WO2011138050A1

WO2011138050A1 - Method for vaccination

Info

Publication number: WO2011138050A1
Application number: PCT/EP2011/002294
Authority: WO
Inventors: Claus-Michael Lehr; Steffi Hansen; Ulrich Friedrich SCHÄFER; Carlos Guzman
Original assignee: Helmholtz-Zentrum für Infektionsforschung GmbH; Universität des Saarlandes
Priority date: 2010-05-07
Filing date: 2011-05-09
Publication date: 2011-11-10

Abstract

The present invention relates in a first aspect to means, in particular, to transdermal or transcutaneous systems, for delivery of a compound, like an antigen for eliciting or modulating an immune response or a therapeutical effect in an individual. Said means or systems comprise a carrier or particle containing a compound, like said antigen optionally in combination with an adjuvant. In another aspect, the present invention relates to methods for transcutaneous or transdermal application of compounds, like vaccination, in particular, transappendageal vaccination. Further, the present invention relates to a pharmaceutical composition comprising carrier or particles containing the compound, like antigen and, optionally, adjuvant, whereby the compound, like antigen is released by the carrier or particles in situ. Finally, the present invention relates to the use of said pharmaceutical composition in transdermal or transcutaneous delivery of compounds, like for vaccination of an individual, in particular, for use in transappendageal vaccination of an individual.

Description

Method for Vaccination

Prior art

Most microbial pathogens enter the body through mucosal surfaces or the (damaged) skin, causing local infections or transiting across them before systemic dissemination. Generally, it is desirable to promote not only systemic but also a local immune response after vaccination in order to block disease (i.e. symptoms) as well as infection (i.e. colonisation), thereby reducing the risk of horizontal transmission from infected individuals to susceptible hosts.

Unfortunately, conventional systemic vaccines are suboptimal, since they failed to induce a local response able to block the earliest stages of infection. This roadblock can be eliminated by administering antigens by the mucosal route. In vaccine development, activating the immune system is the major challenge. The muscle, a traditional site, is not considered to be an efficient site for antigen presentation due to the lack of suitable quantities of antigen presenting cells (APCs), whether dendritic cells (DCs) or macrophages. In contrast, the skin is one of the largest organs and is rich in potent APCs such as Langerhans cells (LCs) in the epidermis and DCs in the dermis.

Mucosal vaccination is also characterised by an easy administration logistics and being highly accepted by the public. Unfortunately, antigens administered by the mucosal route are usually poor or non immunogenic. This may be due to accelerated antigen elimination by the non-specific host clearance mechanisms, antigen degradation by local enzymes, antigen alteration and antigen modification as a result of the environment, poor antigen penetration through the mucosa, limited access of vaccine antigen to antigen presenting cells and local peripheral tolerance, respectively.

A strategy to overcome this bottleneck is their co-administration with mucosal adjuvants. Substances referred to as "adjuvants" are those which are added into or co-formulated with the actual antigen, i.e. the substance which provokes the desired immune response, in order to enhance the humoral and/or cell mediated immune response. Adjuvants are compounds having immunopotentiating properties, in particular, when coadministered with antigens. While the adjuvant itself does not initiate an immune response, the adjuvant promote the immune response against the antigen administered with the vaccine.

However, there is no mucosal adjuvant available for human use, and the most promising candidates tested so far, e.g. derivatives of bacterial toxins, led to concerning neurological side effects. Thus, there is an urgent need for human safe strategies to stimulate the mucosal immunity. The transcutaneous or transdermal vaccination seems to be a valid alternative. However, the approaches tested so far were not robust and it was not possible to tailor the elicited responses.

Recently several methods for transcutaneous vaccine delivery have been developed, including minimal invasive techniques such as micro-needles, the gene gun, the PowderJect, or skin-surface-stripping techniques. However, as these strategies reduce the protective barrier function of the stratum corneum for a significant time they will be suboptimal for certain applications, such as mass vaccination campaigns in third world countries with critical hygienic conditions

In recent years, polymeric nanoparticles or microparticles have been extensively studied as antigen delivery systems. Immunization with various types of antigens entrapped in different polymeric systems elicited enhanced and long-lasting humoral and cellular immune responses. Based on in vitro skin permeation studies, nanoparticles have shown to accumulate in the hair follicles. Further, it has been speculated to use the transfol- licular route for drug delivery, e.g. by liposomal delivery systems, see e.g. Ciotti S.N. and Weiner, N., J. Liposome Res., 2002, 12 (1-2), 143-148.

The present invention aims to provide new means, new methods and uses allowing transcutaneous or transdermal delivery of compounds, e.g. for vaccination, in particular, transappendageal vaccination.

Summary of the present invention

In a first aspect, the present invention relates to means for delivery of a compound, like an epitope-containing compound, in particular, of an antigen, comprising a carrier or particle containing said compound, like epitope-containing compound, optionally, in combination with an adjuvant, for eliciting or modulating an immune response or a therapeutical effect in an individual. The carrier or particle are characterised in being carrier or particles releasing in situ the compound, like an epitope-containing compound, upon triggering. Triggering events include humidity, pH and/or salt concentration. Other triggering events include changes in temperature or energy irradiation, like energy, like electromagnetic irradiation, e.g. UV, IR etc but also applying a magnetic field or ultra-sound. In particular, triggering occurs at a desired place and at a desired time-point, thus, releasing the compounds contained in the carrier or particles.

It is particular preferred that the means are in form of a transappendageal system for delivering of a compound, like an epitope-containing compounds via the microenvironment of hair follicles or the sweat gland. The triggering event may be changes in salt, humidity or pH e.g. by sweat or sebum.

In another aspect, the present invention relates to a method for transcutaneous or transdermal delivery of a compound, in particular for vaccination, in particular, for transappendegeal vaccination. Said method comprises the step of administering transcutaneously or transdermal^, in particular, transappendageally to an individual means for delivery a compound, like an epitope-containing compound, in particular, an antigen, and, optionally, an adjuvant, present in the carrier or particle to an individual whereby the carrier or particle release the compound, like the epitope-containing compound in situ. It is preferred that the release is triggered by a triggering event, like exposure to humidity, pH and/or salt concentration as well as temperature or energy rich irradiation.

Moreover, the present invention relates to a pharmaceutical composition comprising said means or systems or comprising carrier or particles containing the compound, like the epitope-containing compounds, like antigens, and, optionally, an adjuvant, whereby the carrier or particles release the compounds, like the epitope-containing compound in situ upon trigger- ing.

Finally, another embodiment of the present invention relates to a pharmaceutical composition for use in transdermal or transcutaneous delivery of compounds, in particular, for vaccination of an individual, in particular, for use in transappendageal vaccination of an individual whereby said pharmaceutical composition contains the means and/or systems according to the present invention, in particular, the antigen containing carrier and particles according to the present invention.

Detailed description of the present invention

The present inventors recognised that transcutaneous or transdermal delivery of compounds, like vaccination, in particular, transappendageal vaccination is possible when using carrier or particles containing said compounds, like an epitope-containing compound, in particular, antigens. Said carrier or particles are characterised in releasing the compounds in situ only. Thus, the present invention relates in a first aspect to means for delivery of a compound, like an epitope-containing compound, in particular, of an antigen, comprising a carrier or particle containing said compound, like the epitope-containing compound, optionally, in combination with an adjuvant, for eliciting or modulating an immune response or a therapeutical effect in an individual whereby the carrier or particles are characterised in releasing said compound, like the epitope-containing compound in situ upon triggering. The triggering event may be based on humidity, pH, and/or salt concentration. Said triggering events include temperature or irradiation with energy, like electromagnetic irradiation but also ultra-sound or the use of magnetic-field.

It is preferred that the means according to the present invention are in form of a transdermal or transcutaneous system, in particular, in form of a transappendageal system, for delivering of the compounds like the epitope-containing compound into an individual.

As used herein, the terms "transdermal" or "transcutaneous" are used synonomously.

The term "transappendageal" refers to transfoHicular as well as transglan- dular application. The application route include the route via the hair follicle or the sweat glands.

The term "compound" as used herein refers to any type of compounds including therapeutically or cosmetically active compounds. Preferably, the compound is a immune response eliciting or modulating compound or a therapeutically effective compound, e.g. a drug or prodrug, unless otherwise indicated.

The term "carrier or particle containing a compound " or "carrier or particle containing epitope-containing compound" refers to carrier or particles which encapsulate compounds, like epitope-containing compounds and/or which carry the compounds, like epitope-containing compounds, on their surface or adsorb the compounds in a way that the compounds, like the antigen/epitope, are not released before triggering or not inactivated or do not elicit or modulate an immune response or is therapeutically effective before triggering. In addition, the carrier or particles containing the compounds, like the epitope-containing compound do release the compounds, like the epitope-containing compound, after exposure to the triggering event.

As used herein, the term "epitope-containing compound" refers to any compound or molecule either naturally or artificially, preferably provided in isolated form, containing an epitope allowing eliciting a desired immune response e.g. vaccination, in an individual. The epitope may be combined with foreign moieties depending on the desired characteristics. The skilled person is well aware of suitable epitope-containing compounds useful for eliciting an immune response in an individual. Preferably, the epitope- containing compound is an antigen which may be modified. Modification includes solvatisation, the antigen in form of suitable salts, etc. as well as modifications improving stability, bioavailability, etc. Further, the compound may encode an antigen containing the desired epitope which will be expressed in the cells. That is, the epitope-containing compounds include DNA/RNA based vaccines as well.

Moreover, the compounds present in the carrier or particles may be immune modulators. These immune modulators modulate, e.g. direct the immune response in an individual, e.g. from a Th1 to a Th2 response or vice versa. In addition, the modulators may modulate the response from a humoral to a cellular or vice versa.

It is particular preferred that the carrier or particles are nanometer-sized carrier or particles. It is particularly preferred that the particles or carriers are composed of biodegradable and biocompatible material, like polymers. Of course, other materials are possible including organic or inorganic polymers, lipids or natural occurring, isolated material.

Among the various biodegradable polymers, the most studied polymers for vaccine delivery are undoubtedly poly(lactic-co-glycolic acid) (PLGA) and chitosan (derivatives). Both polymers share the properties to be safe, biodegradable and suitable to prepare nanoparticles.

The means and systems according to the present invention are particularly adapted to deliver the compound, like the epitope-containing compounds transappendageally, that is, into the microenvironment of hair follicles, in particular, hair bulbs, or into the microenvironment of sweat glands.. The present invention relates in a further aspect to a method for transcutaneous or transdermal delivery of compounds, like vaccination comprising the step of providing a compound, e.g. an antigen and, optionally, an adjuvant present in a carrier or particle whereby the carrier or particle releases the compound, e.g. the antigen and, optionally, adjuvant in the microenvi- ronment of a hair follicle, like hair bulbs, or sweat glands.

The transappendageal, like the transfollicular or the transglandular, route appears as a very attractive and safe strategy for the delivery of compounds, like vaccine antigens to enhance dendritic cells accumulating around hair bulbs of glands. Hair bulbs are also an excellent reservoir being only slowly cleared by hair growth and sebum production. However, in the upper regions the follicles are covered by an epidermal barrier and in deeper regions tight junctions prevent even small nanoparticles having a size of below 30 nm from transfer into the living skin tissue. It is known that μιη-large pollen accumulates in the follicle opening, sebaceous gland or dermatoglyphs very rapidly so that pollen antigens can enter the sebum- filled follicle. The process is enabled by pollen-bursting in a moist atmosphere with sufficient humidity, e.g. provided by sweat, and the subsequent delivery of antigens into the follicles. The antigens are then ingested by Langerhans cells that are found in the surrounding of the hair follicles. This in turn triggers immune responses in susceptible persons, being the central mechanisms of allergic contact dermatitis.

Hence, the underlying strategy of the present invention is to mimetic pollen delivery to vaccinate across the intact skin barrier. That is, a pollen- mimetic carrier of a size of typically 50 to 5,000 nm allows to transport the antigen optionally together with adjuvants into the lower parts of the skin to allow delivery of the vaccine antigens to Langerhans and dendritic cells. The carrier is typically composed of a biocompatible and biodegradable polymer whereby the optimal size for penetration into hair follicles is about 300 to 1 ,000 nm, like 400 to 700 nm. This size matches the size of hair cuticula surface structures. The carrier maintains its integrity during manufacturing, storage and application to the skin surface, but will release the antigen and, optionally, the mucosal adjuvants, in situ. The release can be triggered e.g. upon contact with sweat (80 to 185 mosmol/kg pH 6.5) which creates swelling and/or bursting of the carrier or upon contract with sebum.

The triggering event is in particular the exposure to changes in humidity, pH, temperature and/or salt concentration. Another possibility to trigger the release of the epitope-containing compound is energy-rich irradiation, e.g. electromagnetic irradiation. Typical examples of irradiation include visible and non-visible light, like UV, IR, e.g. laser induced release. In addition, triggering may a applying a magnetic field or ultra-sound. The triggering event may be one or more of the events described above. Exposure to pH include change of the pH either into more basic or more acid pH range. Changes in salt concentration may include decrease or increase of the salt concentration. The triggering event may be exposure to sweat or sebum as present on the skin and in hair follicles or sweat glands, e.g. as described for pollen. Thus, a pollen mimetic transcutaneous vaccination allows vaccination of individuals. This is in particular useful for antigens showing low mucosal vaccination properties. Further, the antigen presenting cells present in the skin represents useful cells for eliciting or modulating an immune response allowing vaccination against a desired antigen.

This type of carrier or peripheral system allows transcutaneous, in particular, transappendageal vaccination using antigens typically being poor or non-immunogenic when administered by the mucosal route. The particles or carriers are composed of biodegradable polymers, like polylactide gly- colic acid polymers (PLGA), starch derivatives or other types of polysaccharides, like chitosan. In addition, polyacrylates may be used or other polymers known to be biocompatible and biodegradable. The particles or carriers are characterised in showing a swelling, burst-out or triggered re- lease upon contact with the specific microenvironment in hair follicles or sweat glands, e.g. when be in contact with sweat. The microenvironment in the hair follicle is characterised in the specific conditions of humidity, pH, salt concentration etc. In addition, sebum is present. The microenvironment present in the hair follicle or the sweat glands triggers the particles or carriers containing the antigen and, optionally, the mucosal adjuvants, thus, enabling transdermal, in particular, transapendageal vaccination. The particles containing the antigen and, optionally, the mucosal adjuvants, are triggered upon entry into the hair follicle, thus, enabling to cross the stratum corneum and eliciting or modulating an immune response or a therapeutic effect similar to the immune response which can be observed in case of collagenic contact dermatitis. The present invention provides a method which does not harm the skin resulting in injuries of the stratum corneum, as it is the case when using needles or other types of stratum corneum destructing measures.

Delivery of the antigen optionally together with a mucosal adjuvant to Langerhans and dendritic cells present in the skin around the hair bulb or hair follicle or the sweat glands allows to trigger an immune response. Typically, said immune response includes a local as well as a systemic immune response, thus, providing protection against the antigen.

The method of the present invention relates in another aspect to means for transdermal vaccination comprising a composition of the particle or carrier of biodegradable and biocompatible polymers having a size of typically 50 to 5,000 nm containing antigen and, optionally, adjuvants. Said particles or carriers present in said composition allows to cross the skin barrier through the hair bulbs after triggering the hair bulbs resulting in a swelling and/or bursting of said particles and carriers. Preferably, the triggering event is the contact with sweat and/or sebum. Alternatively, the contacting medium may be a suitable medium providing the necessary humidity, pH and/or salt concentration for bursting said particles and carriers. In a further aspect, the present invention relates to a transdermal system comprising the particles describes particles or carriers containing the antigen. Said transdermal system may be in form of a transdermal system containing adhesive components, e.g. in form of a plaster or patches or pavement e.g. silicone-based transdermal system. Alternatively, the particles or carriers containing the antigen and, optionally, the adjuvants may be semi-liquid form e.g. in form of ointments, salves, cream or gel, etc. or in a liquid form or as a dispersion, like suspension, emulsion, solution, shampoo, etc allowing to migrate into the hair follicle of the individual to be treated. Further, the present invention relates to the use of the particles or carriers as described herein for transcutaneous vaccination whereby said particles or carriers contain an antigen and, optionally, an adjuvant, like a mucosal adjuvant.

The skilled person is well aware of methods for providing said particles or carriers containing the compounds, like the antigens and adjuvants, respectively. In addition, the skilled person is well aware of suitable methods for administering and applying the same on the skin of the individuals to be treated.

The present invention is characterised in that the skin of the person to be treated is not impaired or damaged by applying the composition containing the particles or carriers. Moreover, the particles or carriers are composed of biodegradable and biocompatible material, like polymers, which do not irritate or harm the skin or which would elicit or modulate an immune response or a therapeutical effect. Rather, said particles or carriers are suitable transdermal systems for delivery of compounds, like vaccine compositions containing the compounds, e.g. the antigen and an adjuvant, preferably a mucosal adjuvant, respectively.

Hence, in another aspect, a pharmaceutical composition e.g. in form of a transcutaneous or transdermal system, in particular, in form of a transap- pendageal system, is provided. Said pharmaceutical composition comprises carriers or particles containing epitope-containing compounds and, optionally, an adjuvant, characterised in that the carrier or particles release the compound, like an epitope-containing compound upon triggering in situ. The triggering event may be any event as described above.

The pharmaceutical composition may be provided solid, semi-solid or liquid form, e.g. in form of a plaster, film, patch or pavement or in form of an ointment, salve, lotion, cream or gel, as a dispersion, suspension, emulsion, solution or shampoo.

The present invention is particularly useful for vaccination including vaccination against infection, cancer or autoimmunity e.g.for hyposensibilisation against allergy inducing compounds. It may be envisaged to use the means and systems as well as the pharmaceutical compositions according to the present invention to treat or prevent atophic dermatitis or other antigen, in particular, allergy induced diseases or conditions.

Experiments: Ovalbumin was used as a model antigen due to the availability of reagents for performing an in depth characterisation of immune responses including known dominant, subdominant and cryptic epitopes; TCR transgenic animals; specific antibodies; LPS free antigen; etc. As adjuvant a Toll like receptor agonist promoting mixed T helper (Th1) and Th2 responses was included, since it promotes broader responses and target receptors and mechanisms of action are well-known. After optimisation of the initial formulation into an in vitro model of artificial skin/ sweat/ sebum, the particles or carriers according to the present invention, the pollen- mimetic carriers or particles, were evaluated for the in vitro capacity to affect the activation, maturation, and antigen processing/ presentation functions of bone marrow-derived murine DC. The process of differentiation was followed by analysing the expression of surface molecules by FACS. The secreted cytokines and chemokines were evaluated by using flow cytometric bead arrays or ELISA. The functional capacity was assessed by measuring DC ability to take up, process and present antigens to CD4+ and CD8+ T cells from TCR transgenic animals for OVA (ovalbumin). The selected prototypes formulated in an appropriate vehicle were used to immunise transcutaneously mice via the skin where the hair has been clipped before treatment. The control animals received pollen-mimetic empty carriers or carriers containing only antigen. As controls, standing groups of mice received transcutaneous vaccines based on A-B moieties toxins as adjuvants, and antigens co-delivered with established mucosal adjuvants by either oral (CTB) or intranasal (CpG, TLR agonists) route. Immunisation protocols based on one or multiple doses were tested. Humoral and cellular immune responses were characterised at systemic and mucosal level, e.g. lungs, nasal cavities, vaginal tract, intestinal tract. The OVA-specific humoral and cellular responses based on IgM, IgG, IgG subclasses, slgA in different mucosal tissues and Th1 , Th2 and Th17 as well as CTL responses, respectively, were evaluated using established protocols based on ELISA for antibodies, ELISPOT, proliferation test, in vivo CTL test, intracellular staining for cytokines and tetramers. Suitable methods are described for example in EP 1973571 to which it is referred to. The tests allowed determining the most suitable particles and carriers for transdermal vaccination.

With appropriate tests, the encapsulation into the bursting capsule, the association to the carrier surface and cleavage upon contact with sweat, integration into and release from a matrix-type system etc. was tested to optimise loading and release of antigen/ adjuvants from the nano-carrier; said tests were performed with known methods. The most promising particles and carriers were tested to stimulate response of the different mucosal niches by incorporating vaccine-relevant antigens. Said vaccine- relevant antigens may be derived from hemagglutinin from the influenza virus and PspA/ PscB from Streptococcus pneumoniae for respiratory pathogens; VP6/ VP4 from Rotavirus and Psn/ LcrV for Yersinia spp; for enteric pathogens; Tat/ Gag of HIV and HBsAg from HBV for sexual transmitted pathogens. Further, the immune response can be evaluated and tailored further due to incorporation of different adjuvants allowing promotion of polarised Th1 , Th2 or Th17 responses. Suitable mucosal adjuvants are described e.g. in EP 1 973 571. Further, the responses are improved by incorporating DC/ Langerhans cells targeting moieties. If appropriate, the transcutaneous vaccination may be accompanied by a mucosal challenge or boosts.

Loading of the carrier and particles with the epitope-containing compounds, e.g. the antigen, may be effected by known methods for encapsulation or adsorption of compounds.

Suitable adjuvants include known systemic and/or mucosal adjuvants, e.g. MALP-2 or derivatives thereof or cyclic-dinucleotides, like c-diAMP.

Material & Methods

Preparation and Optimization of Ova loaded PLGA nanoparticles

Preparation of Ova loaded PLGA nanoparticles

PLGA nanoparticles were prepared by an adapted double emulsion method. Briefly, 100 mg of PLGA were dissolved in 2.5 ml of ethyl acetate at room temperature. Then 5 mg (in 200 μΙ, 400 μΙ, 600 μΙ) of Ova solution in water was added to it and homogenized (Ultra-Turrax TP18/10, IKA, Germany) at 18.000 rpm for 2 min. Then 5 ml of an aqueous phase containing the stabilizer (1 %, 2%, 3% w/v) was added to it and again homogenized at 18.000 rpm for 2 min. The resulting w/o/w emulsion was poured into 25 ml of water under constant stirring to diffuse and finally evaporate the organic solvent. This resulted in nanoprecipitation and formation of nanoparticles. The nanoparticles were freeze-dried after mixing with trehalose (0.5% w/v). Optimization of formulation and process parameters

Screening of suitable stabilizer

Nanoparticles of PLGA were prepared by using different types of stabilizers like PF-68, sodium cholate, and PVA. The right stabilizer was identified based on the optimum particle size and entrapment efficiency.

Screening of optimum stabilizer concentration

The best suitable stabilizer in the above steps was then screened for the concentration of the stabilizer used in the preparation of the nanoparticles. The optimum concentration was determined on the basis of particle size, size distribution, zeta potential.

Screening of Internal phase volume

200 μΙ, 400 μΙ, 600 μΙ of internal phase (each containing 5 mg OVA) were screened. The optimum internal phase volume was identified based on the size and entrapment efficiency.

Optimization of drug loading

Different drug loading rates 2.5%, 5%, 10% (w/w of polymer) were used and its effect on particle size and entrapment efficiency was studied. The other experimental parameters like homogenization time, final volume of dilution, and aqueous to organic phase ratio were kept constant.

Characterization of nanoparticles

Particle size, size distribution, entrapment efficiency, morphology and integrity of OVA in PLGA nanoparticles

The nanoparticles were characterized for their particle size and size distribution (mean diameter and polydispersity index) as well as their electro- phoretic mobility using a Zetasizer nano ZS (Malvern Instruments Ltd., Worcestershire, UK).

The entrapment efficiency of the nanoparticles with OVA was determined after their hydrolysis using the QuantiPro bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. Briefly, lyophilized samples of purified nanoparticles were hydrolyzed in 0.1 N NaOH (37°C) for 2 h and neutralized with 0.1 N HCI containing 3.3 m M Na₂HP0₄ and KH₂P0 and determined with the QuantiPro BCA method.

For integrity studies, a total of 0.5 g OVA extracted from lyophilized nanoparticles (similarly extracted as in determining entrapment efficiency) was loaded on a 10% poly(acrylamide) gel under reducing conditions. Protein bands were visualized with Coomassie Blue according to the manufacturer's instructions.

The surface morphology of nanoparticles was analyzed by atomic force microscope (AFM) and scanning electron microscopy (SEM).

Results

Optimization of formulation and process parameters

Screening of suitable stabilizer

(Table 1) PVA and PF-68 are the choice among the tested stabilizers as they provide higher entrapment efficiency coupled with optimum particle size and polydispersity.

Table 1

Effect of stabilizer type on particle size, PDI and entrapment efficiency

Surfactant (2% w/v) Size (nm) PDI % EE

PF-68 207.4 ± 1.31 0.095 ± 0.024 37.05 ± 1.62

PVA 240.9 ± 2.49 0.070 ± 0.015 40.79 ± 1.81

Na cholate 251.5 ± 0.98 0.196 ± 0.007 33.46 ± 2.15

PVA- polyvinyl alcohol, PF-68- Pluronic F- 68, Na Cholate- sodium cholate, EE- entrapment efficiency; values are meant standard deviation

(S.D) (n=3).

The particles obtained after freeze drying were re-dispersed in double distilled water (dd. water) to see the effect on size and PDI after freeze dry- ing. PVA was identified as a good stabilizer for the freeze drying with respect to stability in size and PDI.

Screening of optimum concentration of stabilizer

(Table 2) There is no significant effect of PVA concentration on the size and PDI of the nanoparticles when increasing the concentration from 2% to 3% w/v while keeping the theoretical loading constant (5% w/w of polymer). So 2% w/v PVA was found to be an optimum surfactant concentration.

Table 2

Effect of PVA concentration

Surfactant concentration (%w/v) Size PDI

PVA 1% 269.8 ± 3.23 0.1 14 1 0.023

PVA 2% 240.9 ± 2.49 0.070 + 0.015

PVA 3% 238.0 ± 2.14 0.049 + 0.018

Values are mean±S.D (n=3).

Screening of optimum internal phase volume

(Table 3) 400μΙ of internal phase was found to be optimum in terms of size, PDI and entrapment efficiency

Table 3

Effect of Internal phase volume

Internal phase volume Size (nm) PDI % EE

200 μΙ 240.9 ± 2.49 0.070 ± 0.015 40.79 ± 1 .81

400 μΙ 244.0 ± 4.55 0.084 ± 0.016 47.31 ± 1 .64

600 μΙ 254.0 ± 3.21 0.106 ± 0.018 46.92 ± 1 .92

Values are meaniS.D (n=3).

Optimization of compound loading

Table 4

Effect of initial compound loading on particle size, PDI and entrapment loading (% _w/w

of polymer) P.S.(nm) PDI % EE

2.5% 241 .6 ± 3.64 0.064 ± 0.023 35.59 ± 3.34

5% 244.0 ± 4.55 0.084 ± 0.016 47.31 ± 3.64

10% 256.1 ± 2.54 0.147 ± 0.015 20.54 ± 2.41

Values are mean±S.D (n=3). As shown in the Table 4, the initial compound loading had no major effect on particle size. From 2.5% to 10% (w/w of polymer) initial compound loading, particles obtained were very much similar in size but there is increase in the PDI when compound loading increased from 5% to 10%.

Entrapment efficiency of the compound in the nanoparticles showed an increase with increase in compound loading up to 5% loading after which possibly the polymer was saturated with the compound.

Morphology of nanoparticles

SEM & AFM image of nanoparticles shows distinct spherical and particles with a narrow size distribution and a smooth surface.

Antigen intergrity:

Encapsulation of OVA in PLGA nanoparticles did not adversely affect the integrity of OVA. j 2

Characterization of Optimized Ovalbumin loaded PLGA Nanoparticles

Table 5

Characterization of optimized Ovalbumin loaded PLGA nanoparticles

Initial drug load¬

Nanoparticle ing P.S.(nm) PDI Z.P. (mV) % EE

(w/w of polymer)

47.31 ±

Drug loaded 5% 244.1 ± 4.55 0.084 ± 0.016 -20.58 ± 0.17

3.64

The optimized nanoparticle batch with initial drug loading (5 % w/w of polymer) resulted in particles with a mean diameter of 244.1 ± 4.55 nm, narrow size distribution (PDI) of 0.084 ± 0.016, zeta potential of -20.58 ± 0.17 with % EE of 47.31 ± 3.64.

Preparation and Optimization of Chitosan PLGA particles

Chitosan-PLGA nanoparticles were prepared by an adapted double emul- sion method. Briefly, 100 mg of PLGA were dissolved in 2.5 ml of ethyl acetate at room temperature. Then 5 mg (in 200 μΙ, 400 μΙ, 600 μΙ) of Ova solution was added to it and homogenized (Ultra-Turrax TP18/10, IKA, Germany) at 18.000 rpm for 2 min. Then 5 ml of an aqueous phase containing the PVA stabilizer (2% w/v) and 0.3% w/v Chitosan (Protasan™ UP, NovaMatrix, USA) was added to it and again homogenized at 18.000 rpm for 2 or 4 min. The resulting w/o/w emulsion was poured into 25 ml of water under constant stirring to diffuse and finally evaporate the organic solvent. This resulted in nanoprecipitation and formation of nanoparticles.

Release studies of OVA from PLGA nanoparticles.

The in vitro release of OVA from the nanoparticles has been carried out by a centrifugation method. Briefly, for each pre-determined time point in the release profile an Eppendorf cup containing 1 ml of nanoparticles suspension in PBS (pH 5.5) was incubated at 37 °C for 3 days under continuous stirring at 100 rpm. At those pre-determined time points, the respective eppendorff cup was centrifuged for 20 min at 24,000 g and the supernatant wasd analyzed by using the QuantiPro BCA assay.

Evaluation of antigenicity of antigen in SHK-1 mice (OVA encapsulated in PLGA and Chitosan-PLGA nanoparticles)

The optimized batches of OVA encapsulated PLGA and Chitosan-PLGA nanoparticles with or without adjuvants was evaluated on the efficacy of the particulate system in provoking the immune response in SHK-1 mice. A dose of 10 pg OVA (encapsulated in nanoparticles) was applied as aqueous gel formulation (1.5% w/v Natrasol Typ 250 M) to the skin of SHK-1 mice.

Evaluation of different parameters demonstrate a humoral and cellular immune response against the model antigen OVA.

To conclude, the present invention provides suitable methods for trans- dermal vaccination, suitable systems and means for transdermal vaccination as well as its use for the same. The method comprises administering or applying particles or carriers containing the antigen and, optionally, adjuvants, like mucosal adjuvants, on the skin of an individual. Said particles or carriers are suitable to enter into hair follicles. Moreover, said particles or carriers are characterised in being composed of biodegradable and biocompatible polymers. Said polymers are characterised in displaying release of the epitope-containing compounds, like antigen, e.g. after swelling and bursting after coming in contact with sweat or sebum, etc., i.e. the mi- croenvironment present in the hair bulbs or hair follicles of said individuals. After swelling and/or bursting, the antigens and adjuvants cross the skin barrier and can be delivered to Langerhans and dendritic cells which are present around the hair bulbs, thus, eliciting a local as well as a systemic immune response.

That is, the vaccination according to the present invention by tropical application (transcutaneous vaccination, like transfollicular vaccination) represent an approach having the following advantages: needle-free method which ensures patient compliance

obviates complications related to physical skin penetrative

no trained personnel required

no serious adverse effects

elicit systemic and mucosal immune responses

co-administration of adjuvants allow orchestration of specific humoral and cellular responses.

Thus, the vaccines according to the present invention stimulates both systemic and mucosal protective immune responses in contrast to most state of the art vaccine formulations. Embodiments of the present invention include:

1. A method for transcutaneous or transdermal vaccination of an individual comprising the step of providing an antigen and, optionally, an adjuvant present in a carrier or particle whereby the carrier or particle releases the antigen and the adjuvant in the microenvironment of a hair follicle, like hair bulbs.

2. The embodiment of claim 1 wherein the carrier or particles containing the antigen and, optionally, the adjuvant after exposure to humidity, in particular, sweat.

3. Means, in particular, a Transdermal System, for delivery of a vaccine comprising a carrier or particle containing the antigen and, optionally adjuvant for eliciting an immune response, the carrier or particle are characterized by showing swelling and/or bursting upon triggering with humidity, pH and/or salt concentration, in particular, after triggering with sweat, e.g. in the microenvironment of hair follicles.

4. The transdermal system of embodiment in form of a plaster, patch or pavement or in form of an oitment, salve etc.

5. The use of a pollen mimetic particle or carrier containing antigen and, optionally, adjuvant, for transdermal vaccination of an individual.

Claims

1. A means for delivery of compounds, in particular, an epitope- containing compound, comprising a carrier or particle containing said compound, in particular, the epitope-containing compound, optionally in combination with an adjuvant, for eliciting or modulating an immune response or a therapeutical effect in an individual, whereby the carrier or particle are characterized in releasing said compound, in particular, epitope-containing compound upon triggering with humidity, pH, salt concentration, energy including temperature, irradiation, magnetic field, ultra-sound and/or sebum.

2. The means according to claim 1 in form of a transdermal or transcutaneous system, in particular, in form of a transappendageal system, for delivering of the compounds, in particular, the epitope- containing compound.

3. The means according to claim 1 or 2 in form of a patch, film, an ointment, salve, lotion, cream, gel, suspension, emulsion.

4. The means according to claim 3, wherein triggering is with sweat or sebum.

5. The means according to any one of the preceding claims adapted to deliver the compounds, in particular, the epitope-containing isolated compound in the microenvironment of hair follicles, in particular, hair bulbs, or the microenvironment of sweat glands.

6. Means according to any one of the preceding claims wherein the carrier or particles are nanometer-sized carrier or particles.

7. The means according to any of the preceding claims wherein the particle or carrier is composed of a biocompatible and/or biodegradable material.

8. A method for transcutaneous or transdermal delivery of compounds, in particular, for vaccination of an individual comprising the step of providing cutaneously or dermally to an individual means for delivery of a compound, in particular, an epitope-containing compound and, optionally, an adjuvant, present in a carrier or particle whereby the carrier or particle release the compound, in particular, the epitope-containing compound and, if present, the adjuvant, in the skin, preferably, in the microenvironment of a hair follicle, in particular, of a hair bulb, or in the microenvironment of the sweat glands.

The method according to claim 8 wherein the carrier or particle containing a compound, in particular, an epitope-containing compound and, optionally, the adjuvant, releases said compound, in particular, epitope-containing compound and the adjuvant after exposure to humidity, pH salt concentration, energy including temperature, irradiation, magnetic field, ultra-sound and/or sebum, in particular, sweat or sebum.

The method according to any one of claims 8 or 9 wherein the administration of the carrier or particle containing the epitope- containing compound is needle-free and/or wherein the skin of the person to be treated is not impaired or damaged by applying the composition, in particular, the administration is transappendageal.

11. A pharmaceutical composition comprising carriers or particles containing a compound, in particular, an epitope-containing compound and, optionally, an adjuvant, characterised in that the carrier or par- tide swell and/or burst upon triggering with humidity, pH salt concentration, energy including temperature, irradiation, magnetic field, ultra-sound and/or sebum for releasing the compound.

12. The pharmaceutical composition according to claim 11 wherein the carrier or particle swell and/or burst upon triggering with sweat or sebum.

13. The pharmaceutical composition according to claim 11 or 12

adapted for appendageal application, in particular, for application into the microenvironment of hair follicles or sweat glands.

14. The pharmaceutical composition according to any one of claims 11 to 13 in form of a patch, film, an ointment, salve, lotion, cream, gel, suspension, emulsion.

15. The pharmaceutical composition according to any one of claims 11 to 14 wherein the epitope is an epitope of an allergy inducing compound.

16. The pharmaceutical composition according to any one of claims 11 to 15 for use in transdermal or transcutaneous vaccination of an individual, in particular, for use in transappendageal vaccination of an individual.

17. The means according to any one of claims 1 to 7 or the pharmaceutical composition according to any one of claims 11 to 15 in form of a vaccine.