ES2349672T3 - PROCEDURE FOR MEASURING THE ENZYMATIC ACTIVITY OF ADSORBED ALLERGEN ENZYME. - Google Patents

Abstract

A method for measuring the immunological activity of a vaccine preparation in the form of a mixture of one or more allergenic enzymes and aluminum hydroxide, in which the mixture comprises a liquid phase and a solid phase, and in which at least a part of the allergenic enzyme (s) is adsorbed in the solid phase, a process comprising the steps of measuring the enzymatic activity of the mixture and / or measuring the enzymatic activity of the allergenic enzyme in the liquid phase after separation of the liquid phase from the solid phase, and / or measure the enzymatic activity of the allergenic enzyme in the solid phase after a separation of the liquid phase from the solid phase, in an enzyme activity test, and use the measurement obtained as an indication of the immunological activity of the vaccine preparation.

Description

The present invention relates to an in vitro method for measuring the enzymatic activity of one or more allergenic enzymes in a vaccine preparation and thus obtaining an indication of the immunological activity and / or a quantification of the amount of allergenic enzyme in the preparation of vaccine. BACKGROUND OF THE INVENTION

The interaction of proteins with surfaces is a widely recognized phenomenon of both physiological and technological importance. An important example is the adsorption of protein allergens to the aluminum hydroxide adjuvant in allergy vaccines. An adjuvant is a compound that acts by potentiating the immune response after vaccination. The effect of aluminum hydroxide adjuvant has been extensively investigated and numerous theories have been proposed regarding the mechanism.

Allergy vaccines for, for example, subcutaneous injection can be prepared by mixing an aqueous solution of an allergen and a solid phase vehicle, for example, aluminum hydroxide gel, to produce a mixture in which at least a part of the allergen it is adsorbed in the solid phase and part of or none of the allergen is in the liquid phase. The solid phase vehicle may serve as an adjuvant, that is, it enhances the allergen's immune response, although the potentiation mechanism is not always fully understood. Similarly, the mechanism and nature of the adsorption of the allergen in the solid phase vehicle is not always fully understood and may depend entirely on the type of allergen involved. Theoretically, however, adsorption on aluminum hydroxide gels partially involves electrostatic forces. For proteins, it is believed that phosphorylated protein phosphate groups also interact with the aluminum hydroxide gel and possibly replace to some extent the hydroxide groups in the gel structure.

The protein adsorption capacity of aluminum hydroxide has been studied deeply with ovalbumin (OA) and bovine serum albumin (BSA) model proteins. Recently, studies have been carried out concerning the structural impact of protein adsorption on aluminum hydroxide. Emission fluorescence measurements along with differential scanning calorimetry indicate that significant structural alterations occur after adsorption of OA and BSA in aluminum hydroxide (Jones et al., Effects of Adsorption to Aluminum Salt Adjuvants on the Structure and Stability of Model Protein Antigens, The Journal of Biological Chemistry, vol. 280, p. 13406-13414, 2005). Another study indicates by the contario that the presence of aluminum hydroxide in an ELISA experiment helped maintain OA in the native conformation (Houen et al., A Non-denaturing Enzyme Linked Immunosorbent Assay With Protein Preadsorbed Onto Aluminum Hydroxide, Journal of Immunological Methods, vol. 200, p.

99-105, 1997). The OA adsorbed on aluminum hydroxide prior to transfer to the plastic surface of the well of a microtiter plate maintained its ability to bind monoclonal antibodies produced against the native form of OA. In contrast, OA was not pre-incubated with aluminum hydroxide bound to monoclonal antibodies produced against heat-denatured albumin. However, these techniques do not provide information on individual proteins present in a protein mixture.

The effect of aluminum hydroxide on the structure and stability of allergens is important from several perspectives. Conformational epitopes may be lost during adsorption and immunogenicity may be altered as a result of storage for a longer period of time.

The degree of adsorption varies with the nature of the specific allergen in question. In the case of an allergen in the form of an extract of a biological material, for example, an extract of grass pollen allergens, the extract contains several different ions and molecules that possibly interfere with the binding of the allergens to the solid phase vehicle. .

The house dust mite (HDM) Dermatophagoides pteronyssinus is an important source of inhaled allergens. Der p 1 and Der p 2 protein allergens are considered the two most potent allergens of Der p allergens. The structure and enzymatic activity of Der p 1 have been well characterized. Several in vitro studies suggest that Der p 1 cysteine protease activity enhances the potency of the allergen, for example, by cleaving proteins from the occlusion zone in the epithelial cells of the lung and cleavage CD23 (low affinity IgE receptor) in human B cells (Jacquet et al., Biochemical and Immunological Characterization of a Recombinant Precursor form of the House Dust Mite Allergen Der p 1 produced by Drosophila cells, Clinical and Experimental Allergy, vol. 30, p. 784-793, 2000). Vaccines against HDM based on the aluminum hydroxide adjuvant contain purified HDM extract as a pharmaceutical active ingredient (PAF).

The allergenic activity and the possibility of inducing allergic reactions can be tested, for example, by intradermal injection in sensitized animals and by measuring the change of various symptoms (Kildsgaard et al., Assessment of the in vivo allergenic potency of new allergy vaccines by intradermal testing in sensitized mice, Clinical Immunology and Allergy in Medicine, Proceedings of the 21st EAACI Congress 2002, Naples, Italy). However, such in vivo procedures are laborious and time-consuming and need the use of test animals, which is not desired.

Until now it has been a common practice to evaluate the immunological activity of an in vitro vaccine based on a measurement of the immunological activity of the allergen solution used for the preparation of the ready-to-use solid phase vehicle vaccine.

WO2005 / 022157 discloses an in vitro method for evaluating the immunological activity of a vaccine preparation in the form of a mixture of a molecular antigen and a vehicle, in which the mixture comprises a liquid phase and a solid phase at which At least a part of the antigen is bound, a method comprising the steps of i) subjecting the vaccine to a measurement of the immunological activity selected from the group consisting of a) antibody binding capacity using an immunoassay employing an antigen specific antibody bound to a solid phase of antibody, b) ability to activate effector cells and c) possibility of inducing anaphylaxis; and ii) use the measurement results to assess the immunological activity of the vaccine. WO2005 / 022157 does not disclose the measurement of the enzymatic activity in a product containing aluminum hydroxide as an adjuvant and does not disclose the use of the measured enzymatic activity to evaluate the immunological activity of the vaccine.

The nature of the adsorption of allergens to aluminum hydroxide adjuvants is very complex and widely unknown and is also expected to vary between different allergens. The object of the present invention is to provide a new in vitro method for evaluating and quantifying the immunological activity of allergy vaccine preparations based on aluminum hydroxide adjuvants such as ready-to-use vaccines. SUMMARY OF THE INVENTION

According to one aspect of the invention there is provided a method for measuring the immunological activity of a vaccine preparation in the form of a mixture of one or more allergenic enzymes and aluminum hydroxide, in which the mixture comprises a liquid phase and a solid phase, and wherein at least a part of the allergenic enzyme (s) is adsorbed to the solid phase, a process comprising the steps to measure the enzymatic activity of the mixture and / or measure the enzymatic activity of the Allergenic enzyme in the liquid phase after a separation of the liquid phase from the solid phase, and / or measuring the enzymatic activity of the allergenic enzyme in the solid phase after a separation of the liquid phase from the solid phase, in an activity test enzymatic, and use the measurement obtained as an indication of the immunological activity of the vaccine preparation.

According to another aspect of the invention there is provided a method for quantifying the amount of allergenic enzyme in a vaccine preparation in the form of a mixture of one or more allergenic enzymes and aluminum hydroxide, in which the mixture comprises a liquid phase and a solid phase, and in which at least a part of the allergenic enzyme (s) is adsorbed to the solid phases, a process comprising the steps of measuring the enzymatic activity of the mixture and / or measuring the enzymatic activity of the allergenic enzyme in the liquid phase after a separation of the liquid phase from the solid phase, and / or measuring the enzymatic activity of the allergenic enzyme in the solid phase after a separation of the liquid phase from the solid phase, in an enzyme activity test, and use the measurement obtained to quantify the amount of allergenic enzyme. BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 shows a diagnostic algorithm for the purification of Der p 1.

Fig. 2a shows the fluorescence of the synthetic substrate Z-FR-AMC and the relationship between the fluorescence signal and the substrate concentration.

Fig. 2b shows AMC standard curves: a) AMC standard curve up to 500 µM and b) linear range of a).

Fig. 3 shows a study of the optimal enzyme and substrate concentration in which a) shows the activity as a function of the substrate concentration and b) shows the activity as a function of the enzyme concentration.

Fig. 4 shows A280 of 100 µg / ml of papain ± 1.14 mg / ml of aluminum hydroxide and 1.14 mg / ml of aluminum hydroxide alone.

Fig. 5 shows the temporal evolution of the sedimentation of 1.14 mg / ml of aluminum hydroxide.

Fig. 6 shows the effect of aluminum hydroxide on AMC. A350 of a control sample was compared with A350 of a supernatant sample.

Fig. 7 shows a study of AMC time in the presence and absence of aluminum hydroxide.

Fig. 8 shows the absorbance spectra of the Boc-QAR-AMC and Z-FRAMC substrates.

Fig. 9 shows the influence of aluminum hydroxide on the Z-FR-AMC substrate measured as a) endpoint measurement at A325 and b) measurement of papain activity.

Fig. 10 shows the influence of aluminum hydroxide on the Boc-QAR-AMC substrate measured as a) endpoint measurement at A325 and b) measurement of papain activity.

Fig. 11 shows the influence of aluminum hydroxide on papain activity with a constant concentration of the C64-specific protease inhibitor E64.

Fig. 12 shows an overview of samples of the adsorption experiment with papain and aluminum hydroxide.

Fig. 13 shows an overview of samples of the adsorption experiment with Der p 1 and aluminum hydroxide.

Fig. 14 shows the activity of Der p 1 of different samples in the presence and absence of aluminum hydroxide.

Fig. 15 shows a Michaelis-Menten curve for Der p 1 in the presence of hydroxide

of aluminum.

Fig. 16 shows the inhibition of IgE binding. Dashed line: Der p 1 eluted aluminum hydroxide (Elu), continuous line: Der p 1 control in the absence of aluminum hydroxide (With 1). DEFINITIONS

The term "in vitro procedure" as used herein means a procedure that can be carried out outside a living organism.

The term "immunological activity" as used herein means any specific allergen response of the immune system that includes immunoglobulin-mediated immune responses.

The terms "solid phase" and "liquid phase" of a vaccine preparation as used herein means the phases resulting from a method of separating a suspension of aluminum hydroxide into a liquid solvent, for example, water in a phase. solid and a liquid phase, the separation process being, for example, simple centrifugation, extraction or sedimentation.

The term "adsorbed" as used herein means any non-covalent bond, coupling, adhesion or bond that includes adsorption by electrostatic forces. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an in vitro method for measuring the enzymatic activity of an allergenic enzyme and thus obtaining an indication of the immunological activity and / or a quantification of the amount of an allergenic enzyme in a vaccine preparation.

In one aspect of the invention, the term "at least a part of" refers to at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% of the allergenic enzymes are adsorbed to the solid phase.

In one aspect of the invention, immunological activity is the ability of the vaccine preparation to elicit an immune response mediated by an allergen-specific immunoglobulin that includes any class, subclass or combination thereof that includes IgA, IgA1, IgA2, IgD , IgE, IgG, IgG1, IgG2, IgG3, IgG4, IgM, in particular IgG and / or IgE.

Although aluminum hydroxide is the most commonly used adjuvant in vaccines, what happens to allergens when adsorbed to the surface of the vaccine has never been fully characterized. An understanding of how adsorption to aluminum hydroxide affects the structure and activity of allergens is essential for use in vaccines, as well as for understanding the mechanism of immune stimulation with adjuvant.

Enzymatic activity is in general directly related to the structure of the enzyme. If the structure is altered, for example, by exposure to heat or acidic conditions, the activity will probably be affected. A population of homogeneous proteins can be described by the following simplified equilibrium of two states:

image 1

in which N is the protein in the native form, D is the denatured form, ku and kf are the velocity constants of the unfolding and refolding kinetics, respectively. Several intermediate transition states can occur between the native and the denatured form. From this simple model, the definition of denaturation can be established as: any temporary or permanent change in the three-dimensional structure of a protein. Therefore, when the physicochemical properties of the surrounding environment are changed, a change in the free energy landscape that represents the available configuration space for the protein could occur depending on how persistent the change is.

Electrostatic attraction to any surface of a solid phase can lead to adsorption of the protein. Adsorption of a protein to a surface can induce conformational changes in the protein, thus displacing the minimum overall energy of the protein. In the case of an enzyme, this may result in a change in its enzymatic activity. Therefore, a change in enzyme activity could be a sensitive measure of structural changes resulting from adsorption to a solid phase.

The aluminum protein-hydroxide system has been thoroughly investigated due to the adjuvant effect of aluminum hydroxide. The literature shows that acidic proteins bind aluminum hydroxide, but the effect of binding on enzymatic activity has never been investigated, to the extent that the present inventors are aware.

The present invention is based on the finding that it is possible to make measurements of enzymatic activity in a vaccine preparation comprising allergenic enzyme (s) and aluminum hydroxide.

The present invention is further based on the recognition that said measurement of the enzyme activity of the vaccine preparation can be used as an indication of the immunological activity of the vaccine preparation, since a change in enzyme activity can be linked to a change in the conformation of the enzyme molecule as it is present in the vaccine preparation, which is again linked to the immunological activity of the vaccine preparation.

The ability to perform this measurement makes it possible to assess the impact of the adsorption of the allergen to aluminum hydroxide by measuring the enzymatic activity before and after adsorption to the solid phase vehicle and thus obtaining a measurement of the immunological activity after adsorption since As explained above, a change in enzyme activity is expected to have an impact on immunological activity.

The process according to the invention can also be used to quantify the amount of allergenic enzyme (s) in a vaccine preparation.

In one aspect of the invention, the quantification of the allergenic enzyme is performed by comparing the measured enzyme activity with a well characterized pattern. In another aspect of the invention, quantification of the enzyme allergen is performed by titration of active sites. The assessment of active sites requires the use of an inhibitor of that particular enzyme activity that binds to the enzyme irreversibly or at least with a very high affinity. Vaccine Preparation

The vaccine preparation submitted to the process of the present invention may be any preparation ready for use in the form of a mixture comprising one or more allergenic enzymes and aluminum hydroxide, wherein the mixture comprises a liquid phase and a solid phase at the that at least a portion of the allergenic enzyme is adsorbed, or any vaccine preparation such to prepare a ready-to-use formulation. The vaccine preparation may additionally comprise one or more allergens that do not have an enzymatic activity.

The ready-to-use preparation may be for parenteral administration or for mucosal administration.

Parenteral administration includes intravenous, intramuscular, intraarticular, subcutaneous, intradermal, epicutaneous / transdermal and intraperitoneal administration. Vaccines for administration by injection may be formulated so that they are suitable for needle injection or for needleless injection.

Administration in the mucosa includes oral, nasal, vaginal, sublingual, ocular, rectal, urinal, intramammary, pulmonary, otic (i.e., by ear) or oral administration.

The vaccine may be in the form of a spray, an aerosol, a mixture, a suspension, a dispersion, an emulsion, a gel, a paste, a syrup, a cream, an ointment, implants (ear, eye, skin, nose, rectal and vaginal), intramammary, vagitorial, suppository or uterine preparations. Allergenic enzyme

In the present context, the term "allergenic enzyme" is any protein that induces allergic reactions, that is, mediated by IgE after repeated exposure to an individual and has an enzymatic activity, that is, that can catalyze or accelerate a chemical reaction.

Like all catalysts, enzymes work by decreasing the activation energy of a reaction, thus allowing the reaction to proceed much faster. Enzymes can accelerate reactions by a factor of many thousands. An enzyme, like any catalyst, remains unchanged by the completed reaction and, therefore, can continue to function. Because enzymes, like all catalysts, do not affect the relative energy between products and reagents, they do not affect the equilibrium of a reaction. However, the advantage of enzymes compared to most other catalysts is their stereo, regal and chemoselectivity and specificity.

Examples of naturally occurring allergens include pollen allergens (tree, grass, weed and grass pollen allergens), insect allergens (inhalant, saliva and poison allergens, for example, mite allergens, cockroach allergens and of mosquitoes, hymenoptera venom allergens), animal and dandruff hair allergens (from, for example, dog, cat, horse, rat, mouse, etc.) and food allergens. Important pollen allergens of trees, lawns and herbs are those that originate from the taxonomic orders of Fagales, Oleales, Pinales and platanaceae that include, among others, birch (Betula), alder (Alnus), hazelnut (Corilus), carpe (Carpinus) and olive (Olea), cedar (Cryptomeria and Junipers), banana (Platanus), the order of Poales that includes, among others, lawns of the genera Lolium, Phleum, Poa, Cynodon, Dactilis, Holcus, Phalaris, Secale and Sorghum, the orders of Asterales and Urticales that include, among others, herbs of the genera Ambrosia, Artemisia and Parietaria. Other important inhalation allergens are those of house dust mites (HDM) of the genus Dermatophagoides and Euroglyphus, storage mites, for example, Lepidoglyphus, Glycyphagus and Tyrophagus, those of cockroaches, mosquitoes and fleas, for example, Blatella, Periplaneta, Chironomus and Ctenocephalides, and those of mammals such as cat, dog and horse, poison allergens that include those that originate from biting insects or snappers such as those of the taxonomic order of the hymenoptera that includes bees (Apidae superfamily), wasps ( Vespidea superfamily) and ants (Formicoidae superfamily). Important inhalation allergens of fungi are, among others, those that originate from the Alternaria and Cladosporium genera.

In one aspect of the invention, the allergenic enzyme (s) is selected from the group consisting of tree pollen allergens, grass pollen allergens, grass pollen allergens, mite allergens, poison allergens, animal and dandruff hair allergens and food allergens. In another aspect of the invention, the allergenic enzyme (s) is (are) allergen (s) of house dust mites.

A non-exhaustive list of allergenic enzymes is shown in Table 1.

Enzyme activity Allergen Organism

Cysteine proteases

Der p 1 Dermatophagoides pteronyssinus HDM

Der f 1 Blo t 1

Dermatophagoides farinae Blomia tropicalis HDM Mite

Act c 1

Actinidia chinensis Kiwi

Car p 1

Carica papaya Papaya

Serine proteases

Der p 3 Dermatophagoides pteronyssinus HDM

Der p 6

Dermatophagoides pteronyssinus HDM

Der p 9

Dermatophagoides pteronyssinus HDM

Cla h 9 Blo t 6

Cladosporium herbarum Blomia tropicalis Mite Mushroom

Asp fl 13

Aspergillus flavus Fungus

Asp f 13

Aspergillus fumigatus Fungus

Asp p 18

Aspergillus fumigatus Fungus

Tri r 4

Trichophyton tonsurans Fungus

Rho m 2

Mucilaginous Rhodotorula Fungus

Epi p 1

Epicoccum purpurascens Fungus

Api m 7

Apis mellifera Honey bee

Pol d 4

Dominulus Polistes Wasp

Cuc m 1

Cucumis melo Cantaloupe

Metalloproteases

Asp f 5 Aspergillus fumigatus Fungus

Aspartic proteases Asp f 10 Aspergillus fumigatus Fungus Bla g 2 Blattella germanica German cockroach

Enolasas Cyn d 22w Alt a 6 Cla h 6 Asp f 22w Pen c 22w Rho m 1

Cynodon dactylon Alternaria alternata Cladosporium herbarum Aspergillus fumigatus Penicillium citrinum Rhodotorula mucilaginosa

Bermuda Grass Lawn Mushroom Lawn Mushroom

Amylases

Der p 4 Dermatophagoides pteronyssinus HDM

Blo t 4

Blomia tropicalis Mite

Hor v 16

Hordeum vulgare Barley

Hor v 17

Hordeum vulgare Barley

Enzyme activity Allergen Organism

Glutathione transferases

Der p 8 Bla g 5 Dermatophagoides pteronyssinus Blatella germanica HDM German Cockroach

Arginine kinases

Der p 20 Pen m 2 Dermatophagoides pteronyssinus Penaeus monodon HDM Black Tiger Shrimp

Phospholipases

Api m 1 Bom p 1 Dol m 1 Pol a 1 Apis mellifera Bombus pennsylvanicus Dolichovespula maculata Polistes annularis Honey Bee Bumblebee White-faced Hornet

Vesp c 1 See m 1 See v 1

Vespa crabro Vespula maculifrons Vespula vulgaris Wasp European Hornet Yellow Jacket Wasp

Yellow jacket wasp

Dehydrogenases

Alt to 8 Alt to 10 Hala f 4 Alternaria alternata Alternaria alternata Malassezia furfur Fungus Fungus Fungus

One of the main sources of allergens is HDM. In 2000, a total of 13 different species of HDM were identified on all continents, except in Antarctica. The HDM belong to the Arthropoda edge, such as spiders and scorpions. Three species constitute 90% of the fauna of HDM, specifically Dermatophagoides pteronyssinus, Dermatophagoides

5 farinae and Euroglyphus maynei.

For D. pteronyssinus, allergens that mediate the allergic response are found in the feces and dried body debris of D. pteronyssinus. 14 different groups of D. pteronyssinus allergens are identified (Table 2). Although not all are fully characterized, the size and function of the majority is established and immunological tests

10 have determined the reactivity of IgE in vitro. As shown in Table 2, several of the HDMs are allergenic enzymes such as, for example, Der p 1, Der p 3, Der p 6 and Der p 9.

5

10

fifteen

eleven Table 2. Dermatophagoides pteronyssinus allergen groups - molecular weight and function.

Pm Allergen (kDa) Function

Group 1

25 Cysteine protease

Group 2

14 Epithelial secretion

Group number 3

25 Trypsin

Group 4

57 Amylase

Team 5

fifteen ND

Group 6

25 Chymotrypsin

Group 7

31 ND

Group 8

26 Glutathione-S-Transferase

Group 9

30 Collagenolytic Serine Protease

Group 10

37 Tropomyosin

Group 11

92 Paramyosin

Group 12

14 ND

Group 13

fifteen Fatty acid binding protein

Group 14

189 Apolipoforin

ND: Not available

The formal definition of a major allergen is any antigen that binds to human IgE sera in more than 50% of patients in a clinically sensitive group. HDM allergens Der p 1 and Der p 2 are the main allergens and are considered the most potent of HDM allergens.

Several in vitro experiments indicate that the proteolytic activity of Der p 1 could play an essential role in the development of the allergic reaction to HDM. Der p 1 is believed to break the occlusion zones between epithelial cells by excision that occludes and increases the permeability of the bronchial mucosa by degrading α-antitrypsin. This could facilitate an increase in access to subepithelial antigen presenting cells, which could lead to an increase in the allergic response. In addition, Der p 1 cleaves CD23 (the low affinity IgE receptor that regulates the production of IgE) and CD25 (the IL-2 receptor) on the surface of B and T cells. This directs the T cell response to a Th2 response and finally high levels of IgE and a more severe allergic response.

Proteolytic enzymes, hereinafter referred to as proteases or synonymously peptidases, mediate protein breakdown. This is done either by limited proteolysis in which a limited number of peptide bonds are cleaved or by non-limited proteolysis in which proteins degrade into their amino acid constituents. Proteolytic enzymes, like most other enzymes, are classified by the Enzyme Commission (EC) numbering system with a number that indicates the function and specificity of the substrate. Proteolytic enzymes are divided, according to the EC numbering system, into two sub-subclasses, specifically exopeptidases and endopeptidases. The latter are also referred to as proteinases.

Exopeptidases, for example, amino and carboxypeptidase, cleave individual amino acids from both the N and C end of the protein, while endopeptidases cleave bonds within the protein. For endopeptidases, the particular cleaved bond depends on the specificity or preference towards different amino acids in the protein substrate. Thus, an endopeptidase may have a preference towards cleavage of neighboring peptide bonds that are near a bulky hydrophobic residue, while others prefer long charged residues or even two or more chemically or structurally related residues. The actual structure around the active site of the protease dictates the specificity or preference. Residues around the substrate cleavage site are denoted -P3-P2-P1-P1'-P2'P3'-, with the section P1-P1 'being the cleavage site. Similarly, the protease residues aligned to the substrate are denoted -S3-S2-S1-S1'-S2'-S3'-.

Four different types of endopeptidases have been described. These are serine proteases, cysteine protease, aspartyl proteases and metalloproteases. In each case, the proteases generate a nucleophile that attacks the carbonyl of the protein substrate peptide.

Cysteine proteases are active hydrolases for peptide bonds by means of a cysteine residue, belong to sub-class 3.4.22 in the EC numbering system. Currently, 40 cysteine proteases are classified in this system, covering enzymes such as caspase 1, separase, some cathepsins and papain (Car p 1). Several other cysteine proteases have been identified and characterized, but have not yet been classified in the EC numbering system.

In addition to the EC numbering system, proteases are also classified into clans and families based on the phylogenetic relationship, that is, their molecular structure and sequence homology. At this time, the MEROPS database contains detailed information on 1816 different proteases. In this system, proteases are noted by a letter indicating the catalytic type (S, C, T, A, G, M or U for serine, cysteine, threonine, aspartic, glutamic, known metalloprotease or protease, respectively) followed by an arbitrary number Cysteine proteases are divided into five clans. By this system, Car p 1 belongs to the CA clan, family C1, and is given the name C.01.001, unlike 3.4.22.2 in the EC numbering system.

The catalytic remains responsible for the activity of cysteine proteases are highly conserved: a cysteine (Cys), a histidine (His) and an asparagine (Asn) constitute the so-called catalytic triad. These three residues generate a nucleophilic thiolate anion from Cys. A pair of thiolate-imidazolium ions is generated from His and Cys, which attacks the carbonyl of the substrate peptide. The Asn helps to orient the imidazolium ion of His in favorable positions for the various stages of the catalytic mechanism. Catalysis occurs sequentially. First, the enzyme is temporarily acylated by the protein substrate by the thiolate anion. Second, a part of the protein substrate is cleaved followed by deacilation and addition of water. Finally, the residues of active sites of the cysteine protease are reconstituted in their original form.

In addition to the catalytic remains, several other remains perform important functions. A glutamine (Gln) is part of what is known as the oxoanion hole. This structure helps to stabilize the intermediate transition state of the substrate during catalysis. Several hydrophobic moieties establish a non-polar environment around Asn, protecting from the external solvent. These are two tryptophan (Trp), two valine (Val) and one phenylalanine (Phe), all conserved residues.

Catalysis performed by cysteine proteases strongly depends on a reducing environment since reactive cysteine has a tendency to oxidation. For this reason, enzymatic assays with cysteine proteases can be performed with a reducing agent, for example, dithiothreitol (DTT), free cysteine or β-mercaptoethanol.

The cysteine protease Car p 1 of the Carica papaya plant is the most studied and well understood cysteine protease. Car p 1 is a member of the C1 family of cysteine proteases that are normally secreted and are produced as inactive pro forma. Car p 1 consists of a single 212 amino acid polypeptide chain with three disulfide bridges. The polypeptide chain is folded to form a globular protein consisting of two interaction domains that delimit a cleft between them. The residues of active sites Cys25 and His159 are located in this slit in opposite domains. The domain that hosts Cys25 is dominated by structural α-helix motifs while the domain that hosts His159 is dominated by β-sheet structural motifs. The third Asn175 catalytic residue is residing in close proximity to His159 in sequence and tertiary structure. Apart from the Cys25 that coordinates with the carbonyl of the substrate, Asn175 and Gln19 help to keep the substrate in place for catalysis by hydrogen bonds and constitutes the core of the mentioned oxoanion hole. The optimum pH of the proteolytic activity of Car p 1 is 6.0-7.0.

Der p 1, an important HDM allergen that originates from your feces, is also a member of the C1 family of cysteine proteases. Although it is not granted a site in the EC numbering system, it has been classified in the MEROPS peptidases database with the number C.01.073. Der p 1 is secreted as a proenzyme in the gastrointestinal touch of HDM and is activated by the proteolytic elimination of the pro-peptide that forms the mature enzyme that is made up of 222 amino acids with three disulfide bridges. The open reading frame encodes an 18 amino acid signal peptide, in addition to an 80 amino acid pro-peptide. Der p 1 is structurally very similar to Car p 1 and show a structural homology of 80%, despite a sequence homology of 26%. The optimal pH of the proteolytic activity of Der p 1 is 7.0 -8.0.

In one aspect of the invention, the allergenic enzyme (s) is (are) one or more selected from the group consisting of Der p 1, Der p 3, Der p 6 and Der p 9.

In another aspect of the invention, at least one of the allergenic enzyme (s) is a cysteine protease such as Der p 1.

In a further aspect of the invention, at least one of the allergenic enzyme (s) is a serine protease such as one or more selected from the group consisting of Der p3, Der p 6 or Der p 9.

In a further aspect of the invention, the vaccine preparation comprises at least two different species of allergens that originate from both the same allergenic source and that originate from different allergenic sources, for example, group mite allergens 1 and group 3 of different mites.

The allergenic enzyme incorporated in the vaccine preparation may be in the form of an extract, a purified allergen, a modified allergen, a recombinant allergen or a mutant of a recombinant allergen. An allergenic extract may contain, in addition to allergens, several other ions and molecules. An allergenic extract can naturally contain one or more isoforms of the same allergen, while a recombinant allergen usually only represents an isoform of an allergen. The vaccine preparation may additionally comprise one or more allergens that do not have an enzymatic activity and / or one or more enzymes that do not have allergenic activity.

In one aspect of the invention, the allergenic enzyme (s) is in the form of an extract. In another aspect of the invention, the enzymatic activity of an important allergen in the extract is measured.

In a further aspect of the invention, the enzymatic activity of one or more allergenic enzymes can be measured in a complete extract.

In another aspect of the invention, the allergen is a recombinant allergen. In another aspect of the invention, the allergen is a naturally occurring low IgE binding mutant or a recombinant low IgE binding mutant.

In another aspect of the invention, the low IgE binding allergen is an allergen according to

WO 99/47680, WO 02/40676 or WO 03/096869.

Enzymatic inhibitors

Enzymatic activity can be affected by other molecules such as inhibitors that are molecules that decrease or suppress enzymatic activity.

A large number of protease inhibitors, both natural and synthetic, have been described. The inhibitors inactivate the enzyme by different mechanisms, for example, direct covalent modification of the catalytic moieties or protecting the active site for substrate entail. The first type of mechanism is frequently represented by small molecules with a reactive group towards catalytic moieties, thus irreversibly blocking the activity. An example of such an inhibitor is the specific cysteine protease inhibitor E-64 that covalently binds to the catalytic cysteine by a reactive epoxy group. The last type of inhibitor is often a macromolecular structure that is associated with the enzyme by multiple non-covalent interactions. An example of such an inhibitor is the soy trypsin inhibitor (SBTI) specific for serine proteases. SBTI is a naturally occurring 190 amino acid protein that covers the cleavage of the active site and, therefore, catalytic debris through hydrogen bonds, electrostatic and hydrophobic interactions with the enzyme surface.

In one aspect of the invention, the vaccine preparation comprises several allergenic enzymes. In order to measure only the activity of one of the allergenic enzymes, it may be necessary to use relevant inhibitors depending on the type of enzyme activity that one wishes to inhibit. In another aspect of the invention an inhibitor is used to inhibit one or more of the allergenic enzyme (s) in the vaccine preparation.

The use of specific inhibitors for the allergenic enzyme in question is useful for obtaining a better characterization and identification of the enzymatic activity, in addition to quantifying the amount of active enzyme present in the preparation (by, for example, titration of active sites ). In one aspect of the invention, the cysteine protease inhibitor used is selected from the group consisting of E64 (L-trans-epoxysuccinyl-L-leucylamide (4-guanidino) butane) and other epoxides. In another aspect of the invention, the inhibitor is E64. Substrates

In order to be able to measure the enzymatic activity of an allergen enzyme adsorbed to the solid phase in an enzyme activity assay, a substrate and, if necessary, a specific inhibitor for the allergenic enzyme in question must be identified.

In one aspect of the invention, the substrate in the enzyme activity assay is specific to the enzyme in question, which means that no other enzyme in the same allergenic source can convert this substrate into a product. As an example, the Z-Leu-Leu-Glu-MCA substrate is specific for cysteine proteases, for example Der p 1, and is not cleaved by other proteases known to be present in that allergenic source (HDM extracts) such as serine proteases Der p 3, Der p 6 or Der p 9. In one aspect of the invention a specific substrate for an allergenic enzyme is used to measure the enzymatic activity of the allergenic enzyme. In another aspect of the invention, the substrate used is Z-LeuLeuGlu-MCA. Aluminum hydroxide adjuvant

An adjuvant is a compound that acts by potentiating the immune response after vaccination.

Aluminum hydroxide can be characterized by a variety of physicochemical parameters such as adsorption, solubility and dissolution properties, ionic charge measured as the isoelectric point pI (pH at which the net charge of the substance is zero for a dissociable compound), constants of dissociation, coordination of complexes, electronic configurations, valence, binding orbitals and anti-union orbitals, prolonged release properties, adhesion properties, surface characteristics, particle characteristics and adyuvanticity.

It is believed that the biologically active substance is adsorbed (or coupled) to aluminum hydroxide and this adsorption contributes to the efficacy of the vaccine. Several factors may be important or influence adsorption between the active substance and aluminum hydroxide (see, for example PM Callahan et al., Pharmaceutical Research vol. 8, No. 7,851-858 (1991), and Vaccine Design. The Subunit and Adjuvant Approach). These factors include pH, the duration of time during which the adsorption reaction is carried out, mixing conditions, concentrations of the various components in the vaccines, containers, temperature, storage, buffer and excipients. It has been further found that the adsorption of the active substance can be influenced by the net / global charge of the metal salt and the load of the active substance, both pH dependent. Another characteristic that is believed to be of importance is the solubility of aluminum hydroxide.

Aluminum hydroxide may additionally have a prolonged release effect. A prolonged release effect means that the active substance will gradually be released from the vaccine. Therefore, the active substance will be retained by aluminum hydroxide and gradually released from it. It is believed that this has several beneficial effects, for example, prolonged stimulation, beneficial drug release and protection of biological interactive substances against environmental conditions. In addition, it is believed that aluminum hydroxide may possess certain entrapment properties, thereby retaining the active substance to be administered.

Another characteristic of aluminum hydroxide is the protection of the active substance both maintaining the ideal pH for the active substance in the microenvironment, thus preventing the degradation of the acid, as well as protecting the active substance from enzymatic degradation, thus allowing the substance to be administered .

In addition, aluminum hydroxide may have a buffer capacity. This can result in an in vivo microenvironment within the vaccine formulation that protects the active substance from the degradable environment. This can be an advantage, for example, in the stomach

or the intestine in which there is a risk of acidic and enzymatic degradation, respectively.

Aluminum hydroxide can be in the form of a gel suspended in a solvent, usually water. When the gel is stirred, that is, the solid phase, it will be distributed evenly throughout the volume of the suspension, hence enclosing all the water, that is, the liquid phase, present. If allowed to stand or undergo a separation procedure such as centrifugation, a part of the water will separate from the gel. The amount of water separated will depend on the separation process used, in addition to the type and concentration of aluminum hydroxide used.

The molecular formula of aluminum hydroxide is Al (OH) 3. However, it underestimates the true complexity of the compound. The molecular structural composition is an octahedron. Aluminum is at the center of the plane of symmetry of the bipyramid, hydroxides at the intersections of connection and water molecules in all others. The individual octahedron is combined to give macromolecular structures of octahedra. The more octahedra combined, the ratio of aluminum to oxygen asymptotically approaches 1: 3 the more hydrogen is displaced by water.

The physical aspect of aluminum hydroxide is a gel suspension with decreasing fluidity as the aluminum content increases. The gel is added and, therefore, sediments when stored due to its high density, leaving an aqueous phase on it. The typical particle size of an aluminum hydroxide aggregate is in the range of two to three µm. Aluminum hydroxide has a zero charge point (PZC) of 9.1. PZC is equivalent to pI in proteins, which is the pH value at which the molecule has a global net charge of zero. The pI of Car p 1 is 8.75 and for Der p 1 4.6-6.6 (depending on the different isoforms of Der p 1).

In addition, it has been shown that the microenvironment surrounding the aluminum hydroxide adjuvant has a different pH from that of the mass solution. This is due to the attraction of anions that include hydroxyls that form a double layer that surrounds the adjuvant particles.

The base of protein adsorption to aluminum hydroxide is mainly mediated by its difference in charge. A substantial difference in the PZC with respect to the pI at constant pH is needed in order to establish the electrostatic interactions required for adsorption. This is supported by Nernst's potential or the surface potential of aluminum hydroxide given by

Surface potential = 59 mV · (PZC - pH)

Proteins with a pI lower than PZC may bind aluminum hydroxide. The greater the difference between PZC and pH, the greater the potential of the aluminum hydroxide adjuvant and the stronger the electrostatic interaction between aluminum hydroxide and protein, since the protein pI is less than the PZC of aluminum hydroxide.

Other interactions include hydrophobic, van der Waals and hydrogen bonds, but by themselves they are not sufficient to drive adsorption if there is no significant difference in charge between the molecules. Therefore, in theory, Car p 1 should not be adsorbed, or at least to a low degree, to aluminum hydroxide, while Der p 1 should be adsorbed due to the large difference in charge compared to aluminum hydroxide. Enzyme activity test

The purpose of the enzyme activity test is to obtain a progress curve of an enzyme catalyzed reaction. The initial velocity is estimated as the initial rate of product formation or the initial decrease in substrate concentration. This can be obtained in different ways depending on the enzymatic reaction, with absorbance, fluorescence or pH change being the most popular. An enzyme progress curve is the concentration of product as a function of time. In the present context, the term "enzyme activity assay" refers to any appropriate assay that depends on the allergenic enzyme to be measured. Through the chosen procedure it must be possible to follow and quantify the substrate consumption and / or the generation of the product through procedures that are compatible and that are not altered by the presence of the solid phase vehicle, for example, by spectroscopic procedures such as absorbance, fluorescence , FTIR (Fourier transform infrared spectrometry), by immunological procedures, for example ELISA, and the like. Before deciding on an assay, it is common practice for an expert within the field to verify that the presence of the solid phase does not interfere with enzymatic catalysis, with the test conditions (for example, joining the substrate and / or the product, altering the pH conditions, etc.) or with the measurement procedure itself in such a way that the measured results are affected as exemplified, for example, in the examples herein.

In one aspect of the invention, the assay is a fluorescence assay in which product formation is measured over time. The substrate may be made of a synthetic peptide linked to a fluorescent group that is inactivated by the peptide and has relatively low fluorescence intensity while bound to the peptide, such as the cysteine-protease substrates Boc-Gln-Ala-Arg-MCA , Z-Leu-Leu-Glu-MCA or Z-Phe-Arg-MCA, in which MAC is the fluorescent group. If an enzyme cleaves the link between the fluorescent group and the peptide sequence, the fluorescence increases dramatically. The peptide can be designed to meet the specificity requirements of the enzyme.

If in one aspect of the invention the allergenic enzyme is a cysteine protease, the rest of the active cysteine needs to be in its reduced form in order for the protease to be enzymatically active. In this aspect of the invention, the allergenic enzyme is incubated with a reducing agent to activate the allergenic enzyme. It is important that the reducing agent be present in a sufficient concentration so that the rest of the active site cysteine is completely reduced, but not in an excess concentration such that the enzyme disulfide bridges are reduced.

In one aspect of the invention the enzymatic activity of the mixture of the liquid phase and the solid phase of the vaccine preparation is measured. In another aspect of the invention, the vaccine preparation is subjected to a separation process to separate the liquid phase and the solid phase in order to make it possible to measure the enzymatic activity of the solid phase and the liquid phase separately. The separation can be done by any appropriate procedure. In one aspect of the invention, the separation process is performed by centrifugation, extraction or simple settling. Depending on the specific enzyme test used and the quality of the solid phase sample it may be necessary to use, for example, buffer solutions in order to obtain an appropriate sample before measuring the enzymatic activity of the allergenic enzyme (s). (s)

In one aspect of the invention, the vaccine preparation is subjected only to a measurement of the enzymatic activity of the allergenic enzyme in the mixture of the liquid phase and the solid phase (measurement 1).

In another aspect of the invention, the vaccine preparation is subjected only to a measurement of the enzymatic activity of the allergenic enzyme in the liquid phase after a separation of the liquid phase from the solid phase (measurement 2).

In another aspect of the invention, the vaccine preparation is subjected only to a measurement of the enzymatic activity of the allergenic enzyme in the solid phase after a separation of the liquid phase from the solid phase (measurement 3).

In a further aspect of the invention, the vaccine preparation is subjected both to a measurement of the enzymatic activity of the mixture of the liquid phase and the solid phase (measurement 1) and to a measurement of the enzymatic activity of the allergenic enzyme in the liquid phase (measurement 2).

In a further aspect of the invention, the vaccine preparation is subjected both to a measurement of the enzymatic activity of the allergenic enzyme in the liquid phase (measurement 2) and to a measurement of the enzymatic activity of the allergenic enzyme in the solid phase. (measurement 3).

The distribution of allergenic enzymes between the liquid phase and the solid phase is a parameter that is characteristic for each allergenic enzyme, and hence it can be used to characterize the state and immunological activity of a vaccine preparation. Accordingly, the purpose of the above aspects of the invention that involve measurements of the enzymatic activity of various combinations of different phases of the vaccine preparation and / or the complete vaccine preparation is to provide additional information on the immunological activity of the preparation of vaccine.

In another aspect of the invention the enzymatic activity of an allergenic enzyme solution used to prepare the vaccine preparation with adjuvant is measured, and the measurement of said solution is compared with the measurement obtained from the vaccine preparation with adjuvant in order to evaluate the effect on the immunological activity of the preparation of the vaccine preparation with adjuvant.

In a further aspect of the invention, the vaccine preparation is subjected to the measurement of enzyme activity immediately after preparation and after one or more storage periods, and the indication of the immunological activity of the vaccine preparation is based on a comparison of the previous and subsequent measurements.

In another aspect of the invention, the indication of the immunological activity of the vaccine preparation is based on a comparison of the measurement obtained for vaccine preparation with adjuvant and previous corresponding measurements of the same type of vaccine preparation with adjuvant or otherwise. of vaccine preparation. PROCEDURES AND MATERIALS Preparation of allergen vaccines with aluminum gel adjuvant

Lyophilized allergen is dissolved in an aqueous buffer and diluted to a desired concentration. "Alhydrogel" (1.3%) is added to the allergen solution obtained while stirring and then sterile water is added. The resulting solution is allowed to stand until the next day and then buffer is slowly added while stirring to produce the final allergen aluminum hydroxide gel. Rocket Immunoelectrophoresis Objective

This procedure was used to quantify a given protein by measuring the spread of the protein-antibody complex after electrophoresis on an agarose gel containing antibodies directed against the protein under investigation. Theory

This procedure is based on the mobility of the protein-antibody complex on a

agarose gel during electrophoresis. The antibodies are incorporated into the agarose gel during polymerization and the sample protein is then applied to the wells. The proteins move according to their electrophoretic mobility meeting the antibodies in the gel and forming complexes. These complexes grow in size as the antigen encounters more and more antibodies, thus limiting migration through the pores of the gel until no further migration occurs. The complexes are visualized by staining the gel. The area delimited by the complexes is proportional to the amount of protein applied to the well. The quantification is performed with respect to an internal standard preparation applied in a dilution series on the same gel. Apparatus:

Heated water bath controlled by thermostat 56 -60ºC

Electrophoresis apparatus (2 buffer containers, 2 electrodes, cooled surface and

camera)

Power supply, Immuno Power 320, Kebo Lab A / S

Hot air fan, Team International HL2 Materials and reagents:

Glass plate: 7 x 10cm

Paper wicks: filter paper, standard size: 21 x 10cm, Watman Buffer for electrode containers and agarose gel:

5,5-diethylbarbituric acid 0.1 M, Veronal, Tris 0.40 M, Sigma,

2 mM calcium lactate, Purum agarose gel containing antibodies:

1% (w / v) agarose, type HAS, Litex

Antibody: Rb-a-Derp1, ALK-Abelló A / S Staining solution:

Coomasie bright blue 6 mM R-250, Pierce, 10% acetic acid, Bie & Berntsen in

43.2% ethanol Bleaching solution:

10% acetic acid, Bie & Berntsen in 43.2% ethanol Experimental procedure

A glass plate was placed on a level surface and cleaned with ethanol. 11 ml of agarose was pipetted into a test tube in a 56 ° C water bath, 15 µl of antibody was added and the solution was carefully mixed by inversion. The agarose was carefully poured onto the glass plate avoiding the formation of air bubbles. After gelation, a series of wells were drilled 1.5 cm from the bottom edge of the plate. The plate was placed on the cooled surface of the electrophoresis apparatus. Connection bridges of 5 layers of filter paper were established and the voltage across the gel was set at 2 V / cm. 10 µl of sample was applied to the wells. Another glass plate was placed on top of the connection bridges of the filter paper to prevent condensation of water on the gel and electrophoresis continued overnight.

After electrophoresis, the glass plate was placed on filter paper and the wells were filled with distilled water. Then, the gel was covered with wet filter paper and pressed under several layers of dry filter paper, a thick glass plate and a load of 3-4 kg. After ten minutes the procedure was repeated. Then, the plate was placed in a vessel with 0.1 M NaCl for 15-30 minutes followed by compression as described above. After this, the plate was dried in a stream of hot air and the plates were stained for 5 min in Coomassie staining solution. The plate was immersed in distilled water for a few seconds in order to remove the excess staining solution. Finally, the plate was discolored for 2 minutes in successive baths until the desired discoloration was achieved. The plate was dried with hot air and the gel scanning was done by Gel-Pro Analyzer 3.1 software. Purification of Der p 1 Objective

The purpose is the purification of Der p 1 from extract of D. pteronyssinus. Theory

The purification of Der p 1 involves several stages. The application of two types of affinity chromatography steps leads to purified Der p 1. The first chromatography was performed on a SBTI-agarose column. The purpose of this stage is the elimination of contaminating serine proteases present in the extract, making the extract more stable.

The second stage in the purification is performed on a 4C1B8-Sepharose column. 4C1B8 is a mouse monoclonal antibody specific for Der p 1 (from Martin Chapman). Der p 1 is eluted by applying a pH gradient.

The diagnostic purification algorithm is shown in Figure 1.

Further purification is performed on a SBTI column in order to completely remove traces of the Der p 3 serine protease that is copurified with Der p 1 in the previous step. Fractions containing Der p 1 were collected and concentrated by ultrafiltration. Apparatus:

ÄKTA explorer FPLC system, Amersham Biosciences

Sorval RC 3B Plus Centrifuge, Du Pont Materials and reagents:

Affinity Purification: Columns: column for Der p 1 SBTI-agarose (SBTI-agarose), column volume (VC) 1 ml Column for Der p 1 of CNBr-Sepharose-Acm 4C1B8 (4C1B8-Sepharose), VC 5 ml D. pteronyssinus house dust mite extract Buffers for chromatographic purification:

A11: Phosphate buffered saline (PBS), Bie & Berntsen A2: PBS, 0.5 M NaCl, Merck B1: 0.1 M glycine, pH 11, Sigma, 0.5 M NaCl, Merck

Protein concentration: 15 ml centrifugal filtration devices Amicon Ultra-15, Millipore. Buffer Exchange:

Desalination column PD 10, Amersham Biosciences Experimental procedure Sample preparation

120 mg of Der p extract was dissolved in 10 ml of buffer A11. The sample was filtered with a low 0.22 µm protein binding filter. In order to reduce the possible proteolysis of Der p 1, all operations were carried out at 5 ° C. All buffers used were also cooled to 5 ° C. SBTI-agarose column affinity chromatography

The SBTI-agarose column was equilibrated with buffer A11 and 5 ml of the prepared sample was injected into the column. Samples of 50 µl of fractions were taken for further investigation and frozen separately. The continuous flow fractions containing Der p 1 were pooled for subsequent purification. 4C1B8-Sepharose column affinity chromatography

The column was equilibrated with buffer A11 and 5 ml of the sample (purified combination in SBTI-agarose) was injected into the column. The non-specifically bound material was eluted with A2 buffer. Then Der p 1 was eluted with a gradient with respect to buffer B1. 800 mM phosphate buffer, pH 7, was pipetted into the collection tubes (200 µl / ml fraction) intended for the collection of Der p 1 in order to neutralize the alkali eluate. Samples of 50 µl of fractions were taken and frozen separately for further investigation. Fractions of the elution peak were pooled and frozen. Fractions containing Der p 1 were pooled and subjected to a second SBTI-agarose chromatography under the same conditions described above. Postpurification Procedures

The approximately 140 ml combination of the purification of the second SBTI column was concentrated by ultrafiltration using Amicon Altra-15 15 ml centrifuge filters. The filters were washed with PBS buffer and the combined Der p 1 was centrifuged at 3,500 rpm for 15 minutes, reducing the volume to 5 ml.

The buffer was changed to 50 mM Tris, pH 7, using the PD-10 desalination column filled with Sephadex G-25 designed to separate substances from high (MW> 5000) to low molecular weight (MW <1000). Absorbance Objectives

This procedure is used to evaluate the total protein concentration. Theory

The aromatic amino acids tryptophan, tyrosine and phenylalanine absorb ultraviolet light. However, only tryptophan and tyrosine absorb at 280 nm, and tryptophan absorbs 5 times more light than tyrosine. This is due to the n → n * transition in the indole ring of tryptophan in which phenylalanine and tyrosine contain a phenyl group. The absorbance of a protein is linearly correlated with the amount of tryptophan and tyrosine in the protein, the length of the light path and the protein concentration. This relationship is called the Lambert-Beer law and is given by:

A = image2 · L · c

where A is the absorbance, ε is the molar absorption coefficient of the protein (determined by the amount of tryptophan and tyrosine in the protein), I is the length of the light path and c is the protein concentration. Therefore, the protein concentration can be estimated from an absorbance measurement if the molar absorptivity coefficient is known. Appliances

Lambda 800 UV / VIS spectrometer, PerkinElmer ™

100-QS, quartz cuvette, path length 1 cm, Hellma® Materials and reagents

Bis-Tris 50 mM, pH 6.5, Sigma

50 mM Tris, pH 7.0, Sigma

50 mM phosphate buffer, pH 7.0, Merck

2% Helmanex dissolution, Hellma Experimental procedure

The spectrophotometer was turned on 30 min before use for a warm-up period. The wavelength of the spectrophotometer was adjusted to 280 nm and the instrument was set to zero for a blank sample containing the true sample matrix. The quartz cuvette was washed first with a 2% Helmanex solution and then 4 times with MQ water and then dried with high pressure air. After air drying, the outside of the bucket was rubbed with lens cleaning fabric. This procedure was performed between each sample measurement. After cleaning the cuvette, 200 µl of sample was transferred to the cuvette and the absorbance was measured.

Enzyme activity assays (fluorescence assay)

Appliances

Molecular Devices Spectra MAX GeminlXS

Black Corning 96-well non-junction polystyrene plate

Heraeus Sepatech centrifuge

Mixer, Janke & Kunkel Materials and reagents

Purified p1 der

Papain (Car p1) purified, Sigma

Boc-QAR-AMC 10 mM, Bachem

10 mM Z-FR-AMC, Bachem

E-64 0.70 mM in DMF, Merck

DTT 1 M, Sigma

100 mM EDTA, Bie & Berntsen

50 mM Tris buffer, pH 7.0, Sigma

50 mM Bis-Tris buffer, pH 6.5, Sigma

Phosphate buffer, 50 mM, Merck

6.686 mg / ml aluminum hydroxide, Brenntag Biosector Experimental procedure

Stock solutions of 1M DTT and 100 mM EDTA were prepared at the beginning of the experimental period, frozen at -20 ° C and used throughout the project period. A new buffer containing the reducing agent DTT and EDTA was prepared every day from the stock solutions. For a description of the test conditions see Table 4. The DTT is continuously oxidized by oxygen in the air and, therefore, a new buffer must be prepared every day. To benefit from high performance measurements, a 96-well microtiter plate was used and each well had a test volume of 200 µl. The microtiter plate was opaque to avoid cross contamination of the fluorescence emitted between wells. The substrate was diluted to the final concentration in the newly prepared buffer containing DTT and EDTA and transferred to the microtiter plate. The enzyme was diluted in the

5

10

fifteen

twenty

26

same buffer and incubated 10 min in the case of papain and 20 min in the case of Der p 1 for activation. Mixing, transfer and incubation were carried out at room temperature. After incubation, the enzyme solution was transferred to the microtiter plate and measurements were initiated. The measurements were carried out for 10 min with a total of 36 measurements for each well and automatic mixing between each measurement.

Table 4. Conditions of the enzyme assay.

Variables

Papain Der p 1

Substratum

Z-FR-AMC Boc-QAR-AMC

Inhibitor

E-64 E-64

DTT

1-5 mM 5 mM

EDTA

1-5 mM 1 mM

Tampon

50 mM Bis-Tris 50 mM Tris

Temperature

37 ° C 37 ° C

pH

6.5 7.0

Incubation time, activation

10 minutes 20 min

The test volume was not divided equally for each experiment. Therefore, the specific volumes for enzyme, substrate, buffer and inhibitor were different from experiment to experiment depending on the end of the experiment (Table 5).

Table 5. Enzyme assay volumes.

Variables

Activity Kinetics Valuation of active sites

Enzyme

50 µl 50 µl 50 µl

Substratum

100 µl 100 µl 100 µl

Inhibitor

- - 50 µl

Tampon

50 µl 50 µl -

Total

200 µl 200 µl 200 µl

After measuring the enzymatic activity, the maximum slope of the progress curve was estimated using the SoftMax® PRO Life Sciences Edition software, Molecular Devices, 2001. After estimating the initial velocity of the experiments, they were transferred to Prism and analyzed. IgE binding assay

IgE inhibition test for allergen in solution and for allergen adsorbed and then eluted from an aluminum hydroxide adjuvant gel.

This trial assesses the ability of an allergen to bind to IgE from sera from patients allergic to that source of allergens. In this context, this assay was used to evaluate the influence of the binding of an allergen to aluminum hydroxide on its ability to bind IgE, and therefore, on its allergenic activity. Process

IgE inhibition experiments were performed on an ADVIA centaur instrument. Serial dilutions (made with TECAN (P-05-07F294)) of the inhibitor (solution antigen or vaccine in antigen gel adjuvant) were mixed with a fixed amount of biotinylated antigen and further incubated with an IgE absorbed in the phase solid. The amount of biotinylated allergen bound to the solid phase was estimated as the light emitted after incubation with streptavidin labeled with acridinium ester. Unprocessed data was processed in Excel and transferred to GraphPad Prism v. 4.0 for the final analysis (curve fit, representation and statistical comparisons). The data was adjusted to a four-parameter logistic function:

image 1

and the adjusted curves were considered parallel to each other when the slope (HS) of the individual adjustments did not differ significantly. Experimental procedure

Due to the nature of aluminum hydroxide it is not possible to evaluate the binding to IgE in the presence of aluminum hydroxide. Therefore, the effect of the adsorption of Der p 1 on aluminum hydroxide on Der p 1 was evaluated after elution of Der p 1. A 500 µl sample of Der p 1 165 µg / ml was incubated with 100 µl of aluminum hydroxide 6,868 mg / ml for 1 hour at 4 ° C. After adsorption, the solution was centrifuged for 5 minutes at 13,000 rpm. The pellet was resuspended in 300 µl of 50 mM phosphate buffer and incubated for 2 hours in order to elute the adsorbed Der p 1 from the aluminum hydroxide. In the same way, 300 µl of Der p 1 165 µg / ml control were treated without aluminum hydroxide. A sample of Der p extract was prepared for incubation with IgE of pooled serum. EXAMPLE 1 Substrate and enzyme optimization

1.1 Fluorescence of the substrate

Although AMC fluorescence is extinguished while it is bound to the peptide, some fluorescence can still be measured. The influence of substrate fluorescence on the assay was evaluated by making fluorescence measurements of the substrate at different concentrations. Substrate concentrations of 0 µM to 200 µM were used and the fluorescence of the endpoint was measured (Figure 2a).

The linear regression that describes the relationship between substrate concentration and fluorescence has a slope of:

image 1

This indicates that the fluorescence decreases 39.07 URF each time a µM of substrate is cleaved. As a µM of substrate produces a µM of AMC, the increase in fluorescence from the AMC produced is 4106 URF, which means that the net increase in fluorescence when the

image3

All fluorescence measurements were converted to an AMC concentration produced according to the standard curve presented in Figure 2b.

1.2 Enzyme and substrate concentrations

Preliminary studies of optimal enzyme and substrate concentration were performed for the enzyme activity assay. All experiments were carried out with 1 mM DTT and 5 mM EDTA in test concentrations. From the literature, papain should be present in the nM range and the substrate in the µM range, depending on the substrate (Schulz et al .; A Sensitive Fluorescent Assay for Measuring the Cysteine Protease Activity of Der p 1, a Major Allergen From the House Dust Mite Dermatophagoides pteronyssinus, Journal of Clinical Pathology: Molecular Pathology, vol. 51, p. 222-224, 1998; John et al .; Functional Effects of the Inhibition of the Cysteine Protease Activity of the Major House Dust Mite Allergen Der p 1 by a Novel Peptide-based Inhibitor, Clinical and Experimental Allergy, vol. 30, p. 784-793, 2000; Szabelski et al; Influence of Me2SO and Incubation Time on Papain Activity Studied Using Fluorogenic Substrates, Acta Biochimica Polonica, vol. 48: 4, p. 995-1002; 2001). To optimize the exact conditions, a 3 x 3 experimental design was used (Figure 3).

For the 1 nM enzyme concentration, the signal measured in URF / s was approximately equal to the LOQ for the assay (LOQ = 2.44 URF / s). This makes measurements with 1 nM enzyme very unreliable. For the 2.5 nM papain solution, the measurements were at least 2.3 times the LOQ and for the 10 nM papain solution it was 10.6 times. These results reinforce a linear correlation between activity measurements and papain concentration (see Figure 3 (b)). The progress curve of the 10 nM papain solution with 200 µM substrate (which gave the highest activity) showed that the URF did not exceed 15,000 for up to 4 min. This was a reasonable interval for the URF since the initial velocity is measured during the first two minutes and the correlation between URF and the AMC concentration is still linear. At the same time, the correlation between the substrate concentration and the measured enzyme activity was not linear, indicating that the substrate concentrations used in this experiment were higher than KM. From these results, substrate concentrations below 200 µM and papain concentrations of nM were chosen, since the activity measured was at least an order of magnitude greater than that of LOQ.

5

10

fifteen

twenty

25

30

29

EXAMPLE 2 Preliminary experiments on the adsorption of aluminum hydroxide

2.1 Protein quantification in the presence of aluminum hydroxide

To establish whether it was possible to determine the protein concentration by absorption spectroscopy in a sample containing aluminum hydroxide, papain absorption measurements were carried out in the presence and absence of aluminum hydroxide.

The absorbance of the samples containing aluminum hydroxide showed a high level of light scattering, which was expected given the turbidity of the solution (Figure 4). As dilution of the sample results in protein concentrations below the limit of quantification, this procedure is not valid for the estimation of protein under the given conditions. Therefore, the protein concentration in a sample containing aluminum hydroxide was determined indirectly by subtracting the amount of protein not bound to aluminum hydroxide (in the liquid phase) from the amount of protein in the control preparation (in the absence of hydroxide of aluminum).

2.2 Sedimentation of aluminum hydroxide

To investigate whether aluminum hydroxide sediments during the period of the enzymatic test, sedimentation was measured as A400 over time. The gravitational sedimentation profile showed a sedimentation threshold at 40 min (Figure 5). As the enzymatic assay is completed in 10 min, sedimentation does not occur in the assay. EXAMPLE 3 Test Components

In order to verify whether the test components were adsorbed to aluminum hydroxide, thus affecting the result of the enzymatic test, the following binding experiments were carried out (Table 6).

Table 6. Overview of procedures used to assess the influence of hydroxide of

aluminum over the test components. * E-64 showed no different light absorption

between 200 nm and 900 nm.

Measurement

AMC Z-FR-AMC Boc-QAR-AMC E-64 *

Final point

X X X

Activity

X X X

All enzyme assays describing possible interactions with aluminum hydroxide were carried out with papain at a final concentration of 5 nM. 1 mM EDTA and DTT were added at final concentrations of 5 mM. The concentration of aluminum hydroxide was 1.14 mg / ml. Since the absorption measurement of 1.14 mg / ml of aluminum hydroxide shows a high level of light scattering, endpoint measurements were only carried out on samples without aluminum hydroxide. For all experiments concerning the influence of aluminum hydroxide on the test components, adsorption to aluminum hydroxide was carried out for 15 min, 30 min and 60 min.

3.1 AMC

It was evaluated whether AMC, the test product, adsorbs to aluminum hydroxide over time. Measurements were performed at A350 in triplicate of AMC in a control without aluminum hydroxide and in a sample of supernatant, or liquid phase (Figure 6) over time. The supernatant is obtained from an adsorption experiment in which 2 µM AMC in 1.14 mg / ml aluminum hydroxide were mixed together, and then the solid phase fraction was separated from the liquid phase by centrifugation for 5 minutes at 13,000 rpm. A bilateral ANOVA of the results showed that the two factors, absorbance and time (p value = 0.40 and p value = 0.066, respectively) had no significant effect on the results. The interaction term between factors (p value = 0.70) showed no significant effect. Therefore, AMC was not significantly adsorbed to aluminum hydroxide under the conditions given for a period of up to one hour.

To test whether the presence of aluminum hydroxide extinguished AMC fluorescence, an experiment was performed in which the fluorescence of 1 µM AMC was measured with and without aluminum hydroxide over time. This indicated that there was no extinction during the test time (Figure 7). This concentration of AMC is equivalent to the concentration of AMC of the enzymatic assay generated by papain and Der p 1.

3.2 Substrates

It was examined whether the substrates used, Z-FR-AMC and Boc-QAR-AMC, were adsorbed to aluminum hydroxide. For this purpose, the substrates were mixed with 1.14 mg / ml of aluminum hydroxide. The liquid phase (supernatant) was separated from the solid phase by centrifugation. The substrate concentration in the supernatant was compared with the substrate concentration in a preparation without aluminum hydroxide (control) over time in two ways:

a) Comparing the absorption at 325 nm (for both substrates, the maximum absorption

produced at 325 nm (Figure 8)) in both preparations. The determinations are

performed in triplicate using a substrate concentration of 40 µM (Figures 9a and 10a)

b) Measuring the enzymatic activity of papain when mixed with the control in

absence of aluminum hydroxide, the substrate in the presence of aluminum hydroxide

(mixture) and in the supernatant of the mixture. The determinations were made by

triplicate using substrate concentrations of 30 µM (Figures 9b and 10b)

A bilateral ANOVA of the Z-FR-AMC absorbance results indicated that the absorbance value (p value = 0.88) had no significant effect (Figure 9a). On the other hand, the time factor (p value <0.0001) had a significant effect on the result. However, the inspection of the bar chart shows a great variation at the time 60 min. When only data points were analyzed for 15 min and 30 min, the time factor does not become significant (p value = 0.95). This is a reasonable analysis and confirms the validity of the test system.

For the activity test, the statistical analysis of the aluminum hydroxide factor was carried out as a unilateral ANOVA (Figure 9b). The reason for excluding the time factor is that independent enzyme preparations were used for each moment of time, thus confusing time and enzyme concentration. The unilateral ANOVA showed that there was no significant difference between the means of all activity results (p = 0.11). This further corroborates absorbance measurements that do not indicate adsorption of Z-FR-AMC to aluminum hydroxide.

A bilateral ANOVA of the Boc-QAR-AMC absorbance results indicated that neither the absorbance factor (p value = 0.72), neither the time factor (p value = 0.24) nor the interaction factor had significant effect about the result (Figure 10a).

For the activity test, the statistical investigation was carried out in the same way as for Z-FR-AMC using a unilateral ANOVA referring to the activity response alone (Figure 10b). The unilateral ANOVA showed that there was no significant difference between the means of all activity results (p = 0.98). This corroborates absorbance measurements that do not indicate adsorption of Boc-QAR-AMC to aluminum hydroxide.

3.3 E-64

To determine whether the cysteine protease inhibitor E-64 was adsorbed to aluminum hydroxide, enzymatic tests were performed in a control of E-64 12.5 nM without aluminum hydroxide, a mixture of E-64 and aluminum hydroxide, and a supernatant from the mixture, after being separated from the solid phase by centrifugation. This concentration of E-64 does not completely inhibit enzyme activity. Therefore, it is possible to evaluate the binding of E-64 to aluminum hydroxide by enzymatic activity.

A unilateral ANOVA was performed that did not indicate a significant difference between the means (p value = 0.79), hence no adsorption takes place (Figure 11). Moments of time are confused with enzyme concentration due to separate enzyme preparation.

3.4 Summary of the influence of aluminum hydroxide on the components of the enzymatic assay

As a conclusion to the experiments concerning the influence of aluminum hydroxide on the components of the enzymatic test it was found that aluminum hydroxide does not influence any of the test components, AMC, Z-FR-AMC, Boc-QAR-AMC and E-64, both in endpoint measurements and in enzyme activity measurements.

EXAMPLE 4

4.1 Adsorption of papain to aluminum hydroxide

After the validation of the enzyme test with and without the presence of aluminum hydroxide, an experimental design was made to investigate the kinetic parameters of papain and a possible influence of aluminum hydroxide on them. As it was expected that papain would only adsorb to a lesser extent aluminum hydroxide (pI approximately PZC aluminum hydroxide), it was chosen as a negative control. It will reflect the possible effect of the presence of aluminum hydroxide in the test medium on the kinetic results when the greater proportion of the enzyme molecules is not bound to the adjuvant. An overview of sample preparation is given in Figure 12.

A solution of 3 ml with 100 µg / ml of papain and 1.14 mg / ml of aluminum hydroxide was prepared. This solution was placed for 1 h at 4 ° C to allow adsorption of papain to aluminum hydroxide. After adsorption 500 µl were taken for further analysis and the rest of the mixture was centrifuged for 10 min at 4000 rpm. The rest of the samples analyzed follow the diagnostic algorithm in Figure 12. In addition, a control (Con) containing papain in Bis-Tris buffer was prepared in the absence of aluminum hydroxide. The control was incubated as the aluminum hydroxide mixture for 1 h at 4 ° C and then analyzed.

The samples were analyzed with the following procedures:

•

Determination of protein concentration (enzyme) (A280nm and evaluation of active sites)

•

Measurements of enzymatic activity

4.2 Determination of papain concentration

The protein concentration of the different samples was evaluated in two ways:

4.2.1 A280

These measurements were converted to protein concentration by Lambert-Beer's law using a molar absorption coefficient for papain of 2.46 mg / ml. As it is not technically possible to determine the concentration of protein from A280 in the presence of aluminum hydroxide, the concentration of papain in Mixture 1 and Mixture 2 was indirectly estimated. The protein concentration in Mix 1 was the same as in Con, and the protein concentration in Mix 2 was the difference between Con and Sob 1. An ANOVA of the results showed no difference between Con and Sob 1 (p value = 0.615) which indicates that papain is not significantly adsorbed to aluminum hydroxide. The concentrations of papain estimated in the control and the amount adsorbed to aluminum hydroxide are shown in Table 7.

Protein measurements do not represent the 100 µg / ml concentration

estimated in Con. The loss of protein may be due to the erroneous estimation of the original papain concentration in the stock solution. This concentration was measured by the manufacturer and, therefore, not on the same instrument as the rest of the samples.

Table 7. Determination of the kinetic parameters Vmax, KM and kcat (Vmax / 5 protein concentration), in addition to the protein concentration for papain. a Protein concentration

determined to A280. b Protein concentration determined by assessment of active sites. * The protein concentrations obtained are determined indirectly (see text). The control represents a papain preparation in the absence of aluminum hydroxide. Adsorbed refers to a papain preparation adsorbed to aluminum hydroxide (Mixture 2).

10

Preparation

KM (µM) Vmax (ng / ml · s) Protein concentration kcat a (1 / s) Protein concentration kcat b (1 / s)

Control

22 373.9 70.8 ng / ml 5.13 33.7 ng / ml 11.41

Adsorbed

twenty-one 44.1 3.0 ng / ml * 14.58 3.4 ng / ml 12.18

4.2.2 Valuation of active sites

Active site titration was used to estimate the amount of active enzyme in the

different samples. This is a difference in the protein concentration estimated from

A280 representing the total protein content of the sample. Test conditions

15 used are indicated in Table 8 below. These results show that approximately 7% (Mixture 3) of the active papain was adsorbed to aluminum hydroxide, while 91% (Sob 1 + Sob 2) was in solution. This is in accordance with the results obtained in the evaluation of the enzymatic activity in the samples, see Section 4.3.1.

20 4.3 Enzymatic activity Two types of enzyme tests were performed: (i) activity measurements using a fixed substrate concentration to evaluate the enzymatic activity and (ii) Michaelis-Menten kinetic activity to estimate the Vmax and KM kinetic parameters. All activity tests were performed with Bis-Tris buffer, pH 6.5, 5 mM DTT,

25 mM EDTA and Z-FR-AMC as substrate. An overview of the different enzyme tests performed is given in Table 8. Table 8. Specifications of enzyme tests for the papain adsorption experiment. Activity measurements are individual combinations of enzyme and substrate in order to assess the level of activity in samples. Michaelis-Menten's kinetic measurements of the

Enzymatic activity with different substrate concentrations is used to estimate Vmax and KM.

All measurements were in triplicate.

Analysis Parameter

Activity Michelis-Menten Kinetics Valuation of active sites

Dilution (With, Mix 1, Sob 1)

2048, 1024, 512, 256 1024 1024

Dilution (Mix 2, Sob 2, Mix 3)

2048,1024, 512, 256 128 128

Substrate concentration

30 µM 12.5 µM, 25 µM, 37.5 µM, 50 µM, 75 µM, 100 µM, 125 µM, 150 µM 30 µM

Inhibitor concentration

0.0 nM, 0.25 nM, 0.50 nM, 1.0 nM, 1.5 nm, 2.0 nM, 3.0 nM, 4.0 nM

DTT incubation time

10 minutes 10 minutes 60 min *

* Incubation with both DTT and E-64.

4.3.1 Evaluation of enzymatic activity

Activity measurements were used to quantitatively analyze the amount of papain adsorbed to aluminum hydroxide and the amount freely distributed in the solution. 96% of the activity in Mix 1 was found in solution (Sob 1 + Sob 2), while 8%

5 of the papain activity was adsorbed to aluminum hydroxide (Mixture 3). The activity of the control is 17% lower than the activity of Mix 1. This could be due to a loss of activity of the protein in the control during the time period of the experiment. This loss was avoided by the presence of aluminum hydroxide.

In order to further evaluate this observation, new experiments were performed.

10 in which papain activity was followed over time in the absence of aluminum hydroxide. Papain was mixed with 50 mM Bis-Tris buffer at a final concentration of 10 µg / ml in a total volume of 500 µl. The mixture was incubated at 4 ° C and the samples were taken at 0, 30 and 60 min. The samples were diluted and incubated for 10 min in buffer containing DTT and the activity was subsequently measured after substrate addition. The analyzed results

15 with unilateral ANOVA showed that there was a significant difference between the 3 moments of time (p value = 0.015) and by the Newman-Keuis comparison test it was found that they differed 60 min from the other two time points, and it was 7% lower. These results suggest that during the 60 min incubation between papain and aluminum hydroxide, the papain in the control sample has lost 7% of its activity. An additional loss occurs until the start of the enzymatic assay.

4.3.2 Kinetic parameters of Michaelis-Menten

The parameters of Michaelis-Menten KM and Vmax were evaluated in the different samples.

The estimated KM and Vmax values of the different samples are shown in Table 7 above. The Bartlett test did not indicate a significant difference between the variances of KM samples (p value = 0.758) and a unilateral ANOVA was used to compare means. The unilateral ANOVA test showed no significant difference between approximate KM calculations (p value = 0.999), which indicates that the affinity towards the substrate does not change when papain is in the presence and absence of aluminum hydroxide.

The Vmax in solution (Sob 1 + Sob 2) is 97% of that in Mixture 1, while Vmax in Mixture 3 corresponds to 7%.

Vmax is specific for a defined enzyme and depends linearly on the enzyme concentration in the assay. The normalization of Vmax by the enzyme concentration gives the kcat parameter, which only depends on the characteristics of the enzymatic activity. Table 7 above shows the estimated kcat values for the different samples calculated from the enzyme concentration values obtained from the valuation of active sites and A280.

The kcat values obtained from the determination of A280 of the protein concentration were approximately half of those obtained with the assessment of active sites in the control, which indicates that half of the protein in the samples was inactive. The kcat values of Con, Mixture 1, Sob 1, Mixture 2 and Mixture 3 are comparable when the enzyme concentration was calculated by titration of active sites, suggesting that the presence of aluminum hydroxide does not affect the kinetic properties of papain. The kcat values of Con, Mixture 1, Sob 1 and Sob 2 are also comparable when the enzyme concentration is calculated from A280. However, indirect determination of the protein concentration in Mixture 2 makes the kcat value more inaccurate.

The fact that the kinetic parameters evaluated in the presence and absence of aluminum hydroxide are not significantly different indicates that the presence of aluminum hydroxide did not affect the enzymatic reaction. In conclusion, these results show that it is possible to measure the enzymatic properties of an enzyme in the presence of aluminum hydroxide when most of the enzyme (93%) is not bound to the adjuvant.

EXAMPLE 5

5.1 Adsorption of Der p 1 to aluminum hydroxide

According to the theory of protein adsorption to aluminum hydroxide, Der p 1 was expected to be adsorbed (pI lower than the PZC of aluminum hydroxide). The effect of this adsorption on the activity and structure of Der p 1 was examined.

An overview of sample preparation is given in Figure 13. A solution of 3 ml with 100 µg / ml of Der p 1 and 1.14 mg / ml of aluminum hydroxide was prepared. This solution was placed for 1 h at 4 ° C to allow adsorption of Der p 1 to aluminum hydroxide. After adsorption 500 µl was taken for further analysis and the rest of the mixture was centrifuged for 10 min at 4000 rpm. An elution step was performed for 1 h at 4 ° C in which the sediment of Mixture 2 was resuspended in phosphate buffer. In addition, two controls (With 1 and With 2) were prepared containing Der p 1 in Tris buffer in the absence of aluminum hydroxide. With 1 it was analyzed at the beginning of the experiment and with 2 it was analyzed at the end of the 2 hour experiment.

The samples were analyzed with the following procedures:

•

Determination of protein concentration (A280 nm and RIE)

•

Measurements of enzymatic activity

•

Union to IgE

5.2 Determination of the concentration of Der p 1

The determination of the concentration of Der p 1 in the different samples was evaluated by A280 and RIE.

5.2.1 A280

In order to quantify the protein content, absorbance at 280 nm was measured in samples without aluminum hydroxide (With 1, With 2, Sob and Elu). Absorbance measurements were converted to protein concentration by Lambert-Beer law using a molar absorption coefficient for Der p 1 of 1.72 mg / ml as shown in Table 8a.

Table 8a. Determination of the kinetic parameters Vmax, Km and kcat (Vmax / concentration of

protein), in addition to protein concentration for Der p1. a Protein concentration

determined to A280. Protein concentration determined by RIE. * The concentrations of

Proteins obtained are determined indirectly (see text). The control represents a

Preparation of Der p 1 in the absence of aluminum hydroxide. Adsorbed refers to a

Preparation of Der p 1 adsorbed to aluminum hydroxide (Mixture 2).

Preparation

KM (µM) Vmax (ng / ml · s) Protein concentration kcat a (1 / s) Protein concentration kcat c (1 / s)

Control

47 0.25 10.4 0.024 13.9 0.018

Adsorbed

56 0.15 7.1 * 0.022 7.8 0.019

Eluted

51 0.11 4.0 0.025 7.6 0.014

The protein concentration in Mix 1 and Mix 2 was estimated indirectly. The protein concentration in Mix 1 was considered the same as in Con 1 and the protein concentration in Mix 2 was the difference between Con 1 and Sob. A t test did not show

5 significant difference between protein concentration in Con 1 and Con 2 (p value = 0.092). 32% of the protein content in Con 1 was found in Sob, which indicates an adsorption degree of approximately 70%. Of the adsorbed protein, 56% was eluted from aluminum hydroxide using phosphate buffer.

5.2.2 RIE

10 Another procedure applied to quantify protein content was RIE. Samples With 1, With 2, Sob and Elu were evaluated together with three Der p 1 standards (125 ng, 250 ng and 500 ng). The estimated Der p 1 concentrations of Der p 1 in the control, the amount adsorbed to aluminum hydroxide, in addition to the eluted amount are shown in Table 8a. The estimated values were generated from the linear regression standard curve of the

15 three patterns (concentration depending on the area of precipitate). 44% of the protein content in Con 1 was found in Sob that indirectly indicates an adsorption degree of approximately 56%. Of the adsorbed protein, 98% was eluted from aluminum hydroxide using phosphate buffer. The concentration of Con 1 and Con 2 was outside the prediction area of the standard curve and, therefore, it is likely that the concentration of Der p 1 is underestimated. This

20 can explain the high degree of elution of Der p 1 from aluminum hydroxide since the concentration of adsorbed Der p 1 is estimated as the difference between Con 1 and Sob.

5.3 Enzymatic activity

Different types of enzymatic assays were performed: (i) activity measurements using a fixed substrate concentration to evaluate the enzymatic activity, and (ii) Michaelis-Menten kinetics to estimate the Vmax and KM kinetic parameters.

All activity tests were performed with Tris buffer, pH 7.0, 5 mM DTT, 1 mM EDTA and Boc-QAR-AMC as substrate. An overview of the different enzyme tests performed is given in Table 9. Table 9. Specifications of enzyme tests for the Der p 1 adsorption experiment.

30 Activity measurements are individual enzyme and substrate combinations in order to

Evaluate the level of activity in samples. Michaelis-Menten kinetic measurements of enzyme activity with different substrate concentrations are used to estimate Vmax and KM. All measurements were in triplicate.

Analysis Parameter

Activity Measurement Michelis-Menten Kinetics

Dilution (With 1, Mix 1, Sob, Mix 2, Elu, With 2)

4 and 8 8

Substrate concentration

100 µM 25 µM, 50 µM, 75 µM, 100 µM, 150 µM, 200 µM

Inhibitor concentration

Incubation time

20 min 20 min

* Incubation with both DTT and E-64.

5.3.1 Evaluation of enzymatic activity

5 Measurements of the activity of Der p 1 in the absence and in the presence of aluminum hydroxide were used to quantify the amount of Der p 1 adsorbed to aluminum hydroxide and the amount freely distributed in the solution. The results of the activity measurements are summarized in Figure 14.

The activity of Der p 1 not adsorbed on Sob corresponds to 33% of the activity

10 in Mix 1. 66% of Der p 1 activity was adsorbed to aluminum hydroxide and 67% of this activity was desorbed after elution of Mix 2 with phosphate buffer. In order to monitor the stability of Der p 1 during the experiment period, the activity of Con 1 was measured at the beginning of the experiment and Con 2 at the end. The performance of a unilateral ANOVA in Con 1, Con 2 and Mixture 1 showed no significant difference between the means (p value = 0.76). This

15 indicates that neither time nor the presence of aluminum hydroxide influences the activity of Der p 1 under these conditions.

5.3.2 Michaelis-Menten kinetic parameters

The parameters of Michaelis-Menten KM and Vmax were evaluated in the different samples. Figure 15 shows a typical Michaelis-Menten curve for Der p 1 in the presence of

20 aluminum hydroxide. KM and Vmax values are shown in Table 8a. The Bartlett test did not indicate a significant difference between the variances of KM (p value = 0.11) and a unilateral ANOVA showed no significant difference between the means of the samples (p value = 0.62). This indicates that the affinity of Der p 1 towards Boc-QAR-AMC does not change in the presence of aluminum hydroxide.

25 The Vmax of Con 2 is 14% lower than Vmax in Con 1 (t-test, p value = 0.0024) indicating that Der p 1 has lost activity during the time period of the experiment. A Newman-Keuls multiple comparison test showed no significant difference between Vmax of Con 1 and Mixture 1 (p> 0.05), suggesting that aluminum hydroxide had no influence on the activity of Der p 1. In addition, it seems that aluminum hydroxide has prevented the loss of activity in the observed time from Con 1 to Con 2. Vmax in Sob and Mixture 2 is 30% and 62% Vmax in Mixture 1, respectively. 68% of the activity in Mix 2 was found in Elu.

The kcat values of Der p 1 were estimated from the Vmax values obtained and the protein concentrations obtained from the different procedures (A280 and RIE) as shown in Table 8a. The kcat values of the different samples are comparable when the protein concentration was estimated by A280 and RIE.

In conclusion, the KM and kcat values obtained for Der p 1 were not significantly different in the absence of aluminum hydroxide or when most of the Der p 1 molecules (60-70%) were adsorbed to aluminum hydroxide. These data confirm that it is possible to measure the enzymatic activity of an enzyme adsorbed to aluminum hydroxide by evaluating the impact of an enzyme adsorption on aluminum hydroxide on the activity / structure of the possible enzyme.

5.4 Union to IgE

The influence of aluminum hydroxide on the ability of Der p 1 in the absence of aluminum hydroxide (Con 1) and bound and eluted Der p 1 of aluminum hydroxide (Elu) to bind IgE from sera of patients allergic to HDM

Inhibition of the biotin-labeled Der p 1 standard signal using increasing concentrations of Der p 1 in Con 1 and Elu under evaluation followed a sigmoid downward curve (Figure 16).

Comparing the curve of Con 1 with Elu it is clear that the background levels are identical, therefore complete inhibition is possible in any case. This indicates that all IgE epitopes in Con 1 were still present in Elu. A t-test of a sample comparing the slopes of Con 1 and Elu showed that the means were not significantly different (p = 0.83), which indicates that the affinity of IgE towards epitopes in Der p 1 was retained after aluminum hydroxide bond

Claims (10)

1. A method for measuring the immunological activity of a vaccine preparation in the form of a mixture of one or more allergenic enzymes and aluminum hydroxide, in which the mixture comprises a liquid phase and a solid phase, and in which the at least a part of the allergenic enzyme (s) is adsorbed in the solid phase, a process comprising the steps of measuring the enzymatic activity of the mixture and / or measuring the enzymatic activity of the allergenic enzyme in the phase liquid after a separation of the liquid phase from the solid phase, and / or measuring the enzymatic activity of the allergenic enzyme in the solid phase after a separation of the liquid phase from the solid phase, in an enzyme activity test, and using the measurement obtained as an indication of the immunological activity of the vaccine preparation. 2.-A method for quantifying the amount of allergenic enzyme in a vaccine preparation in the form of a mixture of one or more allergenic enzymes and aluminum hydroxide, in which the mixture comprises a solid phase and a liquid phase, and wherein at least a part of the allergenic enzyme (s) is adsorbed to the solid phase, a process comprising the steps of measuring the enzymatic activity of the mixture and / or measuring the enzymatic activity of the enzyme allergen in the liquid phase after a separation of the liquid phase from the solid phase, and / or measuring the enzymatic activity of the allergen enzyme in the solid phase after a separation of the liquid phase from the solid phase, in an enzyme activity test , and use the measurement obtained to quantify the amount of allergenic enzyme. 3. The method according to any one of claims 1-2, wherein a specific substrate for an allergenic enzyme is used to measure the enzymatic activity of the allergenic enzyme. 4. The method according to claim 3, wherein an inhibitor is used to inhibit one or more of the allergenic enzyme (s) in the vaccine preparation. 5. The method according to any of claims 1-2 and 4, wherein the allergenic enzyme (s) is in the form of an extract. 6. The method according to any one of claims 1-5, wherein at least one of the allergenic enzymes is a cysteine protease. 7. The method according to any one of claims 1-5, wherein at least one of the allergenic enzymes is a serine protease. 8. The method according to any one of claims 1-7, wherein the allergenic enzyme (s) is (are) one or more selected from the group consisting of Der p 1, Der p 3, Der p 6 and Der p 9. 9. The method according to claim 8, wherein the allergenic enzyme is Der p 1. 10. The method according to any one of claims 1-6 and 8-9, wherein the substrate used is Z-LeuLeuGlu-MCA. 11. The method according to any one of claims 1-2, wherein the enzyme activity assay is an absorbance assay, fluorescence assay, FTIR or an immunological procedure. 12. The method according to claim 11, wherein the enzyme activity assay is an ELISA. 13. The method according to claim 11, wherein the enzyme activity assay is a fluorescence assay. 14. The method according to any one of claims 1-13, wherein the vaccine preparation is subjected to both a measurement of the enzymatic activity of the mixture of the liquid phase and the solid phase (measurement 1) and a a measurement of the enzymatic activity of the allergenic enzyme in the liquid phase (measurement 2), or is subjected to both a measurement of the enzymatic activity of the allergenic enzyme in the liquid phase (measurement 2) and a measurement 15 of the enzymatic activity of the allergenic enzyme in the solid phase (measurement 3). 15. The method according to any one of claims 1-13, wherein the vaccine preparation is subjected only to a measurement of the enzymatic activity of the allergenic enzyme in the solid phase after a separation of the liquid phase from the phase. solid (measurement 3). twenty