WO2014044690A1

WO2014044690A1 - Improved vaccines

Info

Publication number: WO2014044690A1
Application number: PCT/EP2013/069322
Authority: WO
Inventors: Thomas LINGELBACH; Michael Möhlen; Robert Seid; Kerstin Westritschnig
Original assignee: Valneva Austria Gmbh
Priority date: 2012-09-18
Filing date: 2013-09-18
Publication date: 2014-03-27

Abstract

This invention relates to booster vaccines, i.e. to vaccines that are administered to primed subjects, by using transcutaneous immunization or immunostimulation of biological products, e.g., polypeptides, peptides, and DNA. The inventive schemes are unique and offer several advantages to current applications. Such schemes include those comprising the use of toxoids such as tetanus toxoid (TTx) and/or bacterial antigens such as Escherichia coli heat labile enterotoxin (LT) antigen. The compositions for use according to the invention are not required to be stored at reduced temperatures, but remain stable for extended periods at room temperature, are safe to be used on skin, and are simple to use. Accordingly, the invention is directed, in part, to methods of administration, and the products derived therefrom, comprising a novel application that re-enhances the immune response of the administered composition such as a vaccine to an unexpected high level.

Description

Improved Vaccines

FIELD OF THE INVENTION

This invention relates to booster vaccines, i.e. to vaccines that are administered to primed subjects, by using transcutaneous immunization or immunostimulation of biological products, e.g. , polypeptides, peptides, and DNA. The inventive schemes are unique and offer several advantages to current applications. Such schemes include those comprising the use of toxoids such as tetanus toxoid (TTx), diptheria toxoid (DTx), HbSAg, Hib polysaccharide -protein conjugate and/or bacterial antigens such as Escherichia coli heat labile enterotoxin (LT) antigen. The compositions for use according to the invention are not required to be stored at cold temperatures, but can remain stable for extended periods at room temperature, are safe to be used on skin, and are simple to use. Accordingly, the invention is directed, in part, to methods of administration, and the products derived therefrom, comprising a novel application that re-enhances the immune response of the administered composition such as a vaccine to an unexpected high level. Additionally, the invention provides for a tetanus toxoid vaccine for priming.

BACKGROUND

Transcutaneous immunization {e.g. , using a topically-administered vaccine) and immunostimulation offer distinct advantages over traditional methods of immunization and immunostimulation, e.g. , methods using intravenous, intradermal or intramuscular injection. Practically, transcutaneous immunization and immunostimulation methods require no invasive and costly equipment, e.g. , needles, thus minimizing costs and chances of cross contamination; require minimal training, allowing widespread and rapid distribution of the vaccines that are potentially self-administrable; and are not associated with any trauma, increasing compliance. However, despite great interest, the transcutaneous immunization and immunostimulation, in particular, with polypeptide molecules, face significant obstacles including low efficacy of the topically-applied formulations.

Skin is the largest human organ and an important part of the body's defense against invasion by infectious agents and contact with noxious substances. Therefore, skin has evolved as an effective screen against large molecules (see Bos, 1997). The skin is composed of three layers: the stratum corneum, epidermis, and the dermis. The epidermis is composed of the basal, the spinous, the granular, and the cornified layers; the stratum corneum comprises the cornified layer and lipid (Moschella and Hurley, 1992). The stratum corneum, a layer of dead skin cells surrounded by an extracellular lipid matrix, has traditionally been viewed as a barrier to the hostile world, excluding organisms and noxious substances from the viable cells below the stratum corneum (Bos, 1997).

Besides the physical restrictions limiting the passage of molecules through the skin, passage of polypeptides was believed to be also limited by chemical and local environmental restrictions. U.S. No. 5,679,647 stated that "it is believed that the bioavailability of peptides following transdermal or mucosal transmission is limited by the relatively high concentration of proteases in these tissues. Yet unfortunately, reliable means of delivering peptides ... by transdermal or mucosal transmission of genes encoding for them has been unavailable."

Since the first immunization by Edward Jenner in 1796 for smallpox, immunization has been recognized as the most efficient medical tool to deal with the spread of infectious disease (see UNICEF press release (2002)). In the past few centuries, immunization has shown tremendous impact on human's health worldwide, as demonstrated by the global eradication of smallpox, the elimination of polio-myelitis from most part of the world, and the elimination of measles in many developing countries (see Stewart et al. (2006)). Even with these successes, significant problems remain. Over 30 million infants born each year worldwide still do not have access to fundamental vaccine and over 2 million children die annually from vaccine preventable diseases (see WHO (2003)).

Transcutaneous immunization and immunostimulation methods offer the potential to significantly improve vaccination and immunization efforts. First, it eliminates the use of needles and syringes: due to reuse of needle and syringe and unsafe injection practices in many parts of the world, the World Health Organization (WHO) estimates that unsafe injections cause 22 million new hepatitis B virus (HBV) infections in developing and transitional countries (see WHO (2001)).

Second, transcutaneous immunization methods alleviate distribution problems such as high cost, lack of infrastructure to distribute vaccines and inherent deficiencies in injection.

Accordingly, efforts focused on sufficient delivery of antigen to induce an immune reaction. Because of the recognized barriers in the skin, efforts focused primarily on novel carrier molecules or systems to overcome the barriers. For example, a report by Paul et al. (1995) disclosed induction of complement-mediated lysis of antigen- sensitized liposomes using transfersomes. The transfersomes were used as a vehicle for antigen, and complement- mediated lysis of antigen-sensitized liposomes was assayed. The limit to passage through the skin by antigen was stated to be 750 daltons. However, despite their success, Paul and Cvec (1995) stated that it is "impossible to immunize epicutaneously with simple peptide or protein solutions without using aids such as liposomes." The subsequent discovery that large molecules can be captured by skin antigen presenting cells such as Langerhans cells (Celluzzi and Falo, 1997) in the skin epidermal layer offered hope that transcutaneous immunization could be effective. Moreover, the ability to immunize through the skin using the crucial concept of a skin-active adjuvant has only been recently described (Glenn et al., 1998). Scientific recognition of this important advance in vaccination was prompt. "It's a very surprising result, and it's lovely," said vaccine expert Barry Bloom of the Howard Hughes Medical Institute and the Albert Einstein College of Medicine in New York, the strategy sounds "very easy, very safe, and certainly inexpensive" (CNN News, February 26, 1998).

Transcutaneous immunization has subsequently been demonstrated as not only plausible, but effective in certain settings. In WO 98/20734 and a subsequent publication (Scharton-Kersten et al., Infect. Immun., 2000), formulations comprising a skin-active adjuvant were demonstrated to induce high levels of systemic and mucosal antibodies to co-administered antigens. For example, mice immunized with such skin-active adjuvant and diphtheria toxin (DT) induced high levels of systemic and mucosal anti-DT antibodies. Such antibodies are known to correlate with protection against diphtheria. Meanwhile, transcutaneous immunization has been demonstrated with a wide variety of protein and DNA antigens, including those derived from bacterial and viral pathogens (Seid Jr. RC, Glenn GM. Advances in transcutaneous vaccine delivery. Chapter 40, pp. 415-429; Karande P, Mitragotri S. Ann Rev Chem Biomol Engineering 2010; 1 : 175-201; Bal SM et al. Journal of Controlled Release 2010; 148(3):266-282). Further, Intercell USA, Inc has multiple patents and patent applications directed to transcutaneous immunization comprising the use of a vaccine patch (see US 7,527,802, US 6,797,276, US 7378,097, US 7,037,499, US 5,980,898, US 5,910,306, US2009/0081244 Al). Further research by Intercell USA and other laboratories indicated that delivery of antigen (or adjuvant or both) to other immunocompetent cells (e.g. dermal dendritic cells, keratinocytes, macrophages, and T lymphocytes) present in the skin layers can contribute ultimately to the overall immune response.

However, despite the promise of recent developments in transcutaneous methods, the problem of inducing very high levels of systemic antibodies e.g. comparable or even superior to standard vaccination approaches such as by injection, remains. Due to limits of the transcutaneous immunization and immunostimulation, most transcutaneous vaccines mentioned in the above-referenced art, require substantial higher amounts of antigens to ensure the required immune response for protection against the pathogen attack. The need to produce high amounts of antigen can present technological and manufacturing challenges, accompanied with economic cost, thus making it impractical to deliver vaccines by transcutaneous immunization or immunostimulation. In many parts of the world it would mean that the vaccines may either not be available or too expensive or the efficacy has been compromised. On the other hand there is a need to replace the current vaccination approaches using injectables, i.e. needles, as they require a cold supply chain. The social cost of failed and inefficient cold chain is difficult to estimate. Ineffective vaccinations and requirement of additional doses would also result in loss of confidence in vaccination programs in the areas that likely need them most. Vaccine formulations and administrations that are less dependent on the cold chain and at the same time still efficacious would greatly facilitate access to affordable and active vaccines in the rural areas.

A pharmaceutical composition for transcutaneous immunization used in a booster setting is disclosed in Glynn et al, 2005. However, said pharmaceutical composition includes as an essential element an adjuvant, i.e. LT(R192G) together with the antigen for transcutaneous immunization. Glynn states on page 1958, 2^nd column that "mucosally and transcutaneously administered proteins are usually not immunogenic and require the presence of an appropriate adjuvant". The use of an antigen/adjuvant system may pose safety concern and thus special guidelines by the regulatory authorities have been published (see e.g. the guidelines on adjuvants in vaccines for human use published by the EMEA on the 20^th of January 2005 - EMEA/CHMP/VEG/134716/2004).

Thus, there is a need to develop pharmaceutical compositions such as vaccines and processes thereto that provide stable compositions that can be stored for a long period at high temperatures such as room temperature and which are similarly or even superiorly efficacious as the current vaccine regimen. Furthermore, they need to be cheap and safe.

BRIEF DESCRIPTIONS OF DRAWINGS

Figure 1: Immunization schedule guinea pig - 2. Immunization (i.e. boost) on day 35 with a) a non-adjuvanted TTx patch, and b) a subcutaneous injection in a guinea pig model.

Figure 2: Strong boost with the TTx patch after a prime with 0.5 Lf TTx s.c.

Figure 3: Schematic of the dry rayon disc sandwiched between two release liners.

Figure 4: Microscopic image of dry pharmaceutical composition, showing the non-woven rayon dressing and glassy amorphous formulation blend.

Figure 5: Comparison of the primary immunizations (* unpaired t-test, two tailed) SUMMARY

The present invention is directed to pharmaceutical compositions comprising an effective amount of a protective antigen, preferably without a co-administered adjuvant, that is administered transcutaneously in a subject that has been efficiently primed with a vaccine comprising said antigen. In a further aspect, the invention is directed to pharmaceutical compositions that are dry, i.e. are compositions that are manufactured according to standard practice in the art (e.g. 1 hour at 45 °C in a conventional convection oven). Alternatively, a dry composition is a composition having a moisture content below 10 (also referred herein as "dry pharmaceutical composition"). Furthermore, the compositions of the invention may be applied to an area of the subject's skin that is pre-treated by physical or mechanical disruption of the stratum corneum, in which the pretreatment does not perforate the skin. In an alternative aspect, the invention provides for a pharmaceutical composition comprising an adjuvant that is able to activate Langerhans cells or other dendritic cells in the skins, characterized in that it is administered transcutaneously in a subject that has been efficiently primed with a vaccine comprising a protective antigen. Said pharmaceutical compositions applied in the manner of the invention exhibit enhanced or in some instances comparable immunogenicity or protection when compared to standard antigen boosting approaches such as an injectable antigen booster while still providing an improved stability even in the absence of dependence on the "cold chain", e.g., when stored at room temperatures, relative to similar compositions known in the art.

The methods and processes of the invention comprise a novel administration procedure wherein a booster composition (i.e. booster vaccine) comprising either a) an antigen with high immunogenicity such as rPA (i.e., recombinant protective antigen of Bacillus anthracis) but even also an antigen with low immunogenicity, e.g. toxoid or flu antigen, preferably without adjuvants (Transcutaneous Immunization), or b) an adjuvant such as a Langerhans activating or other dendritic skin cell activating adjuvants such as bacterial ADP-ribosylating exotoxins (bARE) or less toxic variants thereof, e.g. , cholera toxin (CT) and heat-labile enterotoxin from E. coli (LT), preferably without antigen, (Transcutaneous Stimulation); and wherein said composition is formulated as a dry pharmaceutical composition of the invention such as a patch as described herein, and wherein furthermore said composition of the invention may be applied to an area of the subject's skin that is pre-treated by physical or mechanical disruption of the stratum corneum, in which pretreatment does not perforate the skin.

In a further aspect, the present invention relates to a pharmaceutical composition comprising a dressing and a formulation blend that comprises a carbohydrate and an antigen, wherein the moisture content of the pharmaceutical composition is below 10% or 9%, 8%, or 7 % such as around 6%, wherein said composition is applied to an area of the subject's skin that is pre- treated by physical or mechanical disruption of the stratum corneum, in which pretreatment does not perforate the skin, characterized in that said composition is applied to a subject that is primed against said antigen, i.e. shows a substantial amount (e.g. more than 2 fold, 3 fold, 4 fold, 5 fold or more such as 10 fold rise over un-primed, i.e. non- vaccinated or naive, subjects) in the GMT of antibodies directed against the antigen in the said subject.

In a preferred embodiment, the booster application of the pharmaceutical composition of the invention is administered within the usual time period that is common for the antigen. For example, tetanus boosters are often recommended every 10 years.

In another preferred embodiment, the formulation blend is dried into a glassy amorphous state. When the formulation blend is dried to a moisture content of below 10%, it forms a glassy amorphous state as shown in Figure 3. This distinguishes the present pharmaceutical composition from compositions known in the art that have been dried by lyophilization, spray drying or spray freeze-drying. Such pharmaceutical compositions are recognized to be less stable and assemble in a cake like structure and can be easily distinguished under the microscope from the glassy amorphous state of the formulation blend of the invention.

In accordance with the present invention, the formulation blend of the pharmaceutical composition as used in present invention further comprises at least one polyol. Nonlimiting examples of polyols suitable for use in the compositions and/or methods of the invention include maltitol, mannitol, sorbitol or xylitol.

In a preferred embodiment, the formulation blend further comprises a buffer. Preferably, the buffer should be capable of buffering between pH 4 and pH 9 or more preferable, between pH 5 and pH 9. Such buffers are known in the art and described further below.

In a further preferred embodiment, the formulation blend further comprises an ionic component. Ionic components are known in the art and include, but are not limited to, e.g. NaCl.

The formulation blend of the pharmaceutical composition as used in the present invention can further comprise an adjuvant. Nonlimiting examples of adjuvants suitable for use in the present invention include bacterial ADP-ribosylating exotoxins (bARE), e.g. , cholera toxin (CT) and heat-labile enterotoxin from E. coli (LT). Other suitable adjuvants include IC31^®, CpG, imiquimod and chemical derivatives thereof, and/or any type of mutant LT that are e.g. less toxic. In accordance with the present invention, the pharmaceutical composition further comprises a backing and/or liner such as e.g. a coated backing and/or liner, more preferably a water in- penetrable backing and/or liner, even more preferably a Teflon or Silicon, preferably Teflon coated backing and/or liner such as e.g. a 9022-coated 2 millimeter, St. Gobain liner.

In a preferred embodiment, the antigen of the pharmaceutical composition of the present invention that is boosted either by a further antigen composition (transcutaneous immunization) or by an adjuvant (transcutaneous immunostimulation) is an antigen typically inducing a low immunogenic response in mammals such as a human. Preferably, the antigen is HbSAg, Hib polysaccharide- protein conjugate, a toxoid such as a tetanus toxoid, pertussis toxoid or a diphtheria toxoid. In addition, the antigen can also be a part of a macromolecular assembly. Preferably, the macromolecular assembly is a virus particle (virion) or a fragment of a virus particle (fragment of a virion).

In another preferred embodiment, the adjuvant of the pharmaceutical composition as used according to the present invention is the heat-labile enterotoxin from E. coli (LT).

The present invention also relates to a package comprising the pharmaceutical composition of the present invention. The package seals the pharmaceutical composition and protects it from moisture, dirt and air.

DETAILED DESCRIPTION

Definitions

As used herein, the term "about", when referring to a value or to an amount of mass, weight, concentration or percentage is meant to encompass variations of the amount within the standard error typically accepted in the art for the method used to determine the specific measurement. In one example, the variation is ±20%, ±10% or ±5% from the reported measurement; in another example the variation is ±1 % or ±0.1 % from the specified measurement. Such variations are routinely encountered and expected in the art.

In the context of the invention, the term "pharmaceutical composition" is understood to mean a dressing comprising a formulation blend {e.g. , having a formulation blend applied to the dressing), which formulation blend comprises an active agent or a combination of active agents suitable for transcutaneous immunization and/or immune stimulation. Accordingly, the term "pharmaceutical composition" as used herein does not refer exclusively to the formulation blend comprising the active agent or agents, but rather refers to the formulation blend in combination with any dressing (which may include other support materials, such as backings and liners). In preferred embodiments, the formulation blend comprises at least one polypeptide (e.g. , as the active agent) and one carbohydrate. Thus, at a minimum, the pharmaceutical composition of the invention comprises a dressing, a polypeptide and a carbohydrate.

The pharmaceutical composition encompasses therapeutic and prophylactic dressings (e.g. , comprising a formulation blend having an active agent or agents), optionally having additional backings or liners, that are, in particular, suitable for the transcutaneous delivery of such agents as known in the art and/or as described herein. The invention is particularly directed to pharmaceutical compositions comprising one or more polypeptide and/or polypeptide-based therapeutic as the active agent(s). The backings, liners and dressings, if present, of the pharmaceutical composition need not have active therapeutic or prophylactic properties in themselves, but may serve as physical support or protection for the formulation blend. For example, backings and liners, if present, may protect the remainder of the pharmaceutical composition (e.g. , formulation blend and dressing) during packaging and storage (e.g. , as protection from physical trauma or act as moisture barriers). The backings and liners, if present, may also aid in the application of the remainder of the pharmaceutical composition (e.g. , dressing and formulation blend) to the skin of a subject.

The "pharmaceutical composition" of the present invention comprises a dressing. The term "dressing" as used herein encompasses any material known in the art or described herein (1) suitable for deposition of an effective amount of a formulation blend comprising one or more polypeptides (typically liquid or semi-liquid) and drying to low moisture content percentages (e.g. , below 10%) using standard methods known in the art; and (2) suitable for subsequent application to the skin of a subject (e.g. , an animal including a human) for prolonged periods of time (e.g. , one or more hours to one or more days). In certain embodiments, the dressing is suitable such that the formulation blend can be dried into a glassy amorphous state. The dressing can be a woven or non-woven material and comprised of natural fibers, synthetic fibers or a mixture thereof. The fibers can be composed of any suitable material such as cotton, wool, rayon, nylon, etc and may be selected according to standard practices in the art. Accordingly, the dressing may be cotton gauze, combinations of rayon-nylon or other synthetic materials. In preferred aspects the dressing is a non-woven rayon fabric, including but not limited to Lensx^® 90 (Berkshire Co., Great Barrington, MA, USA).

The "pharmaceutical composition" of the present invention may also comprise one or more backings and/or liners. For the purposes of this disclosure, the terms "backing", "liner" and analogous terms are used interchangeably. Backings and liners may, in non-limiting examples, prevent the transport of air and/or water and/or serve as support or protection for other components of the pharmaceutical composition, e.g. , dressing and formulation blend. Backings and/or liners may also serve to adhere the other components of the pharmaceutical composition, e.g., dressing and formulation blend, to the skin of the subject. Backings and/or liners may be woven or non-woven material and composed of natural fibers, synthetic fibers, combinations of natural and synthetic fibers or formed from polymers formed into sheets, such as plastic sheets or coatings. Backing and liners may be formed from any material known in the art or described herein suitable to provide such support and/or protective properties; and/or suitable to aid in the transcutaneous administration of its composition. Multiple suitable backings and liners are known to one of skill in the art and may be selected using well known and routinely implemented criteria. The pharmaceutical composition may be occlusive or non-occlusive. As used herein, an "occlusive pharmaceutical composition" is a composition that prevents the transport of air and/or water. Non-limiting examples of occlusive composition include OpSITE^® (Smith and Nephew, United Kingdom), plastic film and COMFEEL (Coloplast); non- limiting examples of non-occlusive composition include TEGADERM (3M), DUODERM (3M) and OPSITE (Smith & Napheu). Occlusive compositions are preferred. Non-occlusive compositions can also be made occlusive by the addition of occlusive backings to the dressing material, such as solid backings (e.g. , polyvinyl chloride, suitable plastics), gels, creams, emulsions (e.g. , AQUAPHOR, an emulsion of petrolatum, mineral oil, mineral wax, wool wax, panthenol, bisabol, and glycerin from Beiersdorf, Inc.), waxes, oils, parafilm, petroleum jelly, rubber (synthetic or natural), suitable cloth, and/or other membranes.

The pharmaceutical composition of the invention may also comprise an adhesive. The adhesive may function to hold the composition (i.e. , dressing with any backing and formulation blend) onto the skin of the subject or may function to hold together the one or more components of the dressing, e.g. , material and any backings.

As used herein, the term "polypeptide" means any polymer comprising any of the 20 naturally occurring amino acids, or any of the amino acid analogs, regardless of its size or function. Although "protein" is often used in reference to relatively large polypeptides and "peptide" is often used in reference to small polypeptides, usage of these terms in the art overlaps and varies. The term "polypeptide" as used herein refers to peptides, polypeptides and proteins, unless otherwise noted. Thus, as used herein, the terms "protein", "polypeptide" and "peptide" are used interchangeably. The polypeptides used in connection with the present invention, in particular, encompass molecules used as active agents in the formulation blends and may exhibit antigenic activity, adjuvant activity or both antigenic and adjuvant activities. In the context of the present invention the physical presentation of the polypeptide in the formulation blend is irrelevant so long as the desired activity is not affected by the formulation blend. Thus, the term "polypeptide" as used in the context of the present invention encompasses isolated and/or purified polypeptides as well as polypeptides exhibiting the desired activity contained in a cell culture (e.g. , bacterial, insect or mammalian cell culture) fraction, in an animal sera fraction or in a larger macromolecular assembly (e.g. , a virus particle, bacterial cell wall). Therefore, "polypeptide" as used herein does not necessarily refer to a polypeptide isolated and/or purified from culture, sera or macromolecular components or other culture, sera or macromolecular polypeptides. Thus, in certain embodiments, "polypeptide" as used herein encompasses a polypeptide within a cell culture fraction, an animal sera fraction and/or within a macromolecular assembly, e.g. , a virus particle. As such, the invention encompasses compositions comprising one or more cell culture fractions, animal sera fractions and/or macromolecular assemblies, e.g. , virus particles. With particular respect to viruses, polypeptides may be presented as part of a whole, live virion; a whole inactivated virion; a fragment of a virion (e.g. , within a capsid fragment, e.g. , as used in split vaccines), and/or in combinations thereof. In certain embodiments, the polypeptide may be an allergen. For example, the allergen can be an allergen against bee poison, food allergen or metal allergen. The molecular weight of the polypeptide may be greater than 1 kDa, preferably up to 200 kDa, more preferably between 5 and 200 kDa.

The term "antigen" as used in the invention, is meant to describe a substance that induces a specific immune response when presented to immune cells of a subject. An antigen may comprise a single immunogenic epitope, or a multiplicity of immunogenic epitopes recognized by a B-cell receptor (i.e., antibody on the membrane of the B cell) or a T-cell receptor. The term "antigen" as used in the present invention includes an isolated and/or purified polypeptide as well as a polypeptides that are not isolated or purified from the source material; thus, "polypeptide" as used herein can reference a polypeptide in a culture medium, in a serum components and/or as part of a macromolecular assembly (i.e. , the polypeptide may be a component of a virion or fragment of a virion (as used in split vaccines). An antigen as disclosed herein may also function as an adjuvant (i.e. , have adjuvant or adjuvant- like activity, e.g. , cholera toxin). Thus, in the context of vaccine, the compositions of the invention may contain only one ingredient or component (i.e., one polypeptide) that acts as both antigen and adjuvant. The pharmaceutical composition of the invention may comprise a separate adjuvant in addition to the antigen. Non-limiting examples of suitable antigens for use in connection with the invention are described herein.

The term "adjuvant" or "adjuvants" as used herein, is meant to describe a substance added to boost the immunity of a primed subject or is meant to describe a substance added to the formulation in the context of a vaccine to assist in inducing an immune response to the antigen, but do not in themselves confer immunity. The adjuvant of the present invention may be a bacterial ADP-ribosylating exotoxin (bARE), cholera toxin (CT), heat-labile enterotoxin from E. coli (LT). Further adjuvants may be used as recognized in the art and/or as described herein. An adjuvant as disclosed herein may also have antigenic and/or immunogenic properties. Thus, in some embodiments, the pharmaceutical composition of the invention comprises only one active agent that functions as both antigen and adjuvant (e.g., LT and other bacterial ADP-ribosylating exotoxin (bAREs) as herein described). In some embodiments, e.g., for immunostimulation, the pharmaceutical composition of the invention may comprise only an adjuvant or a compound with adjuvant-like activity. Non-limiting examples of suitable adjuvants for use in connection with the invention are described herein.

The pharmaceutical compositions of the invention do not encompass lyophilized polypeptides or lyophilized formulation blends. Rather, the pharmaceutical compositions of the present invention (i.e. , a dressing comprising a formulation blend, optionally comprising one or more backings or liner sheets) are characterized by having a particularly low moisture content ("MC"), e.g. , below 10%. The low moisture content compositions of the invention are distinct from compositions comprising lyophilized components as described herein and/or as readily discernable by standard methods in the art. In certain aspects the pharmaceutical compositions of the invention are distinct from those comprising lyophilized components in that the formulation blend of the composition is in a glassy amorphous state (Figure 4).

The present invention also relates to a package comprising the solid pharmaceutical composition of the present invention. The package may seal the pharmaceutical composition of the invention from air and moisture. Thus, preferably, the package is an aluminum pouch. Other package materials may be selected by one of skill in the art according to standard criteria known in the art.

The term "effective amount" as used in the invention, is meant to describe that amount of polypeptide which induces a therapeutic and/or prophylactic effect. The therapeutic or prophylactic effect of the composition of the invention may be determined by any method known in the art and/or described herein, and may be made according to clinical parameters established by an attending physician. A therapeutic and/or prophylactic effect need not correlate with a cure of any disease or condition, but may be correlated with a decrease in any diagnostic marker or symptom of said disease or condition. In the context of a vaccine or immunization, the effective amount may be the amount of active agent, e.g., polypeptide antigen, sufficient to induce an antigen-specific immune response detectable subsequent to transcutaneous immunization, and the therapeutic/prophylactic effect may be the detection of antigen specific antibodies, e.g. , detectable 1 week, 2 week, 3 weeks, 1 month, 2 months, 4 months, 6 months, 1 year, 2 years, 5 years or longer after administration. In the context of immune stimulation, the effective amount may be the amount of active agent, e.g. , polypeptide adjuvant, sufficient to elicit a specific immune response directed against an antigen not supplied in the composition of the invention, i.e. , sufficient to allow development of the antigen-specific immune response which response could not be developed absent the composition of the invention. The therapeutic/prophylactic effect elicited by the immunostimulating compounds of the invention may be the detection of antigen specific antibodies, e.g. , detectable 1 week, 2 week, 3 weeks, 1 month, 2 months, 4 months, 6 months, 1 year, 2 years, 5 years or longer after administration. The dosage or amount of a composition of the invention to be typically administered or applied to a site can be readily determined by an ordinarily skilled clinician and will be dependent on various factors, such as the physical characteristics of the patient, the characteristics of the active agent (e.g., the antigen and/or adjuvant) as well as other drugs or treatments the patient is receiving.

The compositions of the invention may also include one or more ingredients that function as a carrier or stabilizer as known in the art. Examples of suitable carriers or stabilizers include carbohydrates and polyols. Examples of carbohydrates known in the art to function as carriers and/or stabilizers and suitable in the context of the present invention include but are not limited to carbohydrates such as sucrose, fructose, trehalose or glucose. Examples of polyols known in the art to function as carriers and/or stabilizers and suitable in the context of the present invention include but are not limited to maltitol, mannitol, sorbitol or xylitol. The composition of the invention may comprise only one carbohydrate or polyol, or may comprise multiple types of carbohydrates, polyols, and/or combinations thereof. The invention may also include compositions comprising liposomes, e.g., to function as carrier molecules effecting transcutaneous transport of the active agent, e.g. , the one or more polypeptides. The transcutaneous transport activity of formulations comprising liposomes is well documented and known in the art, e.g. , as demonstrated by studies using transfersomes. Transfersomes are ultraflexible vesicles that have an extremely low pore penetration resistance. They are recognized to transport pharmacological agents, including large polypeptides, through the permeability barriers, such as the intact skin (see Cevc et al., 1998; Cevc, 2003).

In accordance with the present invention, the solid pharmaceutical composition may further comprise a buffer capable of buffering between pH 4 and pH 9, more preferable, between pH 5 and pH 9. Such buffers are known in the art and include, but are not limited to HC1, Citric acid, potassium, MOPS, PIPES, SSC, TAPSO buffer etc. Further suitable buffers include, but are not limited to, phosphate buffered saline Ca⁺⁺/Mg⁺⁺ free, phosphate buffered saline, normal saline (150 mM NaCl in water), and HEPES or Tris buffer. Active agent, e.g. , one or more polypeptides, not soluble in neutral buffer can be solubilized in 10 mM acetic acid and then diluted to the desired volume with a neutral buffer such as PBS. In the case of an active agent, e.g. , one or more polypeptides, soluble only at acid pH, acetate-PBS at acid pH may be used as a diluent after solubilization in dilute acetic acid. Glycerol may be a suitable nonaqueous buffer for use in the invention. The pharmaceutical composition of the present invention may further comprise an ionic component. Substances suitable as the ionic component of the compositions of the invention are well known in the art and include NaCl. In preferred embodiments, the compositions of the invention are iso- or hypertonic to increase transcutaneous delivery of the one or more active agents of the composition of the invention as known in the art. The ionic strength of the formulation blend was found to have no impact on the stability of the Super Dry compositions of the invention.

In the context of the present invention, a "booster" or "booster dose" is an extra administration of a vaccine after an earlier dose. After initial immunization, a booster or booster dose is a re-exposure to the immunizing antigen. It is intended to increase immunity against that antigen back to protective levels after it has been shown to have decreased or after a specified period. For example, a tetanus booster is often recommended every 10 years.

In the context of the present invention, "transcutaneous booster immunization with a pharmaceutical composition of the invention" (also referenced herein as "vaccine delivery patch boosting" or "VDP boosting") means that a sufficient amount of the one or more active agents of the composition of the invention, e.g. , one or more polypeptides such as an antigen, an adjuvants or both, preferably only an antigen, is delivered through the skin to results in the induction, increase, stimulation or generation of a specific immune response (e.g. , an antibody response against the one or more antigens of the composition of the invention) back to protective levels, in particular in a booster setting, i.e. where such response was already detectable at measurable levels through a first immunization by the injectable route (but possibly the immunity was not sufficient anymore) prior to the administration of the VDP boost. Efficient booster immunization can be achieved by transcutaneous delivery of polypeptide that targets the Langerhans cell and/or dermal dendritic cells. These cells are found in abundance in the skin and are efficient antigen presenting cells leading to induction of T-cell memory and potent immune responses. Because of the presence of large numbers of antigen presenting cells in the skin, the efficiency of transcutaneous delivery in a booster setting may be related to the surface area exposed to antigen and adjuvant.

In the context of the present invention, "transcutaneous booster immunostimulation with a pharmaceutical composition of the invention" (also referred to herein as "vaccine enhancement patch boosting" or "VEP boosting") means that a sufficient amount of the one or more active agents of the composition of the invention, e.g. , one or more polypeptides, in particular, an adjuvant or a compound with adjuvant-like properties (including a compound with both adjuvant and antigenic activity) but different to the antigen to which it will boost the immunity, is able to passively diffuse from the composition of the invention into the skin epidermal and/or dermal layers . In contrast with booster immunization, the compositions of the invention directed to booster immunostimulation essentially contain only compounds having adjuvant or adjuvant-like activity (including compounds that have both antigenic and adjuvant-like properties) but are different to the antigen for which the VEP boosting will raise the immunity. Because the VEP boosting may be dependent on the contacting of a sufficient number of immune cells in the skin, e.g. , Langerhans cells and dermal dendritic cells, the efficiency of the transcutaneous immunostimulation may be related to the surface area of the subject's skin exposed to the composition of the invention.

In certain aspects, the use of the pharmaceutical composition of the invention may encompass the pre-treatment of the skin of the subject in the area to which the composition is to be applied. In particular, pre-treatment in the context of the present invention entails the partial or total disruption and/or removal of the stratum corneum in the application area. As such, cleaning of the application area using standard procedures, e.g. , wiping with an alcohol pad or swab, is not considered "pre-treatment" because such standard cleaning does not sufficiently disrupt and/or remove the stratum corneum. The skin vaccination area is mildly abraded prior to VDP boosting or VEP boosting (also referred to herein as "pre-treated VDP boosting" or "pre-treated VEP boosting"). This mild abrasion not only increases antigen delivery through the skin, but also results in transepidermal water loss (TEWL), which facilitates hydration of the dry patch formulation. In addition, the mild abrasion, itself, can exert an immunostimulatory effect through activation of epidermal keratinocytes. Thus, in some aspects, the pre-treatment of the skin is effected with a "skin pretreatment device." As used in the context of the present invention, a "skin pre-treatment device" is meant to describe a mechanical device that is able to partially or totally remove the stratum corneum without pain, which is used before the application of the compositions of the invention to the subject's skin. Numerous physical techniques and devices to gently remove or disrupt the stratum corneum are known in the art. For example, such pre-treatment methods and devices are disclosed in WO 2007/028167. In short WO 2007/028167 discloses, e.g. a disposable strip-pull device to abrade the stratum corneum, which is a hand-held device with a mask platform containing an aperture over which an abrasive strip is pulled. Over the aperture is a push-button force control dome, which when pushed, allows the abrasive strip to be removed, thus gently abrading the stratum corneum under controlled pressure. The use of this device is simple and painless for the human subject. The device can be packaged together with a composition of invention as an integrated vaccine patch system (VDP system) or vaccine enhancement patch system (VEP system) with the potential for self- administration.

The pharmaceutical compositions of the invention are characterized by having a reduced moisture content percentage (MC ) relative to similar compositions of the prior art. As used herein the term "moisture content" in the present invention means the amount of water by weight of the pharmaceutical composition relative to the entire weight of said composition. Thus, the MC of the pharmaceutical composition is usually expressed as a percentage determined by the weight of water in the composition divided by the weight of the composition. The MC is determined for the pharmaceutical composition of the invention as a whole, thus is determined considering the dressing, formulation blend and any backings or liners added to the composition prior to packaging as a single unit. Thus the weight of the composition subsequent to the drying step(s) and subsequent to the application of backings and/or liners is determined, followed by determination of the weight of the water in total composition (i.e. , including all dressings, blends, backings and liners), allowing calculation of MC percentage. The moisture content can be measured by any method known in the art and/or as described herein. Several methods to determine MC are available in the art and such calculations are routine. One of the most common methods is the determination of water content based by Karl Fischer titration, which method as recognized in the art as particularly suited to automation using, e.g. , coulometric titration. Many commercial vendors offer coulometers suitable for implementation of Karl Fischer titration, e.g. , the DL36KF coulometer from Mettler Toledo, Inc. (Columbus, OH, USA).

As used herein, the term "glassy amorphous state" of e.g. the formulation blend of the invention is intended to mean a state of a solid, such as e.g. the formulation blend, that lacks the long-range order of a crystal and has a glassy appearance (see Figure 3). In the context of the invention a glassy amorphous state of the formulation blend is intended to mean an amorphous thin film consisting of solid layers of a few nm to some tens of micrometers thickness of amorphous formulation blend deposited upon an underlying substrate such as the dressing or fibers composing the patch matrix. In order to have an easy "test", this term "glassy amorphous state" of e.g. the formulation blend of the invention is defined herein as the appearance of a film of the formulation blend deposited upon an underlying substrate such as the dressing of the invention, wherein the lack of any visible crystals under the microscope such as e.g. a 10 times, 50 times, 100 times magnifying microscope, in the film determines the formulation blend as of a glassy amorphous state (again see also Figure 3 for illustration of the glassy amorphous state).

Manufacture

The methods of the invention result in a pharmaceutical composition characterized by decreased moisture content ("MC") relative to similar compositions known in the art, and, in particular, have a MC of below 10%. The decreased MC has surprisingly been discovered to impart prolonged stability at increased handling temperatures, e.g., room temperature, of the Dry formulations (see also WO00/61184). For purposes of distinction from other transcutaneous compositions known in the art, the pharmaceutical compositions of the invention are referenced herein as "Dry" compositions. The Dry compositions of the invention exhibit increased stability of the active agent or agents, i.e. , polypeptide or polypeptides, when subject to accelerated storage conditions relative to compositions manufactured according to standard methods in the art similarly tested. Accelerated storage conditions, for instance, can expose a composition to increased temperatures (e.g. , 25 to 50°C) over prolonged periods, e.g. , days, weeks or months, and are generally used to evaluate the stability of the composition over commercially relevant storage, shipping and handling times (e.g. , one or more years) outside of the cold chain (e.g. , at temperatures greater than 4°C, e.g. , room temperature). The increased stability of the Dry compositions allows them to be stored at room temperature over a prolonged period with little to no loss of activity of the active agent. This offers a primary advantage over polypeptide-based transcutaneous compositions known in the art, which must be stored and transported at reduced, temperatures, e.g. , 4°C. Thus, the Dry compositions of the invention do not require the "cold chain" for transport and storage and can allow for delivery and/ or use in areas previously inaccessible and/or inhospitable for such formulations.

Additionally, the Dry compositions of the invention do not rely on lyophilization and do not comprise lyophilized polypeptides or formulation blends. This not only minimizes production costs and complexity, but also eliminates the disadvantages of lyophilization of the formulation well recognized in the art. For example, it is recognized that a lyophilized powder does not typically adhere to suitable dressings, rendering the pharmaceutical composition unstable in that the powder separates from or falls out of the dressing within the package and is lost when the package is open and the dressing applied to the skin. Similar problems are encountered with spray dried formulations. Lyophilized and spray dried powders may also assemble over time into large complex aggregates, decreasing administration efficiency of the composition. In contrast, the Super Dry composition maintains a glassy amorphous state (see, e.g. , Figure 4), which minimizes the formation of large complex aggregates.

The present invention also relates to a process for making a pharmaceutical composition suitable for use in the form of a dressing for transcutaneous delivery of an active agent, e.g. , one or more polypeptides. The process essentially comprises the following steps:

a) providing a formulation blend comprising a polypeptide and a carbohydrate onto a dressing; and

b) drying said formulation blend and dressing to a moisture content of below 10% such as between 5 to 10%.

Step a) represents standard methods known in the art for the manufacture of therapeutic/prophylactic dressings. The composition may vary according to methods routinely practiced and well known to those skilled in the art. For example, pharmaceutical formulations comprising a dressing are typically prepared by coating the dressing with the wet composition and drying in a suitable oven until the moisture content of the composition is sufficiently reduced. Typical drying methods employ suitable ovens and temperatures as known in the art selected according to routine criteria. For example, Dry compositions may be dried in an oven at 45 °C for about 1 hour. In particular, the drying methods of the present invention allow the formulation blend of the composition to be dried to a glassy amorphous state. Such drying procedures can be determined by one of skill in the art using standard criteria.

The compositions of the invention (e.g. , dressing formulation blend and any backings and/or liners) may be dried in a single step, e.g. , in an oven at, e.g. , 45°C for at least one hour, until the MC of a Dry formulation is achieved. In certain aspects the Dry formulation of the composition is characterized by having a glassy amorphous state. Alterations to drying times and temperatures may be readily determined by one of skill in the art to achieve the required MC and/or to achieve the glassy amorphous state. For example, dependent on weather and/or local climate, it may be necessary to control the relative humidity of the manufacturing facility of the composition and/or the drying equipment, e.g. , oven, such that the composition may be dried to a MC of below 10%. In some embodiments, the relative humidity of the manufacturing facility and/or drying equipment may be controlled to below 30%, preferably below 10% such that a MC of below 10% can easily be achieved. Alternatively or additionally, other steps or components of the manufacturing chain may be altered such that the required MC is achieved. This may include, but not be limited to, pre-heating any component such as preheating the dressing, backing and/or liner, if present, to reduce the MC of the individual component prior to primary drying. Variations in the additional drying step may be implemented using criteria routinely used in the art.

Following the manufacture of the dry formulations, the compositions of the invention may be stored in an environment with low humidity, e.g. , about 1% relative humidity. This may be in a chamber designed to maintain such low humidity or, e.g. , in water-tight packaging having an low humidity internal environment, e.g. , about 1% relative humidity. For example, the solid pharmaceutical composition may be packaged to seal said composition from moisture and air. The package can be, e.g. , an aluminum pouch, or any suitable packaging material known in the art recognized to prevent moisture ingress.

The Dry compositions of the invention and those prepared according to the method of the invention are characterized by enhanced stability at increased temperatures, e.g. , room temperature or temperatures at least above 4°C. This allows the compositions of the invention to be stored and/or transported in the absence of a "cold chain." Stability may be assessed by any method known in the art for determining the stability of the particular therapeutic. For example, the stability of the super dry compositions of the invention can be measured by HPLC-SE based on the amount of the active ingredient, e.g. , one or more polypeptide, of the composition at a given time.

The invention also encompasses Dry compositions that show negligible decrease or decreases of less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5% or less than 1% in active agent content after storage at 40°C for one month as compared to the amount of active agent present at the beginning of the storage. Determination of content of active agent may be determined by a) dissolving the pharmaceutical composition in a suitable buffer, wherein the formulation blend will be reconstituted, and then b) analyzing the obtained solution by HPLC. Nonlimiting conditions for determination of peptide content include SE-HPLC using an Agilent 1100 Series HPLC system with UV detection at 220nm and a Tosoh Biosep TSK-Gel G2000SWXL, 7.8 x 300 nm (5 μιη) column; with a Tosoh Biosep SWXL, 6.0 x 40 nm (7 μιη) as a guard column and the following running conditions: mobile phase of 00.2M Sodium Phosphate Buffer (pH 7.2); flow rate of 1.0 ml/min; autosampler and column temperature of 2-8°C and 30°C + 2°C, respectively; injection volume of 50 μL·, and run time of 17.5 min.

The present invention further pertains to a pharmaceutical composition obtained by the methods and processes of the present invention. The Dry composition obtained by the process of the present invention has a low moisture content, i.e. below 10%, and can be stored at elevated temperatures, e.g. above 4°C, preferably at room temperature, for an elongated period.

The pharmaceutical composition may be manufactured under aseptic conditions with practices acceptable to the appropriate regulatory agencies (e.g., the Food and Drug Administration) for biologicals and vaccines. The relative amounts of active ingredients within a dose and the dosing schedule may be adjusted appropriately for efficacious administration to a subject (e.g., animal or human). This adjustment may also depend on the subject's particular disease or condition, and whether treatment or prophylaxis is intended. To simplify administration of the pharmaceutical composition to the subject, each unit dose contains the active ingredients in predetermined amounts for a single round of immunization. The amount of active ingredient, e.g. , polypeptide, of the pharmaceutical composition is administered as single or unit dose. The amount of active ingredient, e.g. , polypeptide, in the unit dose may be anywhere in a broad range from about 0.1 μg to about 10 mg. The range from about 1 μg to about 1 mg is preferred; the range from about 10 μg to about 500 μg is more preferred. Other suitable ranges are between about 1 μg and about 10 μg, between about 10 μg and about 50 μg, between about 50 μg and about 200 μg, and between about 1 mg and about 5 mg. VDP boosting

In the context of the present invention, VDP boosting means that a sufficient amount of the one or more active agents of the composition of the invention, e.g. , one or more polypeptides such as an antigen, an adjuvant or both, preferably an antigen only, is able to passively diffuse from the composition of the invention into the upper skin layers of a subject who has preferably be pre-treated with the skin pretreatment device (see above pretreated VDP boosting) at the VDP boosting site and who can induce a significant, i.e. around a two-fold increase or more in GMT (Geometric Mean Titer) of specific antibody to the antigen to be boosted (note: depending on the antigen the increase can be much higher) than a subject who was never vaccinated against the particular antigen. There in the skin upper layers, including the epidermis and dermis, the antigens (and adjuvants) are captured and processed by immunocompetent cells (e.g. antigen presenting cells such as Langerhans cells (LCs), dermal dendritic cells, keratinocytes, macrophages, and T lymphocytes), in particular by antigen presenting cells such as LCs and dermal dentritic cells. These antigen presenting cells (APCs) carry the antigen (and adjuvants) to the regional skin-draining lymph nodes to induce antibody and cellular immune responses. For example, it has successfully been demonstrated that LT is effective as an antigen in a VDP system for the prevention of travelers' diarrhea in clinical trials (McKenzie et al., 2007; Freeh et al., 2008).

VEP boosting

In connection with VEP boosting, the composition of the invention comprises essentially active agents that have adjuvant or adjuvant-like properties (including active agents that have both adjuvant and antigenic properties). VEP boosting is also referenced herein as the use of a VEP system in a booster setting. The VEP system works similarly as the VDP system. As mentioned above, the one or more active agents (e.g. , one or more polypeptides) such as an adjuvant, a compound with adjuvant-like properties or a compound with both adjuvant and antigenic properties, is able to passively diffuse from the composition of the invention (VEP) into the skin epidermal and/or dermal layers of a subject who has preferably be pre-treated with the skin pretreatment device (see above pretreated VEP boosting) at the VEP boosting site and who can induce a significant, i.e. around a two-fold increase or more in GMT (Geometric Mean Titer) of specific antibody to the antigen to be boosted (note: depending on the adjuvant and antigen system the increase can be much higher) than a subject who was never vaccinated against the particular antigen. Sensing a 'danger signal' represented by the adjuvant or adjuvant-like activity, the APCs become activated and migrate to the same draining lymph node field as an injected vaccine. It is believed that the adjuvant-activated APCs exert bystander and/or direct immunostimulatory effects on APCs loaded with injected antigen if they targeted the same draining LNs (Guebre-Xabier et al., 2003). TTx prime patch

In a further aspect of the invention, the invention provides a high dose tetanus toxoid patch formulation without the use of any adjuvants for use in a first immunization (also referred to as "transcutaneous primary immunization"), i.e. in an unprimed subject. In particular, the invention provides a pharmaceutical composition for transcutaneous primary immunization, wherein said composition comprises a) a dressing; and b) a formulation blend comprising i) the tetanus toxoid; and ii) a carbohydrate; and wherein the composition is a dry composition; and wherein optionally the skin at the site of transcutaneous primary immunization is pre- treated by a device so that the stratum corneum is disrupted at least partially without perforating the skin.

Antigen

An antigen for use in the compositions and/or methods of the invention may be expressed by recombinant technology, preferably as a fusion with an affinity or epitope tag; chemical synthesis of an oligopeptide, either free or conjugated to carrier proteins, may be used to obtain the antigen of the invention. Oligopeptides are considered a type of polypeptide and should have preferred lengths of 6 to 20 residues. Polypeptides may also be synthesized as branched structures (e.g. , U.S. Pat. Nos. 5,229,490 and 5,390, 1 11). Antigenic polypeptides include, for example, synthetic or recombinant B-cell and T-cell epitopes, universal T-cell epitopes, and mixed T-cell epitopes from one organism or disease. Antigen obtained through recombinant technology or peptide synthesis, as well as antigen obtained from natural sources or extracts, may be purified based on their physical and chemical characteristics, preferably by fractionation or chromatography. Recombinant antigens may be formed by combining B subunits or chimeras of bARE. A multivalent antigen formulation may be used to induce an immune response to more than one antigen at the same time. Conjugates may be used to induce an immune response to multiple antigens, to boost the immune response, or both. Additionally, toxins may be used in form of toxoids. Antigen includes, for example, surface antigen of the hepatitis B virus(HbSAg), Haemophilus influenzae type B vaccine (Hib), toxins, toxoids, subunits thereof, or combinations thereof (e.g., cholera toxin, tetanus toxoid, pertussis toxoid, diphtheria toxoid); additionally, toxins, toxoids, subunits thereof, or combinations thereof may act as both antigen and adjuvant. Such oral/transcutaneous or transcutaneous/oral immunization may be especially important to enhance mucosal immunity in diseases where mucosal immunity correlates with protection. An antigen can also be a high molecular weight protein or a protein within a large complex macromolecular assembly, such as a split and a whole inactivated virus.

The antigen may be solubilized in a buffer or water or organic solvents such as alcohol or DMSO, or incorporated in gels, emulsion, microemulsions, and creams. Suitable buffers include, but are not limited to, phosphate buffered saline Ca⁺⁺/Mg⁺⁺ free, phosphate buffered saline, normal saline (150 mM NaCl in water), and Hepes or Tris buffer. Antigen not soluble in neutral buffer can be solubilized in 10 mM acetic acid and then diluted to the desired volume with a neutral buffer such as PBS. In the case of antigen soluble only at acid pH, acetate-PBS at acid pH may be used as a diluent after solubilization in dilute acetic acid. Glycerol may be a suitable non-aqueous buffer for use in the invention.

A hydrophobic antigen can be solubilized in a detergent or surfactant, for example a polypeptide containing a membrane-spanning domain. Furthermore, for formulations containing liposomes or virus like particles, an antigen in a detergent solution (e.g., cell membrane extract) may be mixed with lipids, and liposomes then may be formed by removal of the detergent by dilution, dialysis, or column chromatography. Certain antigens (e.g., membrane proteins) need not be soluble per se, but can be inserted directly into a lipid membrane (e.g., a virosome), in a suspension of virion alone, or suspensions of microspheres or heat- inactivated bacteria which may be taken up by antigen presenting cells (e.g., opsonization). Antigens may also be mixed with a penetration enhancer as described in WO 99/43350.

Many antigens are known in the art which can be used to vaccinate and to boost human or animal subjects and induce an immune response specific for particular pathogens, as well as methods of preparing antigen, determining a suitable dose of antigen, assaying for induction of an immune response, and treating infection by a pathogen (e.g., bacterium, virus, fungus, or protozoan).

Moreover, antigens may be nucleic acids such as deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) (Somogyi et al., 2011 ; Xu et al., 2008).

The pharmaceutical booster composition may include additional antigens such that application to intact skin boosts an immune response to multiple antigens. In such a case, the antigens may or may not be derived from the same source, but the antigens will have different chemical structures so as to boost an immune response specific for the different antigens.

Adjuvant

The pharmaceutical composition may contain an adjuvant, in particular in the VEP booster setting. As known in the art, adjuvants are substances that are used to specifically or non- specifically potentiate an antigen-specific immune response, perhaps through activation of antigen presenting cells (e.g., dendritic cells in various layers of the skin, especially Langerhans cells) (see also Elson et al. in Handbook of Mucosal Immunology, Academic Press, 1994). Although activation may initially occur in the epidermis or dermis, the effects may persist as the dendritic cells migrate through the lymph system and the circulation. Adjuvant may be formulated and applied with or without antigen, but generally, activation of antigen presenting cells by adjuvant occurs prior to presentation of antigen. Alternatively, they may be separately presented within a short interval of time but targeting the same anatomical region (e.g., the same draining lymph node field). Adjuvants include any material known in the art or described herein that act as an adjuvant, in particular, for transcutaneous delivery and/or vaccination of peptides. For example, chemokines (e.g., defensins, HCC-1 , HCC4, MCP-1 , MCP-3, MCP4, ΜΙΡ-Ια, ΜΙΡ-Ι β, ΜΙΡ-Ιδ, MIP-3a, MIP-2, RANTES); other ligands of chemokine receptors (e.g., CCR1, CCR-2, CCR-5, CCR-6, CXCR-1); cytokines (e.g., IL-Ι β, IL-2, IL-6, IL-8, IL-10, IL-12; IFN-γ; TNF-a; GM-CSF); other ligands of receptors for those cytokines, immunostimulatory CpG motifs in bacterial DNA or oligonucleotides; muramyl dipeptide (MDP) and derivatives thereof (e.g., murabutide, threonyl-MDP, muramyl tripeptide); heat shock proteins and derivatives thereof; Leishmania homologs of elF4a and derivatives thereof; bacterial ADP-ribosylating exotoxins and derivatives thereof (e.g., genetic mutants, A and/or B subunit-containing fragments, chemically toxoided versions); chemical conjugates or genetic recombinants containing bacterial ADP-ribosylating exotoxins or derivatives thereof; C3d tandem array; lipid A and derivatives thereof (e.g., monophosphoryl or diphosphoryl lipid A, lipid A analogs, AGP, AS02, AS04, DC-Choi, Detox, OM-174); ISCOMS and saponins (e.g., QUIL A, QS-21); squalene; superantigens; or salts (e.g., aluminum hydroxide or phosphate, calcium phosphate) (see also Nohria et al., Biotherapy, 7:261-269, 1994 and Richards et al., Vaccine Design, Eds. Powell et al., Plenum Press, 1995) for other useful adjuvants.

Adjuvant may be chosen to preferentially induce antibody or cellular effectors, specific antibody isotypes (e.g., IgM, IgD, IgAl , IgA2, secretory IgA, IgE, IgGl , IgG2, IgG3, and/or IgG4), or specific T-cell subsets (e.g., CTL, Thl , Th2 and/or TDTH)- For example, antigen presenting cells may present Class II-restricted antigen to precursor CD4+ T cells, and the Thl or Th2 pathway may be entered. T helper cells actively secreting cytokine are primary effector cells; they are memory cells if they are resting. Reactivation of memory cells produces memory effector cells. Thl characteristically secrete IFN-γ (TNF-β and IL-2 may also be secreted) and are associated with "help" for cellular immunity, while Th2 characteristically secrete IL-4 (IL-5 and IL-13 may also be secreted) and are associated with "help" for humoral immunity. Depending on disease pathology, adjuvants may be chosen to prefer a Thl response (e.g., antigen-specific cytolytic cells) vs. a Th2 response (e.g., antigen- specific antibodies).

Unmethylated CpG dinucleotides or similar motifs are known to activate B lymphocytes and macrophages (see U.S. Pat. No. 6,218,371). Other forms of bacterial DNA can be used as adjuvants. Bacterial DNA is among a class of structures which have patterns allowing the immune system to recognize their pathogenic origins to stimulate the innate immune response leading to adaptive immune responses. These structures are called pathogen-associated molecular patterns (PAMP) and include lipopolysaccharides, teichoic acids, unmethylated CpG motifs, double-stranded RNA, and mannins. PAMP induce endogenous signals that can mediate the inflammatory response, act as costimulators of T-cell function and control the effector function. The ability of PAMP to induce these responses play a role in their potential as adjuvants and their targets are antigen presenting cells such as dendritic cells and macrophages. The antigen presenting cells of the skin could likewise be stimulated by PAMP transmitted through the skin. For example, Langerhans cells, a type of dendritic cell, could be activated by PAMP in solution on the skin with a transcutaneously poorly immunogenic molecule and be induced to migrate and present this poorly immunogenic molecule to T-cells in the lymph node, inducing an antibody response to the poorly immunogenic molecule. PAMP could also be used in conjunction with other skin adjuvants such as cholera toxin to induce different costimulatory molecules and control different effector functions to guide the immune response, for example from a Th2 to a Thl response.

A further adjuvant is known as IC31^® that comprises as active ingredients (dIdC) i3 (also referred to as ODNla) and KLKL5KLK (also referred to as KLK) e.g. as described in WO04/084938 and PCT/EP2011/052496.

The adjuvant of the invention may also be an ADP-ribosylating exotoxins (bARE). Most bAREs are organized as holotoxins consisting of A and B subunits, and can exist in AB or AB₅ assembly. Typically, the B subunit contains the receptor binding activity and the A subunit contains the ADP-ribosyltransferase activity. Exemplary bARE include cholera toxin (CT) E. coli heat- labile enterotoxin (LT), diphtheria toxin, Pseudomonas exotoxin A (ETA), pertussis toxin (PT), C. botulinum toxin C2, C. botulinum toxin C3, C. limosum exoenzyme, B. cereus exoenzyme, Pseudomonas exotoxin S, S. aureus EDIN, and B. sphaeticus toxin. Mutant bARE, for example containing mutations of the trypsin cleavage site (e.g., Dickenson et al., Infect Immun, 63: 1617-1623, 1995) or mutations affecting ADP-ribosylation (e.g., Douce et al., Infect Immun, 65:28221-282218, 1997) may be used.

CT, LT, ETA and PT, despite having different cellular binding sites, are potent adjuvants for transcutaneous immunization, inducing humoral and cellular immune responses to coadministered antigens. CTB without CT can also function as an adjuvant. Thus, both bARE and a derivative thereof can effectively serve as topical adjuvant when epicutaneously applied to the skin and can be used.

In general, toxins can be chemically inactivated to form toxoids which are less toxic but remain immunogenic. We envision that the transcutaneous immunization system using toxin- based immunogens and adjuvants can achieve anti-toxin levels adequate for protection against these diseases. The anti-toxin antibodies may be induced through immunization with the toxins, or genetically-detoxified toxoids themselves, or with toxoids and adjuvants. Genetically toxoided toxins which have altered ADP-ribosylating exotoxin activity or trypsin cleavage site, but not binding activity, are envisioned to be especially useful as non-toxic activators of antigen presenting cells used in transcutaneous immunization and may reduce the safety concerns regarding the use of native toxins. bARE can also act as an adjuvant to induce antigen- specific CTL through transcutaneous immunization. The bARE adjuvant may be chemically conjugated to other antigens including, for example, carbohydrates, polypeptides, glycolipids, and glycoprotein antigens. Chemical conjugation with toxins, their subunits, or toxoids with these antigens would be expected to enhance the immune response to these antigens when applied epicutaneously. To overcome the problem of the toxicity of the toxins (e.g., diphtheria toxin is known to be so toxic that one molecule can kill a cell) and to overcome the problems of working with such potent toxins as tetanus, several workers have taken a recombinant approach to producing genetically- produced toxoids. This is based on inactivating the catalytic activity of the ADP-ribosyl transferase by genetic deletion. These toxins retain the binding capabilities, but lack the toxicity, of the natural toxins. Such genetically toxoided exotoxins would be expected to induce a transcutaneous immune response and to act as adjuvants. They may provide an advantage in a transcutaneous immunization system in that they would not create a safety concern as the toxoids would not be considered toxic. Activation through a technique such as trypsin cleavage, however, would be expected to enhance the adjuvant qualities of LT through the skin which lacks trypsin-like enzymes. Additionally, several techniques exist to chemically modify toxins and can address the same problem. These techniques could be important for certain applications, especially pediatric applications, in which ingested toxins might possibly create adverse reactions.

Adjuvant may be biochemically purified from a natural source (e.g., pCT or pLT) or recombinantly produced (e.g., rCT or rLT). ADP-ribosylating exotoxin may be purified either before or after proteolysis (i.e., activation). B subunit of the ADP-ribosylating exotoxin may also be used: purified from the native enzyme after proteolysis or produced from a fragment of the entire coding region of the enzyme. The subunit of the ADP-ribosylating exotoxin may be used separately (e.g., CTB or LTB) or together (e.g., CTA-LTB, LTA-CTB) by chemical conjugation or genetic fusion.

Exotoxins with point mutations (e.g., single, double, or triple amino acid substitutions) or deletions (e.g., protease recognition site), and isolated functional domains of ADP- ribosylating exotoxin may also be used as adjuvant. Derivatives which are less toxic or have lost their ADP-ribosylation activity, but retain their adjuvant activity have been described. Specific mutants of E. coli heat-labile enterotoxin include LT-K63, LT-R72, LT (H44A), LT (R192G), LT (R192G/L211A), and LT (Δ192-194) and are for the purposes of this invention included in the term as ADP-ribosylating exotoxin. Toxicity may be assayed with the Y-l adrenal cell assay (see Clements and Finkelstein, Infect Immun, 24:760-769, 1979). ADP- ribosylation may be assayed with the NAD-agmatine ADP-ribosyltransferase assay (see Moss et al., J Biol Chem, 268:6383-6387, 1993). Particular ADP-ribosylating exotoxins, derivatives thereof, and processes for their production and characterization are described in U.S. Pat. Nos. 4,666,837; 4,935,364; 5,308,835; 5,785,971 ; 6,019,982; 6,033,673; and 6,149,919.

Any activator of Langerhans cells or dermal dendritic cells may also be used as an adjuvant. Examples of such activators include: inducers of heat shock protein; contact sensitizers (e.g., trinitrochlorobenzene, dinitrofluorobenzene, nitrogen mustard, pentadecylcatechol); toxins (e.g., Shiga toxin, Staph enterotoxin B); lipopolysaccharide (LPS), lipid A, or derivatives thereof; bacterial DNA; cytokines (e.g., TNF-a, IL-Ι β, IL-10, IL-12); members of the TGF superfamily, calcium ions in solution, calcium ionophores, and chemokines (e.g., defensins 1 or 2, RANTES, MIP-la, MIP-2, IL-8).

Other techniques for enhancing activity of adjuvants may be effective, such as adding surfactants and/or phospholipids to the formulation to enhance adjuvant activity of ADP- ribosylating exotoxin by ADP-ribosylation factor. One or more ADP-ribosylation factors (ARF) may be used to enhance the adjuvant activity of bARE (e.g., ARF1 , ARF2, ARF3, ARF4, ARF5, ARF6, ARD1). Similarly, one or more ARF could be used with an ADP- ribosylating exotoxin to enhance its adjuvant activity.

Undesirable properties or harmful side effects (e.g., allergic or hypersensitive reaction; atopy, contact dermatitis, or eczema; systemic toxicity) may be reduced by modification without destroying its effectiveness in transcutaneous immunization. Modification may involve, for example, removal of a reversible chemical modification (e.g., proteolysis) or encapsulation in a coating which reversibly isolates one or more components of the formulation from the immune system. For example, one or more components of the formulation may be encapsulated in a particle for delivery (e.g., microspheres, nanoparticles) although we have shown that encapsulation in lipid vesicles is not required for transcutaneous immunization and appears to have a negative effect. Phagocytosis of a particle may, by itself, enhance activation of an antigen presenting cell by upregulating expression of MHC Class I and/or Class II molecules and/or costimulatory molecules (e.g., CD40, B7 family members like CD80 and CD86).

Application

The pharmaceutical composition can be applied directly to the skin, e.g. , on the deltoid or thigh. The attending physician or other health care provider can determine the optimal site for placement using standard criteria routinely implemented in the art. Before application of the pharmaceutical composition, the skin may be optionally cleaned by using e.g. water or disinfectant as known in the art. It is noted that such standard cleaning or disinfecting of the skin does not comprise the "skin pre-treatment" as disclosed herein, which requires the purposeful disruption and/or removal (partial or total) of the stratum corneum. With respect to a vaccine of the present invention, the solid pharmaceutical composition may be applied to intact skin overlying more than one draining lymph node field using either single or multiple applications.

The application site may be protected with anti-inflammatory corticosteroids such as hydrocortisone, triamcinolone and mometazone or non-steroidal anti-inflammatory drugs (NSAIDs) to reduce possible local skin reaction or modulate the type of immune response. Similarly, anti-inflammatory steroids or NSAIDs may be included in the patch material, in creams, ointments, etc. and corticosteroids or NSAIDs may be applied after immunization. IL-10, TNF-a, other immunomodulators may be used instead of the anti-inflammatory agents.

The skilled person knows that the effective amount of pharmaceutical compositions administered to an individual may, inter alia, depend on the nature of the polypeptide and the nature of the subject and condition to be treated. For example, the total pharmaceutically effective amount of pharmaceutical composition administered per dose may be in the range of about 0.01 μg/kg/day to 100 mg/kg/day of patient body weight, or 1 μg/kg body weight to about 40 mg/kg body weight per day, or about 1 mg/kg body weight to about 30 mg/kg body weight, or about 1 mg/kg body weight to about 20 mg/kg body weight per day, or about 1 mg/kg body weight to about 15 mg/kg body weight per day, or about 1 mg/kg body weight to about 10 mg/kg body weight per day, or about 10 mg/kg body weight to about 15 mg/kg body weight per day, although, as noted above, this will be subject to therapeutic discretion. However, this dose may be further decreased or increased subject to therapeutic discretion. The particular amounts may be determined by conventional tests which are well known to the person skilled in the art. As a nonlimiting example of a dosing schedule, the composition of the LT VDP against moderate to severe travelers' diarrhea may be 7.5 μg, 22.5 μg, 37.5 μg, or 50 μg LT applied to the deltoid or lower back usually in 2 doses (2^nd does after 2 weeks) and the composition is left on the skin for about 6 hours.

EXAMPLES

Example 1: Manufacture of Dry LT Patch The following example provides an exemplary method for the manufacture of a pharmaceutical composition in the form of a dry patch comprising a polypeptide as an active agent, e.g. , as an antigen or adjuvant. Specifically, the dry patch comprises heat-labile enterotoxin from E. coli (LT) as an active agent. The exemplified manufacturing process comprised four steps:

1) Preparation of final formulation blend;

2) Preparation of patch components (template);

3) Dosing and drying; and

4) Packaging.

As detailed herein, dry patches were manufactured according to standard practice in the art (1 hour at 45 °C in a conventional convection oven).

1. Preparation of Final Formulation Blend

The following section describes the formulation process for preparing the final formulation blend for a dry patch. The particular example described herein relates to a dry patch comprising LT as an active agent. In general, the formulation process involves the manufacture/processing of the active agent into a bulk, stock solution followed by the mixing of the stock solution of active agent and those of any desired stabilizers and/or preservatives.

1.1 Preparation of LT

LT is a heterohexameric protein consisting of one A subunit and five B subunits. The amino acid sequence of the A subunit for production was,

KLYRADSRPPDEIKRSGGLMPRGHNEYFDRGTQMNINLYDHARGTQTGFVRYDDGY VSTSLSLRSAHLAGQSILSGYSTYYIYVIATAPNMFNVNDVLGVYSPHPYEQEVSALG GIPYSQIYGWYRVNFGVIDERLHRNREYRDRYYRNLNIAPAEDGYRLAGFPPDHQAW REEPWIHHAPQGCGNSSRTITGDTCNEETQNLSTIYLRKYQSKVKRQIFSDYQSEVDIY NRIRNEL (SEQ ID NO: l); and that for the B subunit was,

PQSITELCSEYRNTQIYTINDKILSYTESMAGKREMVIITFKSGATFQVEVPGSQHIDSQ KKAIERMKDTLRITYLTETKIDKLCVWNNKTPNSIAAISMEN (SEQ ID NO:2). The LT polypeptide was rccombinantly expressed in the Pscnubmonas fluorescence-based expression system available from Pfcnex, Inc. (San Diego, CA, USA). The polypeptide was isolated and purified using routine methods in the art. i .2 Mixing Of Bulk/Stock Solutions

Three different bulk/stock solutions were used in the preparation of the formulation, blend: A) LT bulk, B) 3X stabilizer bulk and C) NaPi S/P bulk, as indicated in Fig u re 6.

The composition of each bulls stock solution used is listed below:

A) LT bulk;

LT concentration ranged from 3.5 to 4.5 mg/ml, 5% sucrose, 0.1 % .Pluvonie, 75 m.M NaCl and 10 mM NaPi, pH 7.3

B) 3X stabiliser bulk;

26% sucrose, 24% aititoL 3% PVP, 0.1 % Pluronic, 10 m NaPi at pH 7.3

C) NaPi P/S bulk;

5% sucrose.. 0.1 % Pluronic. 10 mM; NaPi, pH 7.3

The specific composition of the bulk/stock solutions are also detailed in Table 1 :

Table 1 : Composition pf Bulk Formulations^'

2S Bulk

3X 100.00 0.00 26.0 24.0 3.0 0.10 0 10.0 7.3 Stabilizer

Bulk

NaPi S/P 170.08 0.00 5.9 0.0 0.0 0.10 0 10.0 7.3 Bulk

Formulation 300.00 0.375 12.0 8.0 1.0 0.10 7.48 10.0 7.3 Blend

The formulation blend was designed to have a final concentration of 0.375 mg/ml. Standard calculations can be used to determine the volumes of the bulk/stock solutions to be mixed to achieve any desired concentration and any desired final volume of formulation blend. For example, in the present experiment 300 ml of final formulation blend with LT concentration at 0.375 mg/ml required 29.92 ml of LT bulk (LT concentration at 3.76 mg LT/ml), 100 ml of 3X stabilizer bulk and 185.04 ml of NaPi S/P bulk as indicated in Table 1. The formulation blend was mixed gently with stir bar until visually uniform. The mixed blend was then filtered through a 0.2 μιη PES membrane into a sterile bottle or Hyclone bag. The entire operation was performed aseptically in a bio-safety cabinet. The final formulation contained 0.375 mg LT/ml, 12% sucrose, 8% maltitol, 1% PVP, 0.1% Pluronic L68, 10 mM NaPi at pH 7.3. This formulation blend was used for the preparation of 7.5 μg LT patches used in the subsequent experiments.

Although formulation blends may be maintained for prolonged periods under proper conditions, for most patch preparation described in these examples, the dosing and drying operations were performed within 24 hours of filtration.

2. Preparation of patch components (Template)

The following section describes the preparation of the template consisting of patch components for manual dosing and drying operation. The template consisted of circular empty rayon discs, a release liner sheet, a paper printout and Plexiglas^® (Figure 3) The materials of the patch components are also listed in Table 2.

Table 2: Materials Used in Patch Components Materials Vendor Product* Lots* Expiration Date

Drug Disc Sheet Berkshire Lensz 90 (rayon) 6010159₄2 NA

Release Liner Sheet St Gobain Supra 9022 coated 2 mil polyester film S2580191 .002 NA

Gaitherbursburg

Plexiglas Plate 13"x20"x1/₄" NA NA

Glass

2.1 Circular Rayon Disc

3 cm² circular rayon discs were punched out of 4 X 6 inch sheets of non-woven rayon fabric Lensx^® 90 (Berkshire Co., Great Barrington, MA, USA) using a swing arm die and ¼ inch steel tubular die. In a typical preparation, 5,000 empty rayon discs were cut.

2.2 Printout

A printout was prepared, with a total area of 17" X 11", consisting of the location of 35 circular rayon discs.

2.3 Template

The template was assembled in the following steps:

i) the printout was laid on top of a Plexiglas^® plate;

ii) a transparent release liner sheet was placed on top of the printout (the release liner sheet is a polyester film of about 2 mm thick that serves as a backing supporting the rayon disc during dosing and drying operations);

iii) the transparent sheet and the printout were taped to a Plexiglas plate; and

iv) 35 circular rayon discs were then placed on top of the transparent release liner according to the layout of the printout.

3. Dosing/Drying Operation

After the templates were prepared, dosing of the final formulation blend onto rayon discs was performed by using an IVEK pump (model#3009). 20-40 μΐ of the final formulation blend was dosed on the empty rayon disc. After all rayon discs were dosed with the final formulation blend, the template containing wet rayon discs was placed in a drying oven (9005L Stability Chamber from Sheldon) for 1 hour at 45 °C. After drying, a release liner sheet was placed on the template where the dried rayon discs were sandwiched between two release liners sheets. A pressure roller was used to press the two release liners together to ensure the dried rayon discs were held tightly between the two release liners. 4. Packaging

After the drying, the sheets containing the pharmaceutical formulation (i.e., the dried rayon disc comprising the formulation blend sandwiched between two release liner sheets) were cut into a 2" X 2" square and packaged into an aluminum pouch (Graphic Packaging) sealed under nitrogen purge.

Example 2: Tetanus toxoid (TTx) booster patch

1. Objectives

Compare the booster effect (immunogenicity) of the non-adjuvanted TTx patch versus subcutaneous injection in the guinea pig model

2. Model and Approach (see Table 3)

TTx (Statens Serum Institut, (Copenhagen, Denmark)) in saline solution was stabilized with 3X stabilizing buffer solution containing sucrose, maltitol, pluronic, and 10 mM NaPi at pH 7.3 but without PVP, before application on patch as similarly outlined in example 1.

Guinea pigs were primed on day 0 by subcutaneous injection with TTx (adsorbed on aluminium hydroxide).

The first bleed was collected on day 35.

Animals were boosted on day 37 with TTx patch or with s.c. injection of TTx (adsorbed on aluminium hydroxide).

The final bleed was collected on day 51.

Animal group - see table 3 (5 animals per group)

Read out: ELISA according to Ph. Eur. 5.1, i.e. measuring total anti-TTx IgGs

Table 3: Tetanus boost study - animal groups

Results

Effect of TTx boost after a prime with 0.5 Lf TTx s.c. (Figure 2):

A highly significant (paired t-test, two-tailed) increase in titers was observed two weeks after the boost in all groups after prime with 0.5 Lf TTx s.c.

No significant (unpaired t-test, two-tailed) difference in the titer was observed after a boost with either 5.0 Lf TTx s.c, 18 Lf TTx or 100 Lf TTx on the patch.

A boost with 0.5 Lf TTx s.c. gave a significantly (unpaired t-test, two-tailed) lower titer than the other boosts.

There was no significant difference in the boost given with 18 Lf or 100 Lf TTx on patch.

As expected, the patch placebo control did not induce any significant antibody response to TTx (data not shown).

Summary

A boostering effect was seen in the groups primed subcutaneously with 0.5 Lf TTx. Boosting with 18 Lf TTx on patch is equivalent to 5.0 Lf TTx s.c. boost, after priming with 0.5 Lf TTx s.c.

The dry TTx patches did not contain any adjuvant.

Little, if any, local reactions were observed on the skin after patch administration (data not shown).

Example 3: Tetanus toxoid (TTx) prime patch

1. Objectives

Compare the immunogenicity of the non-adjuvanted TTx patch versus subcutaneous injection in the guinea pig model in the primary immunization setting (TCI immunization)

2. Model and Approach (see Table 4)

Guinea pigs were primed on day 0 by subcutaneous injection with TTx (adsorbed on aluminium hydroxide) or 100 Lf patch.

The bleed was collected on day 35.

Animal group - see table 4 (5 animals per group)

Read out: ELISA according to Ph. Eur. 5.1, i.e. measuring total anti-TTx IgGs Table 3: Tetanus prime study - animal groups

Sera for ELISA were taken five weeks after primary immunization

The ELISA titers from the groups with the same prime were combined

In the 100 Lf TTx patch group was one low responder, titer >10 fold lower than the second lowest

There was no significant difference in the primary immune response between administration of 0.5 Lf TTx subcutaneously and 100 Lf TTx on a patch (see Figure Example 4: Clinical study - Phase I study investigating an adjuvant patch (Vaccine Enhancement Patch - VEP) containing LT (a heat-labile toxin from E. coli) in combination with a A/H5N1 antigen

The Phase 1 study was performed to confirm the mode of action of transcutaneous applied adjuvants when co-administered with non adjuvanted Influenza A/H5N1 antigen, following different and inconsistent results from previous Phase I and Phase II clinical studies.

The study involved 300 healthy adults and investigated two combinations of H5N1 antigen doses with or without patch in one and two injection regimes. An adjuvanted and licensed H5N1 vaccine was used to provide a positive control arm and a well established and validated H5N1 hemagglutination inhibition (HI) assay was applied to measure the immune response.

The combination of A/H5N1 with VEP met two of three CHMP criteria for Pandemic Influenza Vaccines (GMT fold rise from day 0 and Seroconversion). However, the study endpoint of a 2 or more fold rise in HI titers was not achieved since the immunogenicity was only moderately increased by VEP.

Further analysis revealed that the VEP effect was more pronounced and statistically significant on titer, seroconversion and seroprotection in subjects with existing HI titer (of > 1:10 at day 21) compared to A/H5N1 alone which indicates the potential use of the VEP for booster immunostimulation (see Table 5).

The overall local and systemic adverse event rate was similar across all treatment groups and the local safety profile for the VEP was as expected from previous observations in various clinical studies were LT was administered transcutaneously.

Table 5: Post-hoc Analysis: Comparison of subjects in 2x 15μg HA with VEP and 2x 15μg HA alone who had HI titers of at least 1:10 at Day 21

Seroconversion, Seroprotection and GMT at Day 42:

Seroconversion 25 (55.6) [41.2,69.1] 31 (93.9) [80.4,98.3] 0.0002 Seroprotection 26 (57.8) [43.3,71.0] 31 (93.9) [80.4,98.3] 0.0003

GMT 45.5 [34.5,60.1] 80.0 [65.4,97.9] 0.0026**

Percentages are based on the number of non-missing observations (Total) *: Two-sided 95% confidence intervals calculated according Altman

*: Wilcoxon Test (normal approx.)

REFERENCES

US 4,666,837

US 4,816,249

US 4,886,663

US 4,935,364

US 5,049,387

US 5,068,177

US 5,102,663

US 5,141,742

US 5,227,159

US 5,229,490

US 5,262,177

US 5,308,835

US 5,390,111

US 5,538,866

US 5,552,300

US 5,612,035

US 5,614,192

U.S. 5,679,647

US 5,698,416 US 5,785,971

US 5,910,306

US 5,980,898

US 6,019,982

US 6,033,673

US 6,149,919

US 6,218,371

US 6,797,276

US 7,037,499

US 7,378,097

US 7,527,802

WO 98/20734

WO 99/43350

US 2009/0081244 Al

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Claims

1. A pharmaceutical composition for transcutaneous booster immunization, wherein said composition comprises

a) a dressing; and

b) a formulation blend comprising

i) the antigen; and

ii) a carbohydrate;

and wherein the composition is a dry composition;

and wherein optionally the skin at the site of transcutaneous booster immunization is pre-treated by a device so that the stratum corneum is disrupted at least partially without perforating the skin.

2. The pharmaceutical composition of claim 1 that is administered to a subject with a two-fold or more rise of GMT in specific antibody directed against said antigen compared to the GMT against said antigen before a first immunization of the subject.

3. The composition of any one of claims 1 to 2, wherein said formulation blend is in a glassy amorphous state.

4. The composition of any one of claims 1 to 3, wherein the formulation blend further comprises at least one polyol.

5. The composition of any one of claims 1 to 4, wherein the carbohydrate is a non- reducing sugar, preferably sucrose.

6. The composition of any of claims 1 to 5, wherein the formulation blend further comprises a buffer.

7. The composition of any one of claims 1 to 6, wherein said formulation blend further comprises an ionic component.

8. The composition of any one of claims 1 to 7, wherein said composition further comprises a backing and/or liner.

9. The composition of any one of claims 1 to 8, wherein the antigen is an antigen that is able to active a Langerhans cell or an immunocompetent cell in the skin and does not comprise an adjuvant.

10. The composition of any of claims 1 to 9, wherein said antigen is a surface antigenof the hepatitis B virus(HbSAg), a Hib polysaccharide-protein conjugate (Hib), a toxoid, tetanus toxoid, pertussis toxoid, diphtheria toxoid, or combinations of toxoids, inactivated or attenuated poliovirus, bacterial ADP-ribosylating exotoxin (bARE), cholera toxin (CT), heat-labile enterotoxin from E. coli (LT) or mutant LT, a macromolecular assembly, virus particle or a fragment of a virus particle.

11. The composition of claim 10, wherein said antigen is a toxoid.

12. The composition of any of claims 1 to 11 , wherein the dry composition is a composition with a moisture content of less than 10%.

13. A kit comprising a package comprising the composition of any one of claims 1 to 12, a device able to at least partially disrupting the stratum corneum without perforating the skin, wherein said kit is for use in transcutaneous booster immunization.

14. A pharmaceutical composition for transcutaneous booster immunostimulation, wherein said composition comprises

a) a dressing; and

b) a formulation blend comprising

i) the adjuvant; and

ii) a carbohydrate;

and wherein the composition is a dry composition;

and wherein optionally the skin at the site of transcutaneous booster immunostimulation is pre-treated by a device so that the stratum corneum is disrupted at least partially without perforating the skin.

15. A pharmaceutical composition for transcutaneous primary immunization,

wherein said composition comprises

a) a dressing; and

b) a formulation blend comprising

i) the tetanus toxoid; and

ii) a carbohydrate;

and wherein the composition is a dry composition that does not contain any adjuvants; and wherein optionally the skin at the site of transcutaneous immunization is pre- treated by a device so that the stratum corneum is disrupted at least partially without perforating the skin.