy) - (2) -propyl] - [R] -cysteine (Fmoc-f? F? -Dhc-OH ) carried out, the target compound is obtained as a stereochemically defined derivative consisting of L-amino acids, of the modified amino acid S- [2,3-di (hydroxy) - (2f-J-propyl] - [/!] - cysteine and of two or three Acyl residues is constructed and has a higher biological activity.

A vaccine component according to the invention can also be modified as a so-called "prodrug compound", for example as prodrug ester, prodrug peptides or the like A prodrug compound is understood as meaning a molecule which in some cases is itself pharmacologically inactive, but in In other cases, a prodrug compound is less active than a pharmacologically (fully) active agent formed therefrom in the body of the subject, thus allowing a prodrug compound to function as a precursor molecule A prodrug compound may contain residues that are cleaved from the molecule in vivo, for example, by an enzymatically catalyzed reaction such as hydrolysis or oxidation, Thus, a hydroxy group present in a pharmacologically active compound may be present in a prodrug. Verbindun g may be in the form of an ester, an amine may be in the form of an amide, or a carboxyl group may be in the form of an ester or an amide. A carboxyl group present in a pharmacologically active compound can also be present as a hydroxy group which can be oxidized in vivo. In certain cases, coupling of cell-penetration promoting molecules, e.g. Biotin or maleimidopropionic acid, optionally via suitable spacer molecules, to the primary amino group or the acylation of the amino group, the bioavailability and thus the effectiveness of the compounds of the invention can be improved.

Another object of the invention is the use of a micro- and / or nanoparticle vaccine according to the invention for the preparation of a pharmaceutical preparation for humans and / or animals for a medical application. One such application may be the prophylactic specific immunization against and / or for therapeutic vaccination in a disease such as an infection, cancer, autoimmune disease, or an inflammatory and / or allergic condition, as well as for the support of a conventional vaccine.

Such an immunostimulatory active composition containing a therapeutically effective amount of at least one microparticle and / or nanoparticle vaccine according to the invention may further comprise one or more immunological-action-enhancing adjuvants, pharmaceutically suitable excipients, auxiliaries and / or additives, such as, for example, detergents, Solubilizers, stabilizers or preservatives, as well as otherwise otherwise treatment-relevant agents or skin care products. Pharmaceutically acceptable materials are the substances known to be useful in the pharmaceutical and food technology industries and in adjacent fields, in particular those listed in the relevant pharmacopoeias whose properties do not preclude physiological application.

The effects produced by a micro- / nanoparticle vaccine according to the invention are also dependent on its formulation. A corresponding pharmaceutical preparation should allow the direct application of the drug to the untreated skin. For topical application suitable formulations for the micro- and / or nanoparticle vaccines according to the invention, for example, pastes, ointments, creams, lotions, emulsions, gels, hydrogels, suspensions, solutions, sprays or patches, or for example aqueous or ethanolic gels and suspensions or oil Water emulsions.

Because of the long-term storage (depot effect) of a typical microparticle nanoparticle vaccine according to the invention in the hair follicle, a sustained-release formulation is also suitable which can be prepared by methods of pharmaceutical galenics known to the person skilled in the art.

The preparation of a pharmaceutical preparation according to the invention is carried out in the usual way by methods known to those skilled in the art, as described in the relevant pharmacopoeias, wherein the components can be mixed together, dissolved or associated with a physical or biological carrier. A pharmaceutical preparation according to the invention can additionally be sterilized.

The invention also relates to the use of a micro- and / or nanoparticle vaccine according to the invention or a pharmaceutical preparation containing it for the treatment and prevention of one or more human and animal diseases associated with infections, cancer, autoimmune diseases or inflammatory and allergic conditions stand.

For this purpose, a subject, that is to say a person or an animal requiring such a treatment, has a therapeutically effective amount of a microparticle and / or nanoparticle vaccine according to the invention or a pharmaceutical preparation according to the invention applied directly to the untreated skin. Subsequently, the formulation can be massaged in with a finger or optionally with a massage applicator. For exact dosage, additional templates can be used for the size of the treated area of the skin. The order quantity can be specified by the packaging unit.

By pre-treatment of the skin area to be treated by stripping or a cyanoacrylate tear, in the already dead skin cells, dead corneocytes and dried sebum are removed from the opening of the hair follicles, the efficiency of uptake of the micro- and / or nanoparticle vaccine may optionally be increased. Occlusal volumes or patches are other ways to make the drug application.

The micro- / nanoparticle vaccines and pharmaceutical preparations according to the invention can be used alone, in combination with other micro / nanoparticle vaccines or pharmaceutical preparations contained in the present invention or in combination with one or more active substances which are suitable for the respective medical indication are relevant. The temporal sequence of the application is not restricted. It may be administered either at the same time, before or after the other active substances, as a separate medicinal product or combination preparation and on the same or different routes of administration. The application of different vaccines or parts thereof at different locations and at different times corresponds to the subject matter of the invention.

The exact therapeutically effective amount for a subject to be treated, as well as the molar composition of the vaccine or vaccine combination, will depend on various factors, including the nature and course of the disease, size, stature, age and health of the patient, the route of administration, and the specifics used Antigen determinants and, if appropriate, of the other medicaments used. Thus it does not make sense to specify the exact quantity at this time. However, one can in principle start from a 1 to 3-fold application of the pharmaceutical preparations according to the invention over a period of a few weeks to a half year. The amount of active ingredient used should usually be in the range of 0.5 pg to 2.0 mg per application and vaccine component. The exact dosage specification depends on the specific application and composition of the micro- and / or nanoparticle vaccine and requires further investigation in the clinical trials.

The main advantages of a vaccine and a method or an application of the present invention in which this is used, consist in the non-invasive application, the direct transport of the vaccine components through the hair follicle to the target structures and the amplification of the immune reaction by the lipopeptide Immunomodulators.

With the DCs of the skin an ideal target structure is addressed.

By incorporating the synthetic analogues of bacterial lipopeptides as modulators of the innate immune system, a significant increase in the effectiveness of immunization is possible. This opens up the possibility of enhancing immunization success of conventionally administered vaccines by the additional application of a micro- and / or nanoparticle vaccine.

The topical application form of a microparticle and / or nanoparticle vaccine according to the invention is simple and flexible to handle and, as such, independent of medical personnel. The side effects and risks associated with parenteral administration, e.g. Secondary infections can be avoided, with enteral administration usual metabolism can be avoided.

The restriction to a minimum and smallest amounts of vaccine components reduces the potential risk of incompatibilities with nevertheless good immunostimulatory effect. Negative influences were not observed in the previous in vitro and in vivo models.

Thus, the topical vaccination with a micro- and / or

Nanoparticle vaccine superior to the previously dominant systemic and invasive route of administration.

A micro- and / or nanoparticle vaccine according to the invention is on the one hand suitable for the prevention of infectious diseases. Due to the unspecific approach of the vaccination strategy according to the invention, a wide range of known infectious diseases can be covered by the selection and combination of T cell epitopes and antigens for B cells.

Areas of use are primarily vaccination campaigns against widespread infectious diseases, pandemics and vaccinations, especially in pediatrics. Also, the therapeutic treatment and prevention of diseases such as infectious diseases, cancer, autoimmune diseases, chronic inflammation and allergies are promising fields of application for the illustrated principle of topical vaccination.

Last but not least, a micro- and / or nanoparticle vaccine according to the invention has decisive advantages in the self-medication of chronically ill patients and in the immunization of vaccine intolerances. Likewise, topical vaccination is extremely beneficial for the vaccination of children as it saves the children the painful experience associated with the injection.

In addition, the non-invasive principle of the micro- and / or nanoparticle vaccine significantly reduces the risk of infection in vaccines and avoids significant amounts of clinical waste. With a micro- and / or nanoparticle vaccine according to the invention, a new dimension of vaccine development is opened, which allows a rational vaccine design based on the known immunological knowledge and the specific selection of the epitopes.

The principle underlying the invention for overcoming the skin barrier is also open to further development as a general principle for topically applicable drug delivery systems.

Details of individual embodiments and further features and advantages of the invention will become apparent from the following description of embodiments in conjunction with the subclaims, wherein the above-mentioned and below to be explained individual features are claimed individually and in any combination with each other. The following embodiments are purely illustrative and do not limit the scope of the invention.

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embodiments

Example A - Preparation and Modification of the Vaccine Components

In this embodiment, the general method for preparing the vaccine components according to the invention, in particular the peptidic epitopes and the synthetic analogs of bacterial lipopeptides, as well as their modification with the peptidic charge tags with the methods of solid phase synthesis will be described. Structural modifications can be made by skipping individual synthetic steps or adding steps known to the skilled synthetic chemist. a) Synthesis of the Amino Acid Sequences with Automated Solid-Phase Synthesis The synthesis of the peptides proceeds according to classical techniques known from the literature in solution or in an automated process on the solid phase. The solid phase synthesis procedures are the same for almost all amino acid sequences. For this reason, in the example, only the basic synthesis procedure is described, without going into the peculiarities of individual peptides.

The peptides are synthesized using a synthesis robot starting from a Fmoc-L-Lys (Boc) -loaded synthesis resin according to the Fmoc peptide synthesis method. If other amino acids are used for the C-terminal charge tag, one starts in an analogous manner with the corresponding Fmoc-protected derivatives.

For this purpose, the loaded trityl chloride-polystyrene resin (8 mg, 7 pmol) is weighed into a reactor. The amino acids are dissolved in a 1-hydroxybenzotriazole solution (0.5 M in N, N-dimethylformamide) to 0.5 molar solutions. Subsequently, the filled reactors and vials are placed on the intended positions of the synthesis robot. In the first step of the synthesis, the cleavage of the Fmoc protective group takes place according to the program sequence shown in Tab. Tab. 1: Cycle for cleavage of the Fmoc protective group.

Synthesis step Reagents Volumes [μί] Duration [min]

Start resin swelling N, N-dimethylformamide 800 2

1. Deblocking 30% piperidine in 200 5

N, N-dimethylformamide

2. Washing, 3 x N, N-dimethylformamide 300 1

3. Deblocking 30% piperidine in 200 10

N, N-dimethylformamide

4. Washing, 7 x N, N-dimethylformamide 300 1 Subsequently, the corresponding peptide is built starting from the C-terminal end. This is done by the stepwise linking of the respective amino acids. Immediately after each coupling, the Fmoc protecting group of the just-coupled amino acid is cleaved to allow attachment of the next amino acid to the liberated amino group.

The program sequence of the synthesizer robot is shown in the following table.

Tab. 2: Cycle for the synthesis of the peptides.

Synthesis step Reagents Volumes [μΙ_] Duration [min]

1. Coupling 3 M N.N-Diisopropylethylamine 25 120

3 M N, N-diisopropylcarbodiimide 25

in N, N-dimethylformamide,

Amino Acid Solution 100

2. Washing, 3 x N, N-dimethylformamide 300 1

3. Deblocking 30% piperidine in 200 5

N, N-dimethylformamide

4. Wash, 3 x N, N-dimethylformamide 300 1

5. Deblocking 30% piperidine in 200 10

N, N-dimethylformamide

6. Washing, 7x N, N-dimethylformamide 300 1

7. Continue with step 1. b) Synthesis of N, N-bis (fluorenylmethoxycarbonyl) - [7] -cysteine-bis-uf-butyl ester (Fmoc-Cys-OtBu) ₂ as building block for the lipopeptide component

L-Cysteine-bis-tert-butyl ester (5.5 g, 13 mmol) and Fmoc-N-hydroxysuccinimidyl ester (7.04 g, 21 mmol) are dissolved in tetrahydrofuran (15 mL). With stirring, N-ethylmorpholine (3.83 g, 4.2 mL, 30 mmol) in tetrahydrofuran (7.5 mL) is added.

The reaction solution is stirred for 3 h at room temperature and then evaporated to dryness using the rotary evaporator.

The residue is dissolved in ethyl acetate (100 ml) and washed in a separating funnel with potassium hydrogen sulfate solution (5%, 3 × 80 ml) and water (3 × 80 ml). After drying with sodium sulfate, the organic phase is evaporated on a rotary evaporator and the residue obtained an oil.

This residue is dissolved in dichloromethane / methanol (1: 4, 385 mL) using an ultrasonic bath and recrystallized (18 h, -20 ° C.). The resulting colorless precipitate is filtered through a suction filter, washed with tert-butyl alcohol / 2-propanol (1: 1, 2 x 100 mL) and dried in vacuo. Yield (Fmoc-Cys-OtBu) ₂ : 7.48 g (72%); [M + H] ⁺ : 797 c) Synthesis of Na-fluorenylmethoxycarbonyl-S- [2,3-di (hydroxy) - (2? S) -propyl] - [? J-cysteine-te.i.-butyl ester ( Fmoc-RS-Dhc-OtBu): introduction of Dhc

The (Fmoc-Cys-OtBu) ₂ (4.96 g, 6.2 mmol) is dissolved in dichloromethane (39 mL). Then, zinc (2.84 g, 43.4 mmol) and a solution of methanol, hydrochloric acid (32%) and sulfuric acid (98%) (100: 7: 1, 20.7 mL) are added with vigorous stirring. After 15 min reaction time at room temperature, f-s-2,3-epoxy-1-propanol (45.9 g, 41.3 mL, 62 mmol) is added to the reaction mixture. The whole is further stirred in an oil bath (5 h, 40 ° C). The solution is then evaporated to half the volume on a rotary evaporator, mixed with potassium hydrogen sulfate solution (5%, 5.2 mL) and stored at -2 ° C. for 16 h. The product is extracted with dichloromethane (3x50 mL). After drying with sodium sulfate, the organic phase is evaporated on a rotary evaporator to dryness.

Yield (Fmoc-f? S-Dhc-OtBu): 5.67 g (96%); [M + H] ⁺ : 474

In the synthesis, as described above, the exceptional amino acid S- [2,3-di- (hydroxy) - (2S) -propyl] - [f?] Cysteine (Dhc) is the racemic building block? S-2, 3-epoxy-1-propanol used, to obtain a diastereomer mixture of the target compound. By using the enantiomer R-2,3-epoxy-1-propanol, the immunomodulatory, more active, fibrillar stereoisomer of Dhc is obtained in an analogous manner. d) Synthesis of Na-fluorenylmethoxycarbonyl-S- [2,3-di (hydroxy) - (2 /? S) -propyl] - [?] cysteine (Fmoc - /? S-Dhc-OH): hydrolysis of the fert .-butyl ester

Fmoc-? S-Dhc-OtBu (5.67 g, 12 mmol) is dissolved in trifluoroacetic acid (99%, 142 mL) and the reaction is stirred at room temperature for 1 h. The solution is then evaporated on a rotary evaporator, treated with N-hexane (50 mL) and diethyl ether (30 mL) and evaporated again on a rotary evaporator. The viscous, oily residue is then dissolved in tert-butyl alcohol / water (80%, 100 mL) and lyophilized (64 h).

Yield (Fmoc-f? S-Dhc-OH): 3.03 g (60%); [M + H] ⁺ : 418 e) Synthesis of Na-fluorenylmethoxycarbonyl-S- [2,3-di (hydroxy) - (2? S) -propyl] - [/?] Cysteinyl-GDPKHPKSF-KKKK: Coupling of the amino acid sequence

A side chain protected peptide GDPKHPKSF-KKKK (SEQ ID NO: 2) loaded synthesis resin (5.22 pmol) is added with N, N-dimethylformamide (100 pL) and allowed to swell in a reactor for 5 minutes. After filtering off the N, N-dimethylformamide supernatant, Ν, Ν-diisopropylethylamine (3M in N, N-dimethylformamide, 12.2 pL, 36.6 pmol, 7 equivalents), Na-fluorenylmethoxycarbonyl-S- [2, 3-di (hydroxy) - (2f S?) propyl] - [ft] -cysteine (15.5 mg in 100 μM of a 0.5 M 1-hydroxybenzotriazole solution in N, N-dimethylformamide, 36.6 μΐΎΐοΙ, 7 equivalents) and N, N-diisopropylcarbodiimide (3 M in N, N-dimethylformamide; 12.2 μΙ_; 36.6 pmol; 7 equivalents) were pipetted to the resin. The resin suspension is then incubated for 2 h at 37 ° C in a heating block. Thereafter, the resin is washed with N, N-dimethylformamide (3χ600 μί) and dichloromethane (3χ600 μΙ_). f) Esterification of Na-fluorenylmethoxycarbonyl-S- [2,3-di (hydroxy) - (2 /? S) -propyl] - [?] cysteinyl-GDPKHPKSF-KKKK with palmitic acid

The resin (5.22 pmol) loaded with Na-fluorenylmethoxycarbonyl-S- [2,3-di (hydroxy) - (2f-S) -propyl] - [^] cysteinyl-GDPKHPKSF-KKKK is treated with N, N-dimethylformamide ( 100 pL) and allowed to swell for 5 min in a reactor. After filtering off the N, N-dimethylformamide supernatant, Ν, Ν-diisopropylethylamine (3 M in N, N-dimethylformamide, 24.4 pL, 73.2 pmol, 14 equivalents), palmitic acid (1 M in N, N-dimethylformamide / dichloromethane (1: 1); 104 μΙ_; 104 μιτιοΙ; 20 equivalents) and N, N-diisopropylcarbodiimide (3 M in N, N-dimethylformamide; 24.4 L; 73.2 μmol; 14 equivalents) are pipetted to the resin. After 5 min, add 4-dimethylaminopyridine (1M in Ν, Ν-dimethylformamide; 30 [iL; 30 pmol; 5.7 equivalents). The resin suspension is then incubated for 2 h at 37 ° C in a heating block. Thereafter, the resin is washed with N.N-dimethylformamide (3χ600 μΙ_) and dichloromethane (3x600 [iL).

The esterification is repeated once more under the same experimental conditions. g) Cleavage of the Fmoc and the side chain protecting groups

The resin (5.22 μπιοΙ) loaded with Na-fluorenylmethoxycarbonyl-S- [2,3-bis (palmitoyloxy) - (2RS) -propyl] - [f?] Cysteinyl-GDPKHPKSF-KKKK is treated with dichloromethane (100 μl ) and allowed to swell for 5 minutes in a reactor. After filtering off the dichloromethane supernatant, piperidine (30% in N, N-dimethylformamide, 200 pL) is pipetted to the resin and the mixture is left at room temperature for 10 minutes. Thereafter, the resin is washed with Ν, Ν-dimethylformamide (3 x 600 [iL). Subsequently, piperidine (30% in N, N-dimethylformamide, 200 μl) is pipetted into the resin and the suspension is left at room temperature for 20 minutes. Thereafter, the resin is washed with N.N-dimethylformamide (3 × 600 μΙ_), dichloromethane (3 × 600 μl) and diethyl ether (4 × 600 μl).

The completeness of the reaction is controlled by the ninhydrin test.

In an analogous manner, the cleavage of the Fmoc and the side-chain protective groups is carried out by the amino acid sequences of the epitope peptides. h) Cleavage of the lipopeptide S- [2,3-bis (palmitoyloxy) - (2S) -propyl] - [?] - cysteinyl-GDPKHPKSF-KKKK or the epitope peptides from the synthesis resin

The cleavage solution trifluoroacetic acid / triisopropylsilane / water (92.5: 5: 2.5, 300 L) is pipetted to the resin (2.6 pmol) and the suspension left at room temperature for 2 h. Subsequently, the cleavage solution is filtered with compressed air into a test tube. Subsequently, another 200 μΙ_ cleavage solution is pipetted to the resin and the suspension is left for another 30 minutes at room temperature. After re-filtration of the cleavage solution with compressed air, the resin is washed with dichloromethane (200 μL). The filtrates and the washings are combined and evaporated on a rotary evaporator. The residue is digested with diethyl ether (3 χ 300 μΙ_) and then centrifuged. The sediment is taken up in tert-butyl alcohol / water (4: 1, 2 mL) and freeze-dried (18 h).

Yield (Pam ₂ - (2RS) -Dc-GDPKHPKSF-KKKK) [(SEQ ID NO: 3)]: 5.5 mg (97%);

M: 2179; [M + 2H] ²⁺ : 1091; [M + 3H] ³⁺ : 727 i) Acylation of S- [2,3-bis (palmitoyloxy) - (2? S) -propyl] - [? L-cysteinyl-GDPKHPKSF-KKKK with palmitic acid

The resin (2.6 pmol) loaded with S- [2,3-bis (palmitoyloxy) - (2RS) -propyl] - [/ -] cysteinyl-GDPKHPKSF-KKKK (SEQ ID NO: 3) is labeled with N, N-dimethylformamide (100 μΙ_) and allowed to swell for 5 min in a reactor. After the NN-dimethylformamide supernatant has been filtered off, Ν, Ν-diisopropylethylamine (3M in N, N-dimethylformamide, 12.2 μΐ, 36.6 μητιοΙ, 7 equivalents), palmitic acid (1M in N, N-dimethylformamide / dichloromethane (1: 1); 1), 52 pL, 52 μηηοΙ, 10 equivanylene) and N, N-diisopropylcarbodiimide (3 M in N, N-dimethylformamide; 12.2 [iL; 36.6 μπιοΙ; 7 equivalents) were pipetted to the resin , The resin suspension is then incubated for 2 h at 37 ° C in a heating block. Thereafter, the resin is washed with N.N-dimethylformamide (3 × 600 μΙ_) and dichloromethane (3 × 600 μL).

The cleavage of the product from the resin is carried out according to the method described under h).

Yield (Pam ₃ - (2RS) -Dc-GDPKHPKSF-KKKK) [(SEQ ID NO: 4)]: 5.6 mg (89%);

M: 2417; [M + 2H] ²⁺ : 1210; [M + 3H] ³⁺ : 807

Example B - Reversible immobilization of the vaccine components on the carrier particles

1 000 nm diameter silica particles were coated with the layer sequence PAH / PSS / PAH / PSS / PAH / PMAA using LBL® technology and brought to pH 8 after repeated washing with 10 mM TRIS buffer. Then a sequential loading with 20% DMSO solutions of the different vaccine components was carried out, wherein the adsorbed amount per component was defined by the order and amount of added vaccine components and the incubation time.

To calibrate the loading amount, the vaccine components were labeled with different fluorescent dyes and the decrease in color in the supernatant of the loading solutions was monitored by UV / vis or fluorescence spectroscopy. The results are shown in the following table and Fig. 2.

Tab. 3: Percentage of the adsorbed at pH 8 on the carrier particles and desorbed at pH 5 mass of the vaccine components based on the mass of the carrier particles. (GILG = GILGFVFTL-KKKKK fluorescein, SEQ ID NO: 11 coupled to the fluorescent dye fluorescein, P2C = Pam ₂ Cys-GDPKHPKSF-KKKKK-Tamra, SEQ ID NO: 9 coupled to the fluorescent dye Tamra, Cha = aK -L- Cha-VAAWTLKAAa-Aca-C-KKKKK-Cy5, SEQ ID NO: 10 coupled to the fluorescent dye Cy5). amount adsorbed on particles GILG [%] P2C [%] Cha [%]

Addition GILG, 30 min 2.0 - -

Addition GILG, 120 min 2.09 - -

Addition P2C, 30 min 17.6 16.6 -

Addition P2C, 120 min 16.7 15.3 -

Add Cha, 30 min 12.4 12.0 6.3

Addition Cha, 120 min 8.3 9.5 8.4

Desorption in 10 mM acetate 3.2 4.4 2.5

Desorption in 10 mM acetate + 0.1 M NaCl 2.2 3.6 1.6 1

Desorption in 10 mM Acetate + 20% DMSO 1, 9 6.1 · 3.2 With the loaded carrier particles, release experiments were carried out in aqueous solution, whereby different release media were simulated. On the one hand, the vaccine components were released in a 10 mM aqueous acetate buffer solution at pH 5. To simulate potential solubilizers in the hair follicles, such as lipids or salts, NaCl or 20% DMSO was also added to the solution.

The results in Tab. 3 show that the degree of release and thus the ratio of the released vaccine components are highly dependent on the composition of the release medium and the composition of the micro / nanoparticle vaccine.

Fluorescence spectrometric studies on the kinetics of the release of the three vaccine components in 1% suspensions of the loaded carrier particles in 10 mM aqueous acetate buffer solution with addition of 0.1 M NaCl at pH 5, show the different release kinetics of the vaccine components (FIG. 3). Within 20 min, however, were all three components released to more than 50% of the maximum value. 80% of the maximum value of the release was reached after about 1 h.

For the biological tests, the unlabeled vaccine components were adsorbed on the surface of the carrier particles in analogy to the respectively optimized loading strategy.

To prepare ready-to-use samples for application, the particles were incorporated at pH 8 (10 mM TRIS buffer) in aqueous agarose gel 0.5%. After adding a preservative (ethyl paraben, benzalkonium chloride, ethyl hydrobenzoate), the finished and storage-stable formulations were stored until use.

Example C - Transport of the micro / nanoparticle vaccine into the hair follicles and to the

targets

In this embodiment, the effectiveness of transport of the vaccine components to the target structures will be described depending on the nature of the carrier particles. a) Control of the penetration depth over the particle size

The investigation of the dependence of the depth of penetration achieved in the hair follicle on the size and the material of the particles was carried out in vitro on the model tissue pig ear, which is very similar both in structure and with respect to the physiological properties of the human skin. The determination of the depth of penetration was carried out after topical application of fluorescently labeled carrier particles, removal of punch biopsies and evaluation of the prepared cryosections by means of laser scanning microscopy.

It could be shown that the penetration depth of the particles is controllable by the particle size, wherein the nature of the particles plays almost no role. While particles with a diameter of approx. 500 nm to 600 nm reach penetration depths of> 1000 pm, smaller and larger particles penetrate much less deeply (FIG. 7). Explanation for this phenomenon is a mainly mechanical effect. Since the thickness of the hair shells is also about 500 nm, a gear pump effect can be assumed, which is initiated by the movement of the hair in the hair follicle. This is the more pronounced, the more similar particle size and Haarschuppendicke are. In vitro, this effect can be simulated by massage.

The target structures of the topical vaccine, the DCs, are particularly abundant in the infundibulum area of the hair follicles. This is located at a depth of up to 600 pm and is reached by the vaccine components already after 1 h. Within In the next 24 h a minimal further diffusion takes place, which is of little importance for the vaccination.

For the preparation of a topical vaccine for human use, therefore, spherical silica particles with a diameter of 300 nm to 1,500 nm have proven to be particularly suitable carrier particles. b) Long-term storage of the carrier particles in the hair follicle

To determine the in vivo residence time of the carrier particles in human skin, fluorescence-labeled particles were applied to the calf skin of volunteers. Subsequently, the amount of particles deposited in the stratum corneum or in the hair follicles was recorded by means of differential stripping (Teichmann et al.2005). Almost after one day, almost no particles were detectable in the upper layers of the skin (stratum corneum), while about 80% of the initial amount could be recovered in the hair follicle. Even after 10 days, about 20% of the particles were still detectable in the hair follicle (FIG. 8). c) Kinetics of release

Release kinetics were studied on the pig ear model with Cy5-labeled silica particles loaded with rhodamine-labeled vaccine components. Cryosections were made again and the penetration depths of the two fluorescent dyes were examined by laser scanning microscopy.

On the basis of the different penetration depths it could be shown that the vaccine components are released in the hair follicle and penetrate significantly deeper than the transporting particles (FIG. 6). d) Inclusion of the vaccine components in the Langerhans cells

For these studies, double-labeled micro- / nanoparticle vaccines were topically applied to human excised skin. On the basis of the fluorescence staining of about 80% of the examined Langerhans cells it could be proven that the vaccine components were taken up in the cell interior of the Langerhans cells. In contrast, uptake of the carrier particles by the Langerhans cells could not be observed. This, too, speaks for the effective release of the vaccine components in spatial proximity to the target structures. Example D - Immunological Activity of the Micro- / Nanoparticle Vaccines and Their

components

The immunological activity of a model vaccine with the components:

(i) GILGFVFTL (SEQ ID NO: 5) from the influenza matrix protein M1 (GILG) or

NLVPMVATV (SEQ ID NO: 6) from the cytomegalovirus CMV early antigen pp65

(NLVP)

as models for an epitope for CD8 effector T cells,

(ii) Pan-DR epitope aK-L-Cha-VAAWTLKAAa-Aca-C (PADRE) [SEQ ID NO: 7], where "a" is D-alanine, "Cha" is cyclohexylalanine and "Aca" is 6 -Aminocaproic acid, as a model for an epitope for helper T cells as well

(iii) Pam ₂ Cys-GDPKHPKSF (P2C) [SEQ ID NO: 8]

as a model for an immunomodulator,

was studied in in vitro cell cultures of human cell systems. Depending on the specific question, established T cell clones, peripheral white blood cells (PBMC), subfractions of these PBMCs (monocytes, CD8 T cells, CD4 T cells, naive T cells, memory T cells) were obtained ) or differentiated cell types (immature dendritic cells (iDC), mature dendritic cells (mDC)) generated from these cells. The individual components were tested in free and particle-bound form as well as various combinations of these components on the carrier particles. a) Effect of modification of epitopes with oligoamino acid tags

In a radiochrome release assay, the model epitope GILG was titrated with different variants of C-terminal extension by histidine (H), lysine (K) or glutamate (E) over 12 orders of magnitude and the induction of cytolytic activity in the human cytotoxic T-cell clone 10-21A tested against the B-lymphoblastoid cell line JY. As can be seen from the results shown in Fig. 9, the modification of the C-terminus of the epitope is expected to reduce the stimulatory capacity. The epitope with the H ₄ -tag is 45-fold, that with K _t -tag 131-fold and that with E ₄ -tag 708-fold less potent than the unmodified epitope. This loss of activity is tolerable, at least for the H and the K / p-day, given the very high functional avidity of the epitope of 1.52 pM (peptide concentration required for half-maximal cytolysis). b) Immunological activity of particle-bound vaccine components

To compare the immunological activities of the micro- and / or nanoparticle vaccines with that of the free vaccine components, primary human T cells from peripheral blood of a healthy donor with the free components, mixtures thereof, were harvested individually or in combination with the carrier particles bound components and mixtures of loaded particles over 14 days incubated. All components were provided with an IC tag. The induction of vaccinate-specific CD8 T cells was quantitated in the ELISpot assay for IFN-γ.

The results obtained are representative of the success of prophylactic vaccinations because the T-cell immune responses are induced in the cell culture system without prior in vivo vaccination. It was shown that the particle-coupled epitope GILG is equally active in the cell culture system as the free peptide. That is, the carrier particles as well as the charge tags do not significantly affect the biological activity of the vaccine components (Figure 10). In the titration of mixtures of particle-bound and free vaccine components, a higher activity for the particle-bound vaccines over a mixture of the free components could be detected (Figure 11). c) induction of maturation and activation of iDCs by the immunomodulator

The vaccination effect, i. the stimulation of the adaptive immune system, and especially the effector T cells, depends on the successful induction of the differentiation of iDCs into mDCs and their activation into potent immunostimulatory cells. This process can be monitored by expression of costimulatory surface molecules on the DCs. CD80, CD86 and CD40, as well as the DC activation marker CD83, should be up-regulated.

This immunomodulatory function of the microparticle nanoparticle vaccine was studied with iDCs from the peripheral blood of volunteer donors. For this purpose, PBMC were incubated in plastic vessels and the adherent monocytes were cultured for 5 days in cell culture medium with IL-4 and GM-CSF, whereby they developed into non-adherent iDCs. These were then incubated for maturation and activation with differently loaded carrier particles. After two days, the expression of the costimulatory surface molecules and the activation marker was examined by flow cytometry.

These studies were performed in independent experiments with PBMC of 10 subjects to map the variations in human populations. The data demonstrate that the particle-coupled immunomodulator P2C, alone or in combination with other vaccine components, induces differentiation, maturation, and activation of iDCs (Figure 12 AC). The helper T cell epitope PADRE induces expression of the co-stimulatory molecules CD80 and CD86 and the DC activation marker CD83, but not MHC class II molecules (HLA-DR) and CD40, a costimulatory molecule on DCs required for the primary induction of cytotoxic effector T cells. Complete activation of the DCs, on the other hand, is achieved with P2C (FIG. 13). d) induction of T cells by DCs activated by micro / nanoparticle vaccines

DCs, which were differentiated and activated as described under c) with differently loaded carrier particles or which were not or only partially matured because of the lacking immunomodulator, were labeled with T cells of the same donor from whose PBMC the DCs were generated Incubated for 7 days. The T cells were then examined by flow cytometry for proliferation and differentiation. Separate detection of the CD45RA-expressing naive T cells and the CD45RO-expressing memory T cells were separated from PBMC on the basis of specific antibodies using magnetic separation and incubated separately with the DCs.

Proliferation was detected by CFSE assay. In this assay, the T cells are labeled with the cell-stable fluorescent dye CFSE before stimulation. For each cell division, the dye is diluted 1: 1 so that the rate of proliferation can be determined from the decrease in cell-associated fluorescence. The differentiation of naive to memory T cells could be detected with differentiation markers. In naive T cells (Figure 14), DCs preincubated with particles loaded with the P2C immunomodulator induce proliferation but hardly any differentiation, similar to an inflammatory cytokine cocktail (TNF + IL-4 + IL-6). , The helper T cell epitope PADRE has a similar but much weaker effect. In contrast, DCs pretreated with P2C and PADRE-bearing particles induce both high proliferation and differentiation of a high proportion of naive T cells, and thus exactly the immunological effect expected of a prophylactic vaccine.

For memory T cells (Figure 15), the DCs induce proliferation after incubation with the immunomodulator P2C or the helper T cell epitope PADRE, as well as with the cytokine cocktail. As expected, no qualitative differentiation can be observed here, but a clear enhancement of the effect when PADRE and P2C-bearing particles are combined. This effect reflects the situation of therapeutic vaccination or revaccination of immune individuals. e) Induction of antigen-specific T cell responses by the micro / nanoparticle vaccine

To study the induction of specific immune responses by the micro / nanoparticle vaccines, human peripheral blood iDCs were cultured for two days with carrier particles coated with the T-cell epitopes NLVPMVATV [SEQ ID NO: 6] (Figure 16) or GILGFVFTL [SEQ ID NO: 5] (Figure 17), the immunomodulator P2C, or combinations thereof, and then combined with CD8 T cells and incubated for 7 days. Thereafter, the proliferation of effector T cells with specificity for influenza or CMV was investigated by recirculating tetramerized HC-peptide complexes (HLA-A2.1 molecules carrying the respective peptides GILG and NLVP, respectively) by flow cytometry in the cell cultures. The iDCs and effector T cells were each generated from peripheral blood of the same healthy donor.

Against the CMV epitope, it was possible to induce weak T cell reactivities with epitope-alone particles. Particles simultaneously loaded with the epitope and immunomodulator produced a significantly greater response. This effect demonstrates that the CMV epitope and immunomodulator must be coupled for optimal efficacy.

In the example shown for the influenza epitope, however, the strongest immune stimulation was achieved with a mixture of particles carrying the epitope and the immunomodulator separately. So here is no coupling request. A mixture of the particles is more advantageous.

In any case, induction of antigen-specific T cells, as shown in the previous experiments, depends on the concomitant administration of the specific epitope and the immunomodulator, which, depending on the situation and antigen, should be coupled to the same particles or uncoupled. The few tetramer and CD8-positive cells that have already been detected in the T-cell cultures with or without iDCs show that the donors in these examples have long been in contact with the viruses from which the epitopes originate have had. The experiments carried out thus correspond to the situation of a therapeutic or Revakzinierung.

Example E - Toxicity of the model vaccine

In the cell culture, organ culture and in vivo investigations, the investigated concentration range from 1 f M to 500 μΜ for the model epitopes Pan-DR-E, GILGFVFTL (SEQ ID NO: 5) and NLVPMVATV (SEQ ID NO: 6 ) and for the lipopeptide adjuvants no evidence of a toxic effect of the micro- / nanoparticle vaccine according to the invention.

In studies on the cytotoxicity of the carrier particles on RKO cells, no influence on the survival rate was observed for 2% suspensions of differently coated carrier particles.

Also in the animal experiment in pigs no side effects could be observed after application of 2 mL of a 1% -igen, in Agarose gel formulated micro / Nanopartikel vaccine. Overall, it could be shown in the experimental studies that the carrier particles loaded with the vaccine components are effectively transported into the hair follicles and remain there for about 5 to 10 days, and that the active substances are specifically released only in the hair follicle, then to the DCs in the epidermis the skin diffuse and be absorbed by these. Furthermore, it has been shown that the particle-coupled vaccine components induce a comparable to the free drugs and partially more effective specific T-cell response, which is not affected by the peptidic charge tags.

Claims

claims

1. Micro- and / or nanoparticle vaccine comprising the basic components

(i) one or more antigenic determinants for cytotoxic effector T cells,

(ii) one or more antigenic determinant (s) for helper T cells and

(iii) one or more immunomodulator (s) for activating

antigen-presenting dendritic cells,

wherein the vaccine components are coupled to a micro- and / or nanoscale carrier particle.

2. micro- and / or nanoparticle vaccine according to claim 1, wherein the micro- and / or nanoscale carrier particle is designed so that it is transported to the skin surface after topical application to the hair follicles active and temporarily deposited there, so that the vaccine components induce T cell-mediated immunity via stimulation of the skin's dendritic cells.

3. micro- and / or nanoparticle vaccine according to claim 1 or 2, wherein the micro- and / or nanoscale carrier particle has a diameter in the range of 100 nm to 3000 nm.

4. micro- and / or nanoparticle vaccine according to claim 3, wherein the micro- and / or

nanoscale carrier particles has a diameter in the range of 300 nm to 1500 nm.

5. Micro and / or nanoparticle vaccine according to one of claims 1 to 4, wherein the one or more antigen determinant (s) for cytotoxic effector T cells and / or the one or more antigenic determinant (s) for Helper T cells have / have an amino acid sequence of a suitable epitope.

6. A micro- and / or nanoparticle vaccine according to any one of claims 1 to 5, wherein the one or more antigenic determinant (s) for cytotoxic effector T cells comprises a CTL epitope, a polyepitope, a biopolymer, a protein, DNA / RNA, a polysaccharide, a glycolipid and / or a glycoprotein.

7. Micro and / or nanoparticle vaccine according to one of claims 1 to 6, wherein the

Coupling of the vaccine components to the micro- and / or nanoscale carrier particle based on chemical and / or physical interactions.

8. micro- and / or nanoparticle vaccine according to claim 7, wherein the chemical and / or physical interactions, a covalent interaction, an ionic interaction, a Van der Waals interaction, a hydrogen bond, an adhesive bond, an electrostatic interaction and / or have a magnetic interaction.

9. micro and / or nanoparticle vaccine according to claim 8, wherein one or more

Vaccine components have charge tags and wherein the coupling of the vaccine components to the micro- and / or nanoscale carrier particle is based on electrostatic interaction between the polarized surface of a charged polymer-coated core sequence and the charge tag of the vaccine component (s).

10. A micro- and / or nanoparticle vaccine according to claim 9, wherein the charge tag comprises a series of 4-10 basic or acidic amino acids.

11. A micro- and / or nanoparticle vaccine according to any one of claims 1 to 10, wherein the one or more antigenic determinant (s) for helper T cells comprises a Pan-DR epitope as a degenerate peptide for all HLA-DR cells. Allotypes has / have.

12. The micro- and / or nanoparticle vaccine according to claim 1, wherein the one or more immunomodulator (s) for activating the antigen-presenting dendritic cells has / have a synthetic analog of a bacterial lipopeptide.

13. A micro- and / or nanoparticle vaccine according to any one of claims 1 to 12, wherein two or more of the three basic components are covalently or non-covalently linked together.

14. A microparticle and / or a nanoparticle vaccine according to any one of the preceding claims, wherein the immunomodulator is a lipopeptide having the general structure (I), R1 -O- CH,

in which

R 1 and R 2, which can be identical or different, represent a saturated or unsaturated acyl radical having 7 to 25 C atoms,

R3 is a saturated or unsaturated acyl radical having 7 to 25 carbon atoms or H and

R4 represents a physiologically acceptable peptide which consists of 1 to 25 amino acid residues and is not immunogenic per se in the treated species,

including the corresponding pharmaceutically acceptable salts, tautomers, stereoisomers, stereoisomer mixtures, prodrug esters or prodrug peptides.

15. The microparticulate and / or nanoparticle vaccine of claim 14, wherein the lipopeptide is present as the RR stereoisomer of the dihydroxypropyl cysteine.

16 micro- and / or nanoparticle vaccine according to claim 14 or 15, wherein R1, R2 and / or R3 is a saturated acyl radical having 16 carbon atoms.

17. Micro and / or nanoparticle vaccine according to any one of claims 14 to 16, wherein R4 is a peptide having the amino acid sequence of SEQ ID NO: 1.

18 micro- and / or nanoparticle vaccine according to any one of the preceding claims, wherein each carrier particle carries one, several or all vaccine components in a defined according to the active spectrum of the vaccine molar ratio.

19. Micro and / or nanoparticle vaccine according to claim 18, wherein the vaccine components are coupled individually or as a conjugate to the carrier particle.

20. micro and / or nanoparticle vaccine according to claim 18 or 19, wherein the

Vaccine components to a conjugate according to one of the following general

Structures (II), (III) of (IV) are linked lipopeptide

T cell epitope ₁

T-cell epitope,

in which

n each for a natural number from 0 to 6,

R1, R2, R3, R4, R5 and R6, which may be the same or different, for an amino acid spacer having a length of 0 to 6 amino acids, T cell _epitope! and T cell epitope ₂ , which may be the same or different, for an amino acid sequence of an epitope for cytotoxic effector T cells or an epitope for helper T cells,

Lipopeptide for an immunomodulator according to formula (I) according to one of

Claims 14 and 15 and

Day for a sequence of 4-10 basic or acidic amino acids,

21. Micro and / or nanoparticle vaccine according to one of the preceding claims for medical use.

22. A pharmaceutical preparation containing a therapeutically effective amount of a micro- and / or nanoparticle vaccine according to any one of the preceding claims alone or in combination, wherein the preparation is present in a formulation suitable for topical application.

23. micro and / or nanoparticle vaccine according to claim 21, wherein the medical

Application has that reversibly coupled vaccine components

(i) are deliberately released under the physiological conditions prevailing in the hair follicle,

(ii) by diffusion to the target structures, the dendritic cells in the

Epidermis of the skin, arrive and

(iii) induce a T cell-based vaccine antigen-specific immune response.

24. micro and / or nanoparticle vaccine according to any one of claims 21 or 23 for

Use in the prophylactic and / or therapeutic treatment of

Human and animal diseases related to infections, cancer, autoimmune diseases or inflammatory and allergic conditions

Supporting a conventional vaccine treatment with the

treated subject after application of a therapeutically effective amount of one or more vaccines by massaging into the skin at one or more spatially separated sites and at the same or different times, cause an immune response specific to the antigenic determinants used.