# Patent

## Title
Recombinant allergen with reduced enzymatic activity

## Abstract

The present invention provides a novel treatment for allergy comprising a recombinant allergen with reduced enzymatic activity, thereby reducing the potential for an allergic response upon contact with a wild-type allergen.

## Background
This application claims the benefit of §371 application of PCT/EP98/07521 filed 16 Nov. 1997.
The present invention relates to novel therapeutic formulations, said formulations being effective in the reduction of allergic responses to specific allergens. Further, this invention relates to novel polynucleotides, polypeptides encoded by them and to the use of such polynucleotides and polypeptides, and to their production. In particular, novel vaccines are provided comprising polypeptides and to their use in the treatment of humans suffering from allergies or prevention of individuals at risk from allergies, preferably said vaccines comprising a recombinant mutant Dermatophagoides pteronyssinus allergen Der P1.
Allergic responses in humans are common, and may be triggered by a variety of allergens. Allergic individuals are sensitised to allergens, and are characterised by the presence of high levels of allergen specific IgE in the serum, and possess allergen specific T-cell populations which produce Th2-type cytokines (IL-4, IL-5, and IL-13). Binding of IgE, in the presence of allergen, to Fc receptors present on the surface of mastocytes and basophils, leads to the rapid degranulation of the cells and the subsequent release of histamine, and other preformed and neoformed mediators of the inflammatory reaction. In addition to this, the stimulation of the T-cell recall response results in the production of IL-4 and IL-13, together cooperating to switch B-cell responses further towards allergen specific IgE production. For details of the generation of early and late phase allergic responses see Joost Van Neeven et al., 1996, Immunology Today, 17, 526. In non-allergic individuals, the immune response to the same antigens may additionally include Th1-type cytokines such as IFN-γ. These cytokines may prevent the onset of allergic responses by the inhibition of high levels of Th2-type immune responses, including high levels of allergen specific IgE. Importantly in this respect, is the fact that IgE synthesis may be controlled by an inhibitory feedback mechanism mediated by the binding of IgE/allergen complexes to the CD23 receptor on B-cells (Luo et al., J. Immunol., 1991, 146(7), 2122-9; Yu et al., 1994, Nature, 369(6483):753-6). In systems that lack cellular bound CD23, this inhibition of IgE synthesis does not occur. Current strategies in the treatment of such allergic responses include means to prevent the symptomatic effects of histamine release by anti-histamine treatments and/or local administration of anti-inflammatory corticosteroids. Other strategies which are under development include those which use the hosts immune system to prevent the degranulation of the mast cells, Stanworth et al., EP 0 477 231 B1. Other forms of immunotherapy have been described (Hoyne et al., J. Exp. Med., 1993, 178, 1783-1788; Holt et al., Lancet, 1994, 344, 456-458).
Some common allergens present in bee venom, house dust mite emanations and parasite proteins have been found to induce mast cell degranulation, and to stimulate interleukin-4 synthesis and secretion, even in the absence of allergen-specific IgE (Machado et al, 1996, Eur. J. Immunol. 26, 2972-2980). This non-immunological degranulation by proteolytic allergens, such as bee venom phospholipase A2 or proteases associated with house dust mite emanations is dependent on enzymatic activity.
The present invention provides recombinant mutant allergens having significantly reduced proteolytic activity relative to the wild-type proteolytically active allergen, as well as nucleic acids encoding the same, and their use as a prophylactic or immunotherapeutic agent against allergy. A preferred allergen is the house dust mite allergen Der p1.
The present invention relates to the provision of formulations for the treatment and prophylaxis of allergy, by providing means to down-regulate the production of IgE, as well as modifying the cell mediated response to the allergen, through a shift from a Th2 type to a Th1 type of response (as measured by the reduction of ratio of IL-4: IFN-γ producing DerP1 specific T-cells, or alternatively a reduction of the IL-5:IFN-γ ratio). This is achieved by the provision and use of recombinant mutant allergens with impaired enzymatic activity.
DerP1, a group 1 protease allergen of the house dust mite Dermatophagoides pteronyssinus (Topham et al., 1994, Protein Engineering, 7, 7, 869-894; Simpson et al., 1989, Protein Sequences and Data Analyses, 2, 17-21) is one such allergen. It is a 30 KDa protein and has been cloned and sequenced (Chua et al., 1988, J. Exp. Med., 167, 175-182). It is known to contain 222 amino acid residues in the mature protein. The sequence of DerP1 shares 31% homology to Papain, and importantly shares homology in the enzymatically active regions, most notably the Cys34-His170 ion pair (Topham et al., supra). DerP1 is produced in the mid-gut of the mite, where its role is probably related to the digestion of food. Up to 0.2 ng or proteolytically active DerP1 is incorporated into each fecal pellet, each around 10-40 μm in diameter and, therefore, easily inspired into the human respiratory tract. Overnight storage of purified DerP1 preparations at room temperature results in almost complete loss of enzymatic activity due to autoproteolytic degradation (Machado et al., 1996, Eur. J. Immunol. 26, 2972-2980).
DerP1 has been found to cleave the low affinity immunoglobulin IgE Fc receptor from the surface of human B lymphocytes (CD23, Hewitt et al., 1995, J. Exp. Med., 182, 1537-1544) and CD25 (Schultz et al., J. Exp. Med, 1998, 187(2):271-5) the alpha subunit of the human T cell interleukin-2 receptor. Cleavage of the receptor from the B cell surface was associated with a parallel increase in soluble CD23 in the culture supernatant. It has been suggested that the loss of cell surface CD23 from IgE-secreting B cells may promote and enhance IgE immune responses by ablating the important inhibitory feedback mechanism that normally limits IgE synthesis (Hewitt et al., 1995, J. Exp. Med., 182, 1537-1544). Furthermore, since soluble CD23 has been shown to promote IgE production, fragments of CD23 released by DerP1 may directly enhance the synthesis of IgE. In addition to the effects of CD23 cleavage, the cleavage of CD25 from the surface of T-cells induces a decrease in proliferation and INF-gamma secretion, which, consequently, may bias the immune response toward a Th2 type response. Recent papers which relate to the DerP1 antigen are Machado et al. Eur. J. Immunol. (1996) 26: 2972-2980; Hewitt et al., J. Exp. Med. (1995) 182: 1537-1544; and Schulz et al. Eur. J. Immunol. (1995) 25: 3191-3194.
Other mutant allergens having reduced proteolytic activity which form part of the present invention may be based upon other group I cyteine proteases, such as Der f1 from Dermatophagoides farinae (80% homology to DerP1), as well as the groups III allergens (serine proteases) including DerpIII (Stewart et al., 1992, Immunology, 75, 29-35) and DerpIV (Yaseuda et al., 1993, Clin. Exp. Allergy, 23, 384-390); and the group IV allergens (amylases).
The allergens of the present invention are recombinantly produced. Der p1 proteolytic activity can be impaired by introducing mutations into the cDNA or genomic DNA, either at the enzymatically active site, or at the site of cleavage between the propeptide and the mature molecule. Said mutant allergen having the following advantages over the wild-type allergen: 1) increases the Th1-type aspect of the immune responses in comparison to those stimulated by the wild type allergen, thereby leading to the suppression of allergic potential of the vaccinated host, and 2) having reduced allergenicity thus being more suitable for systemic administration of high doses of the immunogen, 3) will induce DerP1 specific IgG which compete with IgE for the binding of native DerP1.
The allergens of the present invention are also more stable than isolated or recombinant active DerP1, as measured by the lack of autoproteolytic degradation. Thus, the present invention also provides allergens which are stable compared to the wild-type form of the allergen, said allergens having significantly reduced proteolytic activity and being substantially full length proteins, optionally said allergens further comprising the pro-form of allergen.
One aspect of the present invention provides a nucleic acid encoding mutated Der p1 as set out above, and a further aspect of the invention provides mutated Der p1 per se. A yet further aspect of the present invention provides substantially stable recombinant DerP1. Said stable DerP1 being of substantially full length mature protein, or mature protein further comprising the pro-DerP1 section. The term “stable” in the context of the present invention is a product which does not undergo a substantial amount of decomposition by autoproteolysis when incubated overnight at room temperature in comparison to proteolytically active wild-type DerP1, as evidence by SDS PAGE analysis.
A still further aspect of the invention provides a process for the preparation of a mutated Der p1 protein, which process comprises expressing DNA encoding the said protein in a recombinant host cell and recovering the product.
A DNA molecule encoding a mutated Der p1 (or other mutated allergen) forms a further aspect of the invention and can be synthesized by standard DNA synthesis techniques, such as by enzymatic ligation as described by D. M. Roberts et al in Biochemistry 1985, 24, 5090-5098, by chemical synthesis, by in vitro enzymatic polymerization, or by a combination of these techniques.
Enzymatic polymerisation of DNA may be carried out in vitro using a DNA polymerase such as DNA polymerase I (Klenow fragment) in an appropriate buffer containing the nucleoside triphosphates dATP, dCTP, dGTP and dTTP as required at a temperature of 10°-37° C., generally in a volume of 50 ml or less. Enzymatic ligation of DNA fragments may be carried out using a DNA ligase such as T4 DNA ligase in an appropriate buffer, such as 0.05M Tris (pH 7.4), 0.01M MgCl2, 0.01M dithiothreitol, 1 mM spermidine, 1 mM ATP and 0.1 mg/ml bovine serum albumin, at a temperature of 4° C. to ambient, generally in a volume of 50 ml or less. The chemical synthesis of the DNA polymer or fragments may be carried out by conventional phosphotriester, phosphite or phosphoramidite chemistry, using solid phase techniques such as those described in ‘Chemical and Enzymatic Synthesis of Gene Fragments—A Laboratory Manual’ (ed. H. G. Gassen and A. Lang), Verlag Chemie, Weinheim (1982), or in other scientific publications, for example M. J. Gait, H. W. D. Matthes, M. Singh, B. S. Sproat, and R. C. Titmas, Nucleic Acids Research, 1982, 10, 6243; B. S. Sproat and W. Bannwarth, Tetrahedron Letters, 1983, 24, 5771; M. D. Matteucci and M. H Caruthers, Tetrahedron Letters, 1980, 21, 719; M. D. Matteucci and M. H. Caruthers, Journal of the American Chemical Society, 1981, 103, 3185; S. P. Adams et al., Journal of the American Chemical Society, 1983, 105, 661; N. D. Sinha, J. Biernat, J. McMannus, and H. Koester, Nucleic Acids Research, 1984, 12, 4539; and H. W. D. Matthes et al., EMBO Journal, 1984, 3, 801.
Alternatively, the coding sequence can be derived from DerP1 mRNA, using known techniques (e.g. reverse transcription of mRNA to generate a complementary cDNA strand), and commercially available cDNA kits.
The invention is not limited to the specifically disclosed sequence, but includes any proteolytic allergen which has been mutated to remove some or all of its proteolytic activity, whilst retaining the ability to stimulate an immune response against the wild-type allergen. The proteolytic activity of the mutant allergens may be compared to the wild type by a CD23 cleavage assay according to Shultz et al., 1995, European Journal of Immunology, 25, 3191-3194), or enzymatic degradation of substrates described in Machado et al., 1996, Eur. J. Immunol., 26, 2972-2980. The immunogenicity of the mutant allergen may be compared to that of the wild-type allergen by various immunologicals assays. The cross-reactivity of the mutant and wild-type allergens may be assayed by in vitro T-cell assays after vaccination with either mutant or wild-type allergens. Briefly, splenic T-cells isolated from vaccinated animals may be restimulated in vitro with either mutant or wild-type allergen followed by measurement of cytokine production with commercially available ELISA assays, or proliferation of allergen specific T cells may be assayed over time by incorporation of tritiated thymidine. Also the immunogenicity may be determined by ELISA assay, the details of which may be easily determined by the man skilled in the art. Briefly, two types of ELISA assay are envisaged. First, to assess the recognition of the mutant DerP1 by sera of mice immunized with the wild type Der p1; and secondly by recognition of wild type DerP1 allergen by the sera of animals immunised with the mutant allergen. Briefly, each wells will be coated with 100 ng of purified wild type or mutated Der p1 overnight at 4° C. After incubating with a blocking solution (TBS-Tween 0.1% with 1% BSA) successive dilutions of sera will be incubated at 37° C. for 1 hour. The wells are washed 5 times, and total IgG revealed by incubating with an anti-IgG antibody conjugated with Alkaline phosphatase.
The reduction of enzymatically active allergen or DerP1 may be performed by introducing mutations into the native sequence before recombinantly producing the inactivated mutants. This may be achieved by: introducing substitutions, deletions, or additions into the active sites; by inserting, deleting, or substituting residues in regions of processing the inactive pro-enzyme into the active mature protein; or by altering the three dimensional structure of the protein such that enzymatic activity is lost, this may be achieved, amongst others, by expressing the protein in fragments, or by deleting cysteine residues involved in disulphide bridge formation, or by deleting or adding residues such that the tertiary structure of the protein is substantially altered. Alternatively, mutations may be generated with the effect of altering the interaction between the Cys and the His residues, at positions 34 and 170 of the mature protein respectively (corresponding to positions 132 and 268 of the pre-pro-protein respectively) in the resultant fully folded recombinant protein.
The invention is illustrated herein, but not limited to, three specific mutations which are are given as examples of proteolytically inactive DerP1. First, the enzymatic activity of DerP1 is abrogated by substituting a Cysteine residue in the active site for an alanine. This substitution occurs at Cys132→Ala132 of the pro-DerP1 protein sequence, and is set out in SEQ ID NO. 1. Second, the DerP1 allergen is recombinantly expressed and retained in its inactive pro-protein form by deletion of four amino acid residues at the linker region between the pro- and mature proteins. This deletion removes amino acid residues NAET (SEQ ID NO. 31) from the site 96-99 inclusive, from the Pro-DerP1 protein sequence. This sequence is set out in SEQ ID NO. 2. Third, enzymatic activity of DerP1 is abrogated by substituting a Histidine residue in the active site for an alanine. This substitution occurs at His268→Ala268 of the pro-DerP1 protein sequence, and is set out in SEQ ID NO. 3.
The active sites of each wild-type enzymatic allergen may be determined from the literature, or by reference to homologues. For example, the active sites of DerP1, being a cysteine protease, may be putatively inferred by reference to other known cysteine proteases such as Papain. DerP1 shares essential structural and mechanistic features with other papain-like cysteine proteinases, including cathepsin B. The active site thiolate-imidazolium ion pair comprises the side chains of Cys34 and His 170 (Topham et al., 1994, Protein Engineering, 7, 7, 869-894).
Mutated versions of Der p 1 may be prepared by site-directed mutagenesis of the cDNA which codes for the Der p 1 protein by conventional methods such as those described by G. Winter et al in Nature 1982, 299, 756-758 or by Zoller and Smith 1982; Nucl. Acids Res., 10, 6487-6500, or deletion mutagenesis such as described by Chan and Smith in Nucl. Acids Res., 1984, 12, 2407-2419 or by G. Winter et al in Biochem. Soc. Trans., 1984, 12, 224-225.
The process of the invention may be performed by conventional recombinant techniques such as described in Maniatis et. al., Molecular Cloning—A Laboratory Manual; Cold Spring Harbor, 1982-1989.
In particular, the process may comprise the steps of:

    
    
        1. Preparing a replicable or integrating expression vector capable, in a host cell, of expressing a DNA polymer comprising a nucleotide sequence that encodes the said mutant Der p1 protein;
        2. Altering the enzymatic activity of the resultant protein by one of the following techniques: replacing the cysteine or histidine residues (or other residues interacting with other residues within the active site) from the active site with an alanine residue using site directed mutagenesis; replacement of a cDNA fragment by a pair of oligonucleotides whose sequence differ from the natural one; or alternatively, deleting four residues at the junction between the propeptide and the mature enzyme using site directed mutagenesis
        3. Transforming a host cell with the said vector
        4. Culturing the transformed host cell under conditions permitting expression of the DNA polymer to produce the protein; and
        5. Recovering the protein.
    
    


The term ‘transforming’ is used herein to mean the introduction of foreign DNA into a host cell by transformation, transfection or infection with an appropriate plasmid or viral vector using e.g. conventional techniques as described in Genetic Engineering; Eds. S. M. Kingsman and A. J. Kingsman; Blackwell Scientific Publications; Oxford, England, 1988. The term ‘transformed’ or ‘transformant’ will hereafter apply to the resulting host cell containing and expressing the foreign gene of interest.
The expression vector is novel and also forms part of the invention.
The replicable expression vector may be prepared in accordance with the invention, by cleaving a vector compatible with the host cell to provide a linear DNA segment having an intact replicon, and combining said linear segment with one or more DNA molecules which, together with said linear segment encode the desired product, such as the DNA polymer encoding the Der p 1 protein under ligating conditions.
Thus, the DNA polymer may be preformed or formed during the construction of the vector, as desired.
The choice of vector will be determined in part by the host cell, which may be prokaryotic or eukaryotic. Suitable vectors include plasmids, bacteriophages, cosmids and recombinant viruses.
The preparation of the replicable expression vector may be carried out conventionally with appropriate enzymes for restriction, polymersation and ligation of the DNA, by procedures described in, for example, Maniatis et al cited above.
The recombinant host cell is prepared, in accordance with the invention, by transforming a host cell with a replicable expression vector of the invention under transforming conditions. Suitable transforming conditions are conventional and are described in, for example, Maniatis et al cited above, or “DNA Cloning” Vol. II, D. M. Glover ed., IRL Press Ltd, 1985.
The choice of transforming conditions is determined by the host cell. Thus, a bacterial host such as E. coli may be treated with a solution of CaCl2 (Cohen et al, Proc. Nat. Acad. Sci., 1973, 69, 2110) or with a solution comprising a mixture of RbCl, MnCl2, potassium acetate and glycerol, and then with 3-[N-morpholino]-propane-sulphonic acid, RbCl and glycerol. Mammalian cells in culture may be transformed by calcium co-precipitation of the vector DNA onto the cells, by lipofection, or by electroporation. The invention also extends to a host cell transformed with a replicable expression vector of the invention.
Culturing the transformed host cell under conditions permitting expression of the DNA polymer is carried out conventionally, as described in, for example, Maniatis et al and “DNA Cloning” cited above. Thus, preferably the cell is supplied with nutrient and cultured at a temperature below 45° C.
The product is recovered by conventional methods according to the host cell. Thus, where the host cell is bacterial, such as E. coli it may be lysed physically, chemically or enzymatically and the protein product isolated from the resulting lysate. Where the host cell is mammalian, the product may generally be isolated from the nutrient medium or from cell free extracts. Conventional protein isolation techniques include selective precipitation, absorption chromatography, and affinity chromatography including a monoclonal antibody affinity column.
Alternatively, the expression may be carried out either in insect cells using a suitable vector such as a baculovirus, in transformed drosophila cells, or mammalian CHO cells. The novel protein of the invention may also be expressed in yeast cells as described for the CS protein in EP-A-0 278 941.
The vaccine of the invention comprises an immunoprotective amount of the mutated version of the Der p1 (or other) allergenic protein. The term “immunoprotective” refers to the amount necessary to elicit an immune response against a subsequent challenge such that allergic disease is averted or mitigated. In the vaccine of the invention, an aqueous solution of the protein can be used directly. Alternatively, the protein, with or without prior lyophilization, can be mixed, adsorbed, or covalently linked with any of the various known adjuvants. Preferably, the adjuvant may be a preferential inducer of Th1-type immune responses.
An immune response is generated to an antigen through the interaction of the antigen with the cells of the immune system. The resultant immune response may be broadly distinguished into two extreme categories, being a humoral or cell mediated immune responses (traditionally characterised by antibody and cellular effector mechanisms of protection respectively). These categories of response have been termed Th1-type responses (cell-mediated response), and Th2-type immune responses (humoral response). In mice Th1-type responses are characterised by the generation of antibodies of the IgG2a subtype, whilst in the human these correspond to IgG1 type antibodies. Th2-type immune responses are characterised by the generation of a broad range of immunoglobulin isotypes including in mice IgE, IgG1, IgA, and IgM.
It can be considered that the driving force behind the development of these two types of immune responses are cytokines, a number of identified protein messengers which serve to help the cells of the immune system and steer the eventual immune response to either a Th1 or Th2 response. Thus Th1-type cytokines induce a cell mediated immune response to the given antigen, whilst Th2-type cytokines induce a humoral immune response to the antigen.
It is important to remember that the distinction of Th1 and Th2-type immune responses is not absolute. In reality an individual will support an immune response which is describe as being predominantly Th1 or predominantly Th2. However, it is often convenient to consider the families of cytokines in terms of that described in murine CD4 T cell clones by Mosmann and Coffman (Mosmann, T. R. and Coffman, R. L. (1989) TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annual Review of Immunology, 7, p 145-173). Traditionally, Th1-type responses are associated with cell mediated effector mechanisms such as cytotoxic lymphocytes (CTL) and can be characterised by the production of the INF-γ and IL-2 cytokines by T-lymphocytes. Other cytokines often directly associated with the induction of Th1-type immune responses are not produced by T-cells, such as IL-12. In contrast, Th2-type responses are associated, with humoral mechanisms and the secretion of IL-4, IL-s, IL-6, IL-10 and tumour necrosis factor-β (TNF-β).
It is known that certain vaccine adjuvants are particularly suited to the stimulation of either Th1 or Th2-type cytokine responses. This weighting of cytokine production translates into the generation of either a predominantly Th1-type ot Th2-type immune responses. Traditionally the best indicators of the Th1:Th2 balance of the immune response after a vaccination or infection includes direct measurement of the production of Th1 or Th2 cytokines by T lymphocytes in vitro after restimulation with antigen, and measurement of the IgG1:IgG2a ratio of antigen specific antibody responses.
Thus, a Th1-type adjuvant is one which stimulates isolated T-cell populations to produce high levels of Th1-type cytokines when re-stimulated with antigen in vitro, and induces antigen specific immunoglobulin responses associated with Th1-type mechanisms (IgG2a in mice, IgG1 in the human).
Adjuvants include, but are not limited to, aluminium hydroxide, muramyl dipeptide and saponins such as Quil A, 3D-MPL (3-O-deacylated monophosphoryl lipid A), or TDM. As a further exemplary alternative, the protein can be encapsulated within microparticles such as liposomes. Particularly preferred adjuvants which preferentially stimulate Th1-type immune responses are combinations of 3D-MPL and QS21 (EP 0 671 948 B1), oil in water emulsions comprising 3D-MPL and QS21 (WO 95/17210), 3D-MPL formulated with other carriers (EP 0 689 454 B1), or QS21 formulated in cholesterol containing liposomes (WO 96/33739), or immunostimulatory oligonucleotides (WO 96/02555). In yet another exemplary alternative, the protein can be conjugated to a carrier protein which is capable of providing T-cell help to the generation of the anti-allergen immune response, such as tetanus toxoid. Use of Quil A is disclosed by Dalsgaard et al., Acta Vet Scand, 18:349 (1977).



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 illustrates the expression of a 43 kDa protein corresponding to mature Der p1 in fusion with the prepeptide MF-alpha of Pichia pastoris (construct pNIV4811) in yeast cells. The culture supernatants from various Pichia pastoris clones incubated in the absence or presence of methanol (methanol induction for 1 to 5 days indicated on the x axis) have been analyzed by SDS-PAGE and immunoblot analysis using an anti-Der p1 peptide (117-133) polyclonal antibody.
FIG. 2 illustrates expression of mature Der p1 (30 kDa) in fusion with the prepeptide of Pichia pastoris MF-alpha (construct pNIV4817) in yeast cells. The culture supernatants from Pichia pastoris cells incubated in the absence (J0) or presence of methanol for 1 day (J1) have been concentrated 50 times and, then, analyzed by SDS-PAGE and immunoblot analysis using an anti-Der p1 peptide (117-133) polyclonal antibody. Arrows indicate the mature Der p1 doublet at about 30 kDa.
FIG. 3 illustrates expression of Der p1 in fusion with its propeptide (construct pNIV4812) in CHO-K1 cells. The cell extracts from different clones of CHO-K1 cells transfected with pNIV4812 (lanes 3-8) or transfected with the vector pEE14 alone as negative controls (lane 1 & 2) have been analyzed by SDS-PAGE and immunoblot analysis using an anti-Der p1 peptide (117-133) polyclonal antibody. The arrow indicates the mature Der p1 protein.
FIG. 4 illustrates expression of Der p1 in fusion with its propeptide (construct pNIV4840) in drosophila cells S2 (Invitrogen). The cell extracts of different clones of CHO-K1 cells transfected with pNIV4840 (lanes 1 & 4) or transfected with the inducible vector pMT/V5-His alone as negative controls (lanes 2, 3, 5, & 6) have been analyzed by SDS-PAGE and immunoblot analysis using an anti-Der p1 peptide (117-133) polyclonal antibody. The induction has been carried out for 22 hours (1-3) and 28 hours (4-6).
FIG. 5 illustrates expression of non-cleavable, non-activable Der p1 mutant in fusion with its pro-peptide (construct pNIV4842) in drosophila cells S2 (Invitrogen). The cell supernatants from transiently transfected S2 cells with pNIV4842 (lanes 1-4) or transfected with the inducible vector pMT/V5-His alone as negative control (lanes 5) have been analyzed by SDS-PAGE and immunoblot analysis using an anti-Der p1 peptide (117-133) polyclonal antibody. Lanes 1 to 4 correspond to 1, 4, 5, and 6 days of induction, respectively. Arrows indicate the pro Der p1 doublet at about 36 kDa.
FIG. 6 illustrates expression of non-active Der p1 mutant in fusion with its propeptide (construct pNIV4843) in drosophila cells S2. The cell supernatants from transiently transfected S2 cells with pNIV4843 (lanes 6-9) or transfected with the inducible vector pMT/V5-His alone as negative control (lanes 5) have been analyzed by SDS-PAGE and immunoblot analysis using an anti-Der p1 peptide (117-133) polyclonal antibody. Lanes 6 to 9 correspond to 1, 4, 5, and 6 days of induction, respectively. Arrows indicate the mature Der p1 doublet at about 36 kDa.
FIG. 7 illustrates DerP1 restriction map of SEQ ID NO. 6.
FIG. 8 illustrates sequence of full mutant DerP1 including pre-protein. Active site mutation Cys 132→Ala 132, corresponding to Cys34→eAla34 of the mature protein). Sequence includes coding (listed as SEQ ID NO. 6) and complementary DNA, and amino acid sequences (listed as SEQ ID NO. 1).
FIG. 9 illustrates sequence of full mutant DerP1 including pre-protein containing a deletion at the propeptide cleavage site (NAET). Sequence includes coding (listed as SEQ ID NO. 7) and complementary DNA, and amino acid sequences (listed as SEQ ID NO. 2).
FIG. 10 illustrates sequence of full mutant DerP1 including pre-protein. Active site mutation His 268→Ala 268, corresponding to His170→Ala170 of the mature protein). Sequence includes coding (listed as SEQ ID NO. 8) and complementary DNA, and amino acid sequences (listed as SEQ ID NO. 3).
FIG. 11 illustrates amino acid sequence (SEQ ID NO: 4) for the mutant DerP1 as encoded by pNIV4842, and shown in FIG. 5.
FIG. 12 illustrates amino acid sequence (SEQ ID NO: 5) for the mutant DerP1 as encoded by pNIV4843, and shown in FIG. 6.



Vaccine preparation is generally described in New Trends and Developments in Vaccines, Voller et al. (eds.), University Park Press, Baltimore, Md., 1978. Encapsulation within liposomes is described by Fullerton, U.S. Pat. No. 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, U.S. Pat. No. 4,372,945 and Armor et al., U.S. Pat. No. 4,474,757.
The amount of the protein of the present invention present in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and whether or not the vaccine is adjuvanted. Generally, it is expected that each dose will comprise 1-1000 μg of protein, preferably 1-200 μg. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Following an initial vaccination, subjects will preferably receive a boost in about 4 weeks, followed by repeated boosts every six months for as long as a risk of allergic responses exists.
The vaccines of the present invention may be administered to adults or infants, however, it is preferable to vaccinate individuals soon after birth before the establishment of substantial Th2-type memory responses.
A further aspect of the invention provides a method of preventing or mitigating an allergic disease in man, which method comprises administering to a subject in need thereof an immunogenically effective amount of a mutated allergen of the invention, or of a vaccine in accordance with the invention.
The examples which follow are illustrative but not limiting of the invention. Restriction enzymes and other reagents were used substantially in accordance with the vendors' instructions.
EXAMPLE 1
Expression in Pichia pastoris 
Construction of pNIV4811
pNIV4811 is designed to promote the expression of mature Der p1 in fusion with the prepropeptide of Pichia pastoris MFα. Plasmid ATCC87307 contains the sequence for mature DerP1. The full Derpl restriction map is given in FIG. 7.
Ligate with T4 DNA Ligase:

    
    
        SphI-XhoI from pPIC9k (INVITROGEN V175-20)
        XhoI-PstI oligonucleotides whose sequences follow (no 97038 and no 97039)
        PstI-XbaI from pNIV4810 (plasmid ATCC87307)
        AvrII-SphI from pPIC9k
    
    


Sequences of the Oligonucleotides:
no. 97038 (SEQ ID NO. 9)
5′TCGAGAAAAGAGAGGCTGAAGCTACTAACGCCTGCA3′
no. 97039 (SEQ ID NO. 10)
5′GGCGTTAGTAGCTTCAGCCTCTCTTTTC3′
Results
Pichia Pastoris transfected with pNIV4811 leads to the expression of a protein of 43 kD, comprising uncleaved proMFα-mature Der p1 fusion protein, has been detected in several clones (FIG. 1).
Construction of pNIV4817
pNIV4817 is derived from pNIV4811. It is designed to promote the expression of the mature Der p1 in fusion with the prepeptide of Pichia pastoris MFα.
Ligate: BstEII-BamHI from pNIV4811

    
    
        BamHI-PstI oligonucleotides no. 97262 and no. 97263 whose sequence follows
        PstI-BstEII from pNIV4811

Sequences of the Oligonucleotides

    
    


no. 97262 (SEQ ID NO. 11)
5′GATCCAAACGATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGC AGC ATCCTCCGCATTAGCTGCTCCAACTAACGCCTGCA3′
no. 97263 (SEQ ID NO. 12)
5′GGCGTTAGTTGGAGCAGCTAATGCGGAGGATGCTGCGAATAAAACTGC AGTAAAAATTGAAGGAAATCTCATCGTTTG3′
Results
Several clones expressed the mature form of Der p1 protein with an apparent molecular weight of 30 kDa, which was secreted into the supernatant (FIG. 2).
Construction of pNIV4815
Starting from pNIV4811, the following construction is designed to delete four residues [N-A-E-T (T is the first residue of the mature protein)] at the junction between the propeptide and the mature enzyme.

    
    
        Ligate: BinI-BamHI fragment from pPIC9k (the vector used for expression in P. pastoris)
        
            BamHI-EaeI fragment from pNIV4811
            EaeI-EcoRI fragment generated by RT-PCR with primers No 97142 and 97143. Residues: A6 to E74.
            EcoRI-PstI oligonucleotides whose sequence follows (No 97140 and 97141). Residues: F75 to C102 except N96AET99
            PstI—XbaI fragment from pNIV4810.
        
        
    
    










Sequence of the oligonucleotides



allowing the NAET deletion:


No 97140 (SEQ ID NO. 13)
75 bases







5′ AATTCAAAAACCGATTTTTGATGAGTGCAGAAGCTTTTGAACACCTAAA


ACTCAATTCGATTTGAACGCCTGCA 3′


 








No 97141 (SEQ ID NO. 14)
67 bases







5′ GGCGTTCAAATCGAATTGAGTTTTGAGGTGTTCAAAAGCTTCTGCATCA


TCAAAAATCGGTTTTTG 3′


 


RT-PCR Primers








No 97142 (SEQ ID NO. 15)
25 bases







5′ CATGAAAATTGTTTTGGCCATCGCC 3′


                    EaeI


 








No 97143 (SEQ ID NO. 16)
24 bases


5′ CGGTTTTTGAATTCATCCAACGAC 3′


          EcoRI






Construction of pNIV4819

Starting from pNIV4817, an expression plasmid designed to produce the mature form of Der p1 in Pichia pastoris, the following construction is made to replace the cysteine residue from the active by an alanine residue (corresponding to the Cys 34 mutation in the mature protein).
Ligate: Bpu11021-Asel fragment from pNIV4817

    
    
        AseI-TfiI synthetic fragment resulting from hybridization of oligonucleotides no. 97121 and no. 97122 whose sequence follows: corresponding to residues I104 to E142 of the proDerP1 (I6− of mature DerP1 protein)
        TfiI-BstEII fragment from pNIV4810 (ATCC 87307)
        BstEII-Bpu 11021 fragment from pNIV4817
    
    









Sequences of the oligonucleotides








no 97121 (SEQ ID NO. 17)
Ala 113 bases







5′ TAATGGAAATGCTCCAGCTGAAATCGATTTGCGACAAATGCCACTCCCA


TTCGTATGCAAGGAGGCTGTGGTTCAGCTTGGTGTTGCCGCAACTG 3′


 








no 97122 (SEQ ID NO. 18)
114 bases







5′ ATTCAGTTGCGGCAACACCAGAGAAAGCCCA AGCTGAACCACAGCCT


CCTTGCATACGAATGGGAGTGACAGTTCGCATTTGTCGCAAATCGATTTC


AGCTGGAGCATTTCCAT 3′






Construction of pNIV4815

Starting from pNIV4811, the following construction is made to delete four residues [N-A-E-T (T is the first residue of the mature protein)] at the junction between the propeptide and the mature enzyme.

    
    
        Ligate: BlnI-BamHI fragment from pPIC9k (the vector used for expression in PPIChia pastoris)
        
            BamHI—Eael fragment from pIV4811
            EaeI—EcoRI fragment generated by RT-PCR with primers
            No. 97142 and 97143. Residues: A6 to E74.
            EcoRI—PstI oligonucleotides whose sequence follows (No. 97140 and 97141). Residues: F75 to C102 except N96AET99
            PstI—XbaI fragment from pNIV4810.
        
        
    
    









Sequence of the oligonucleotides:


allowing the NAET deletion.








No 97140 (SEQ ID NO. 19)
75 bases







5′ AATTCAAAAACCGATTTTTGATGAGTGCAGAAGCTTTTGAACACCTCAA


AACTCAATTCGATTTGAACGCCTGCA 3′


 








No 97141 (SEQ ID NO. 20)
67 bases







5′ GGCGTTCAAATCGAATTGAGTTTTGAGGTGTTCAAAAGCTTCTGCACTC


ATCAAAAATCGGTTTTTG 3′


 


RT-PCR Primers








No 97142 (SEQ ID NO. 21)
25 bases







5′ CATGAAAATTGTTTTGGCCATCGCC 3′


                  EaeI


 








No 97143 (SEQ ID NO. 22)
24 bases







5′ CGGTTTTTGAATTCATCCAACGAC 3′


          EcoRI






EXAMPLE 2
Expression in Mammalian Cells
pNIV4812, an expression plasmid based on pEE14 (CellTech, Cockett et al., 1990 Biotechnology, vol 8, 662-667) designed to produce the mature form of Der p1 in CHO-K1, codes for a pre-Der p1 followed by the mature Der p1 sequence (no pro-protein).
Ligate: HindIII-XbaI from pEE14

    
    
        HindIII-PstI oligonucleotides no. 97040 and 97041 whose sequence follows
        PstI-XbaI from pNIV4810 (plasmid ATCC 87307)

Sequence of the Oligonucleotides

    
    


no 97040 (SEQ ID NO. 23)
5′AGCTTACCATGAAAATTGTTTTGGCCATCGCCTCATTGTTGGCATTGAG CGCTGTTTATGCTCGTACTAACGCCTGCA3′
no 97041 (SEQ ID NO. 24)
5′GGCGTTAGTACGAGCATAAACAGCGCTCAATGCCAACAATGAGGCGAT GGCCAAAACAATTTTCATGGTA3′
Results
The expression of a protein of an apparent molecular weight of 30 kDa has been detected in several extracts (FIG. 3). No protein has been detected in the culture supernatants (data not shown), which suggests that the protein was not secreted from CHO-K1 cells.
Construction of pNIV4814
Starting from pNIV4812, the following construction is made to replace the cysteine residue from the active site by an alanine residue.

    
    
        Ligate: AflIII-AseI fragment from pNIV4812.
        
            AseI-TfiI oligonucleotides as in pNIV4819 construction (No. 97121 and 97122)
            TfiI—BstEII fragment from pNIV4810 (ATCC 87307)
            BstEII—AflI fragment from pNIV4812.
        
        
    
    


Construction of pNIV4819 and pNIV4814 was made possible, thanks to the discovery that in pNIV4810 the codon encoding isoleucine 6 of the mature protein was ATT instead of ATC as published. This sequence is responsible for the presence of the AseI restriction site.
Construction of pNIV4816
Starting from pNIV4812, designed to expressed in CHO-K1, pNIV4816 has the same deletion as for pNIV4815. This construct results in the production of recombinant properP1 with the deletion of the NAET residues from the junction between the pro and mature protein.

    
    
        Ligate: XbaI—AflII fragment from pEE14
        
            AflII—EaeI fragment from pNIV4812
            EaeI—EcoRI fragment generated by RT-PCR using primers No 97142 and 97143
            EcoRI—PstI oligonucleotides No 97140 and 97141 (same oligonucleotides as used in pNIV4815)
            PstI—XbaI fragment from pNIV4810.
        
        
    
    


EXAMPLE 3
Expression in Drosophila Cells
Construction of pNIV4827
pNIV4827 has been designed to promote the expression and secretion of mature Der p1 from baculovirus infected insect cells.
Ligate: pAcGP67A vector linearized with PstlI

    
    
        PstI fragment from pNIV4810 (ATCC 87307)
    
    


The expression of Der p1 from pNIV4827 has been demonstrated by western blot.
Construction of pNIV4828
pNIV4828 has been designed to promote the expression and secretion of Proper p1 from baculovirus infected insect cells.
Ligate: SapI-BamHI from pAcGP67A (Pharmingen ref. 21220P)

    
    
        BamH1-EcoRI 172 bp synthetic fragment
        EcoRI-BssSI from pNIV4820
        BssSI-SapI from pNIV4827
    
    


Sequence of the synthetic fragment:
a) coding oligonucleotide No. 97520 (SEQ ID NO. 25)

    
    
        5′GAT CCC CGG CCG TCA TCG ATC AAA ACT TTT GAA GAA TAC AAA AAA GCC TTC AAC AAA AGT TAT GCT ACC TTC GAA GAT GAA GAA GCT GCC CGT AAA AAC TTT TTG GAA TCA GTA AAA TAT GTT CAA TCA AAT GGA GGT GCC ATC AAC CAT TTG TCC GAT TTG TCG TTG GAT G3′
        172 mer
    
    


b) complementary sequence No. 97521 (SEQ ID NO. 26)

    
    
        5 AAT TCA TCC AAC GAC AAA TCG GAC AAA TGG TTG ATG GCA CCT CCA TTT GAT TGA ACA TAT TTT ACT GAT TCC AAA AAG TTT TTA CGG GCA GCT TCT TCA TCT TCG AAG GTA GCA TAA CTT TTG TTG AAG GCT TTT TTG TAT TCT TCA AAA GTT TTG ATC GAT GAC GGC CGG G3 
        172 mer
    
    


The expression of Proper p1 from pNIV4828 has been demonstrated by western blot.
Construction of pNIV4832
This plasmid codes for a Der p1 propeptide followed by the mature Der p1 (Proper p1) sequence and is designed to be expressed in drosophila cells.
Ligate: Asp718-BamHI fragment from expression vector pDS47/V5-His (INVITROGEN V4115-20)

    
    
        Asp718-SpeI synthetic fragment resulting from hybridization of 98023 and 98024 oligonucleotides
        SpeI-BgIII fragment from pNIV4828

Sequences of the Oligonucleotides

    
    


no. 98023 (SEQ ID NO. 27)
5′GTA CCC TTA AGA TGC TA3′
no. 98024 (SEQ ID NO. 28)
5′CTA GTA GCA TCT TAA GG3′
NB: pNIV4828 is a plasmid designed for the isolation of recombinant baculoviruses expressing the pro-Derp 1 fused to gp67 signal peptide.
Results
Transitory expression of pro-DerP1 in drosophila cells has been detected (data not shown).
Construction of pNIV4840
pNIV4840 differs from pNIV4832 in that the expression vector used is stable and inducible (pMTNV5-His)
Ligate: Asp718-NotI fragment from pNIV4832

    
    
        NotI—Asp718 from pMTNV5-His (INVITROGEN V4120-20)

Expression of proDerp 1 in drosophila cells has been shown (FIG. 4)

    
    


Construction of pNIV4842
pNIV4842 was designed to promote the expression and secretion of Proper p1 from recombinant drosophila cells. Proper p1 coding sequence was engineered to impair the cleavage of the propeptide. To achieve this goal, four nucleotide triplets coding for NAET including the cleavage site were deleted.
Ligate: NotI-EcoRI from pNIV4840

    
    
        EcoRI-PstI synthetic fragment resulting from hybridization of oligonucleotides no. 98136 and no. 98137
        PstI-BstEII from pNIV4840
        BstEII-NotI from pNIV4840
    
    









Sequence of the synthetic oligonucleotides


a) Coding sequence








No 98136 (SEQ ID NO. 29)
75 mer







5′ AAT TCA AAA ACC GAT TTT TGA TGA GTG CAG AAG CTT


TTG AAC ACC TCA AAA CTC AAT TCG ATT TGA ACG CCT


GCA 3′


 


Complementary sequence








No 98137 (SEQ ID NO. 30)
67 mer


5′ GGC GTT CAA ATC GAA TTG AGT TTT GAG GTG TTC AAA


AGC TTC TGC ACT CAT CAA AAA TCG GTT TTT G 3′






Results

Detection of Der p1 in fusion with its propeptide has been detected in the supernatants after induction (FIG. 5). The sequence of this recombinant mutant DerP1 is given in SEQ ID NO. 4.
Construction of pNIV4843
pNIV4843 has been designed to promote the expression and secretion from recombinant drosophila cells of a Proper p1 form in which the cysteine residue of the active site has been mutated to an alanine.
Ligate: NotI-Asp718 from pMT/V5-His

    
    
        Asp718-PstI from pNIV4832
        PstI-TfiI from pNIV4819
        TfiI-NotI from pNIV4832

Results

    
    


Detection of Der p1 in fusion with its propeptide has been detected in the supernatants after induction (FIG. 6). The sequence of this recombinant mutant DerP1 is given in SEQ ID NO. 5.
EXAMPLE 3
Purification Procedure of Recombinant ProDer p1 Secreted from Recombinant drosophila Cells
Proteins from the spent culture medium (1 liter) were concentrated at 4° C. by overnight ammonium sulfate precipitation to 60% saturation. After centrifugation at 17000 g during 30 min., the precipitate was resuspended in 20 ml of 20 mM Tris-HCl pH8.0 and dialyzed against 5 liters of the same buffer. Insoluble proteins were discarded by centrifugation at 20000 g during 30 min. The dialysate was loaded onto a Q sepharose XL column (3×1.6 cm, Pharmacia) equilibrated in 20 mM Tris-HCL pH8.0. After washing the column with the same buffer, bound proteins were eluted by steps of 100 mM increases of NaCl concentration. Proper p1 mainly eluted at 200 mM NaCl. Enriched Proper p1 fractions were pooled and loaded onto an hydroxyapatite type 1 column (1×1.6 cm, Biorad) conditioned in 5 mM potassium phosphate buffer pH 7.0. Unbound material containing Proper p1 was concentrated by ultrafiltration using Omega membrane (cut-off: 10 kD, Filtron). The concentrate was loaded onto a superdex 75 FPLC column (30×1 cm, Pharmacia) in PBS pH 7.3. Eluted Proper p1 from the gel filtration column was more than 80% pure.
EXAMPLE 4
Vaccine Formulation
Vaccines comprising the mutant DerP1 or allergens may be formulated with many common adjuvants. One preferred adjuvant system is an oil in water emulsion described below:
The oil in water emulsion adjuvant formulations used in the present invention are made comprising following oil in water emulsion component: 5% Squalene, 5% α-tocopherol, 2.0% polyoxyethylene sorbitan monooleate (TWEEN 80). The emulsions are prepared as a 2 fold concentrate. All examples used in the immunological experiments are diluted with the addition of extra components and diluents to give either a 1× concentration (equating to a squalene:QS21 ratio (w/w) of 240:1) or further dilutions thereof.
Briefly, TWEEN 80 is dissolved in phosphate buffered saline (PBS) to give a 2% solution in the PBS. To provide 100 ml of a two fold concentrate emulsion, 5 ml of DL alpha tocopherol and 5 ml of squalene are vortexed to mix thoroughly. 95 ml of PBS/TWEEN solution is added to the oil and mixed thoroughly. The resulting emulsion is then passed through a syringe needle and finally microfluidised by using an M110S Microfluidics machine. The resulting oil droplets have a size of approximately 145-180 nm (expressed as z av. measured by PCS). The other adjuvant/vaccine components (QS21, 3D-MPL and antigen) are added to the emulsion in simple admixture.
The antigen containing vaccines used herein are formulated either with full dose SB62 adjuvant to give a high squalene:QS21 ratio (240:1) or with a lower amount of SB62 to give a low ratio formulation (48:1). Other vaccines may optionally be formulated with the addition of cholesterol to the oil phase of the emulsion.
These vaccines are assayed in groups of Balb/c mice. Briefly, groups of 10 mice are immunised intramuscularly 2 times at 3 weeks interval with 2 μg mutant allergen combined with oil in water emulsion adjuvant. 14 days following the second immunisation the production of cytokines (IL-4, IL5 and IFN-γ) are analysed after in vitro restimulation of spleen and lymph nodes cells with allergen. Antibody response to wild-type allergen and the isotypic profile induced are monitored by ELISA at 21 days post II and 14 days post IV.

## Claims

1. A recombinant mutant pro-DerP1 allergen of the DerP1 pre-pro-protein of SEQ ID NO: 1 wherein SEQ ID NO: 1 comprises a cysteine to alanine substitution at amino acid position 132 and wherein said pro-DerP1 allergen is from Dermatophagoides pteronyssinus.

2. A therapeutic formulation comprising the recombinant mutant pro-DerP1 allergen as claimed in claim 1 and an adjuvant.

3. The therapeutic formulation as claimed in claim 2, wherein the adjuvant is a preferential stimulator of the Th1-type immune response.

4. The therapeutic formulation as claimed in claim 2, wherein the adjuvant comprises at least one selected from the group consisting of QS21 and 3-O-deacylated monophosphoryl lipid A.