Patent 6,830,891

Methods for the diagnosis of ABPA in a human individual comprise determining if the individual carries antibodies reactive with one or more ABPA-related recombinant allergens, which one or more ABPA-related recombinant allergens discriminate between ABPA and allergic sensitization to A. fumigatus. Suitable allergens include rAsp F4, rAsp F6, rAsp F8, and ABPA-related fragments thereof which bind with IgE or IgG antibody.

Description of the Invention

TECHNICAL FIELD

The present invention relates to methods for the diagnosis of allergic bronchopulmonary aspergillosis (ABPA) and recombinant allergens to be used in the methods. During the priority year the recombinant allergen that in the priority application was called rAsp f2 has officially been named rAsp f6. The official name is used in this text.

TECHNICAL BACKGROUND

Allergic bronchopulmonary aspergillosis (ABPA). Allergic bronchopulmonary aspergillosis is the most severe allergic complication caused by Aspergillus species, mainly A. fumigatus. ABPA is the result of hypersensitivity to Aspergillus-antigens mainly in patients suffering from long-standing atopic asthma (8-12) or cystic fibrosis (16-19). Although originally considered as a rare disease (13), ABPA is currently recognized with much greater frequency. ABPA with varied clinical presentations has been reported to occur in about 15% of the asthmatic patients sensitized to A. fumigatus (14,15), while in patients with cystic fibrosis the reported incidence varies from 10 to 35% (16,17). ABPA has been described as an immune disease that ranges from asthma to fatal destructive lung disease with defined clinical, serological, radiological and pathological features (8,18-22). Because of its severity ABPA should be ruled out in patients with chronic asthma or cystic fibrosis exhibiting immediate cutaneous reactivity to A. fumigatus (8). The diagnostic criteria for ABPA are asthma or cystic fibrosis, history of roentgenographic infiltrates (in most cases), immediate cutaneous reactivity to A. fumigatus extracts, elevated total serum IgE, precipitating antibodies to A. fumigatus, peripheral blood eosinophilia, elevated specific serum IgE and IgG to A. fumigatus as compared to sera from patients with asthma and cutaneous reactivity to Aspergillus, but without ABPA, and proximal (central) bronchiectasis with normal tapering of distal bronchi (23-25). In cases where all criteria are present, diagnosis is readily made (26). However, all of the eight criteria are rarely present at the same time even in classic ABPA-patients with central bronchiectasis. With exception of bronchiectasis and to some extent elevated specific serum IgE and IgG to A. fumigatus, none of the diagnostic criteria are specific for ABPA (26). Furthermore, pulmonary infiltrates and central bronchiectasis are commonly detected in patients suffering from cystic fibrosis also in the absence of sensitization to A. fumigatus, which makes a diagnosis of ABPA in patients with cystic fibrosis even more difficult (16). Therefore, serologic identification of ABPA has a greater diagnostic potential, but is, however, hampered by the lack of standardized, reliable fungal extracts (5,7,27-29).

Aspergillus fumigatus antigens. The major problem in the immunodiagnosis of diseases related to A. fumigatus stems from the antigenic complexity of the fungus. Antigen/allergen extracts of A. fumigatus contain hundreds of different proteins (6,30,31), of which a limited subset are able to let bind human serum IgE (6,32,33,35). The fungus has been reported to produce more than 40 IgE-binding components which generate complex IgE-binding patterns when extracts are examined by Western blot analysis using sera from allergic individuals (32,33). To make the picture even more complicated, serum IgE from different patients recognize highly variable patterns of fungal proteins (6,36). In the case of patients suffering from ABPA, depending on the stage of the disease, different allergenic "fingerprints" may be obtained with serum of the same patient taken at different times, even if fungal extract from the same batch is used (36,37).

It has been suggested to use purified native allergenic components instead of crude allergen extracts for diagnosing ABPA (79). Recombinant A. fumigatus allergens with connections to ABPA have been described earlier (71,83).

The inventors are named authors in a number of articles about recombinant allergens from A. fumigatus (cloning and expression: 39,43,49,51,52,82, and diagnostic use: 59,66,32,71,76,81).

Result of International-Type Search During the Priority Year.

The references 66, 79 and 84 have been categorised as being of particular relevance.

Banerjee et al., (84) describes antigens that cannot be intracellular. The described antigens are shown to react with sera of patients with ABPA, but there are no data suggesting that the antigens will not react with sera of A. fumigatus-sensitised patients not having ABPA.

Moser et al (66) and Little et al., (79) describe secreted proteins/antigens that do not allow for differential diagnosis of ABPA because they frequently reacts with sera of A. fumigatus-sensitised patients without ABPA and sera of ABPA patients.

THE OBJECTIVES OF THE INVENTION

The main objective of the invention is to provide improved methods for diagnosis of ABPA.

One subobjective is to provide in vitro diagnostic methods that have the sufficient specificity and sensitivity for diagnosis of ABPA.

A second subobjective is to provide well-defined allergen preparations that can be used for the diagnosis of ABPA both in vitro and in vivo, including immunoassay and skin reactivity measurement methods, respectively.

THE INVENTION

The first major aspect of the invention is a method for diagnosis of allergic bronchopulmonary aspergillosis (ABPA). This aspect is characterised in using as a reagent an ABPA-related recombinant allergen, i.e. a recombinant allergen carrying an epitope against which antibodies of various Ig classes/subclasses, such as of the IgE class or total IgG or IgG subclasses (IgG1, IgG2, IgG3 and IgG4) can be detected so that an ABPA condition in a patient can be differentiated from allergic sensitization to A. fumigatus, which is particularly useful in patients suffering from cystic fibrosis.

The concept of ABPA-related recombinant allergens includes any recombinant allergen, irrespective of origin, having the above-mentioned antibody binding feature permitting the differential diagnosis indicated. It encompasses in particular ABPA-related recombinant allergens derived from A. fumigatus and their ABPA-related fragments. For ABPA-related recombinant allergens cloned from A. fumigatus, the concept encompasses ABPA-related allergens and fragments derived from other sources, having one or more ABPA epitopes in common with an ABPA-related allergen from A. fumigatus. At the priority, date rAsp f4 and rAsp f6 and their fragments, as defined above, were considered to be the most useful ABPA-related allergens. Various derivatized forms retaining the ability to bind antibodies, as defined for ABPA-related recombinant allergens, are also included.

Various subaspects include in vitro and in vivo testing protocols as described below.

The second major aspect of the invention is novel ABPA-related recombinant allergens binding to human IgE present in ABPA patients and useful in the first aspect of the invention.

Various subaspects of this second major aspect of the invention are apparent from the below and encompasses derivatized forms including but not limited to underivatized, insolubilized and labelled ABPA-related allergens. Another aspect of the invention is the use of ABPA-related allergens for hyposensitization treatment as done for other allergens.

Cloning of Allergens from Aspergillus fumigatus.

The cloning strategy utilized phagemid pcomb3 (47) and the ability of the leucine zipper proteins Jun and Fos to associate with each other (74,48,74,75).

A modified gIII product, obtained by fusing the DNA encoding the jun leucine zipper flanked by cysteine residues, N-terminal to the viral coat protein was expressed from a LacZ promotor and secreted into the periplasmic space of E. coli by a pelB leader peptide, thereby being structurally incorporated into phage particles during infection with helper phage (49). Using a second LacZ promotor of the phagemid, the fos leucine zipper domain, flanked by cysteine residues, co-expressed as N-terminal fusion peptide to cDNA protein products of A. fumigatus, was secreted into the 4: periplasmic space of E. coli using the pelB leader peptide (50). Through Jun-Fos heterodimerization and disulfide bond formation, the gIII-Jun fusion protein incorporated into phage particles provides a covalent link to phage surface for random recombinant cDNA products with the Fos leucine zipper attached N-terminally (48,49). The phagemid pJuFo which contains the described elements (49,51,52), allows expression and display of cDNA libraries, in this case encoding shot-gun 19 cloned A. fumigatus peptides/proteins, on phage surface and application of the powerful screening technology based on biopanning procedures used for other filamentous phage systems (46,47). The key of success in cDNA cloning from libraries displayed on phage surface lies in the screening strategy used. The most important factor to be considered is that the ligand used to select phage should be tagged or immobilized in a way allowing the ligand to retain its native conformation (46). It must be taken into account that proteins, when directly immobilized to a solid phase by hydrophobic interaction, may lose biological activity due to alterations in the three-dimensional structure (54,55). In general the known or expected characteristics of the ligand will dictate the procedure used for ligand immobilization. For the isolation of allergens recognized by serum antibodies, the use of capture antibodies has proven to be very effective for different reasons. First, monoclonal antibodies raized against the immunoglobulin .epsilon. constant domains C.beta.2, C.beta.3 or C.beta.4 do not interfere with the antigen binding site of the antibody. Second, a surface coated with such anti-IgE antibodies will be able to immobilize selectively IgE antibodies from serum of allergic patients. Therefore, after washing away interacting and cross-reacting serum antibodies of other isotypes together with all other serum components, a specific surface able to adsorb only phage displaying IgE binding molecules will be obtained (51-53).

The application of pJuFo to display cDNA products and select phages from a library constructed using mRNA from A. fumigatus (39,51,52) yielded a wide variety of phage clones able to bind IgE antibodies from sera of patients sensitized to A. fumigatus (table 1).

Compared with screening of .lambda.-libraries immobilized on solid phase supports, the screening procedure for cDNA libraries displayed on the surface of filamentous phage has several advantages. Capturing serum IgE with an immobilized anti-IgE antibody generates a homogenous surface with immobilized IgE which does not become denatured (56,57) and therefore retain the full antigen binding capacity. The most important advantage results from the fact, that the phage library is kept in a liquid phase, where only phage with affinity to the ligand are retained on the solid phase after washing (47,53). Desorbed phage can be used to infect E. coli in order to amplify phage with affinity for the ligand. Therefore, successive rounds of phage growth and selection allow enrichment of phage displaying proteins with affinity for the ligand (table 3). After selection of candidate phage clones displaying proteins with IgE binding properties, phage particles produced from 10 ml culture can be precipitated (47) and samples of 1010-1013 phage particles of each candidate clone analysed by SDS-PAGE under reducing conditions, followed by transfer to nitrocellulose membranes (49,51). After blocking in order to saturate free binding sites on the nitro-cellulose sheet, membranes are incubated with patient serum diluted 1:10 as "first antibody" and mouse anti-human IgE mAb as second antibody can be used to visualize binding of IgE to the cDNA product originally present on the phage surface. Western blots can easily be developed using non-radioactive systems and horse-radish-peroxidase-conjugated goat-anti mouse Ig as detection system. The apparent molecular mass of the IgE-binding proteins enriched from an A. fumigatus cDNA library displayed on phage surface was in the range of 10 to more than 50 kDa. Nucleotide sequence determination (58) of some cDNA-inserts differing in size and restriction pattern revealed that they encode different proteins as deduced from the open reading frames.

Production of Recombinant Allergens in E. coli.

Illustrative examples of production methods will now be given for two ABPA-related recombinant allergens cloned from A. fumigatus, designated rAsp f4 and rAsp f6.

rAsp f6: DNA encompassing the coding sequence of rAsp f6 was cloned into an expression vector under the transcriptional control of the T7 promoter (78). The construct was designed in such a way that one methionine residue was added at N-terminal end of the allergen amino acid sequence, while at the C-terminus the eight-residue stretch -VEHHHHHH (SEQ ID NO:7) added, of which the six consecutive histidine residues serve as an affinity tag for metal-chelate affinity chromatography (61). After sequence confirmation, the construct was transferred to E. coli BL21[pT7POL23] (77), in which synthesis of the T7 RNA polymerase can be induced by raising the temperature of the growing culture to above 37^oC. To produce rAsp f66, 1 liter of LB medium containing an appropriate complement of antibiotics was inoculated with 1 ml of an overnight starter culture grown at 30^oC. After approximately 3 hrs of growth at 30^oC., at an OD₆₀₀ of 0.7, the temperature of the culture was shifted to 42^oC. in order to induce expression. After 4 hrs of of incubation at inducing temperature, cells were harvested by centrifugation and resuspended in 50 ml of ice-cold 20 mM Tris-HCl pH 8.0 containing 0.5 M NaCl and 5 mM imidazol (resuspension buffer). The cells were disrupted by sonication and insoluble debris removed by centrifugation. The supernatant, containing the overexpressed allergen, was passed through a 0.22 .mu.m filter to remove remaining particulate material and loaded onto an assembly of two serially connected 5 ml HiTrap Chelating columns (Pharmacia Biotech AB, Uppsala, Sweden) previously charged with Ni²⁺ and equilibrated with resuspension buffer. The column assembly was washed first with 50 ml of resuspension buffer, then with 50 ml of resuspension buffer supplemented with Imidazol to 60 mM. To elute rAsp f6, a 30 ml linear gradient of 60-500 mM imidazol in 20 mM Tris-HCl pH8.0/0.5 M NaCl was applied while 1 ml fractions were collected and analysed by SDS-PAGE. Fractions containing rAsp f6 were pooled and subjected to gel filtration through a Superdex 200 column (Pharmacia Biotech AB, Uppsala, Sweden) equilibrated and eluted with 0.15 M NaCl. Fractions containing rAsp f6 were pooled and concentrated using an Amicon cell fitted with a YM5 membrane. Final yield of purified rAsp f6 from one liter of bacterial culture was 23 mg.

rAsp f4: DNA encompassing the coding sequence of rAsp f4 was cloned into an expression vector under the transcriptional control of the T7 promoter (78). The construct was designed in such a way that the 11-residue stretch MRGSHHHHHHM-(SEQ ID no:8) was added to N-terminal end of the allergen amino acid sequence, of which the six consecutive histidine residues serve as an affinity tag for metal-chelate affinity chromatography (61). No amino acid addition was made at the C-terminal end of the protein. After sequence confirmation, construct was transferred to E. coli BL21[pT7POL23] (77), in which synthesis of the T7 RNA polymerase can be induced by raising the temperature of the growing culture to above 37^oC. To produce rAsp f4, 1 liter of LB medium containing an appropriate complement of antibiotics was inoculated with 1 ml of an overnight starter culture grown at 30^oC. After approximately 3 hrs of growth at 30^oC., at an OD₆₀₀ of 0.7, the temperature of the culture was shifted to 42^oC. in order to induce expression. After 4 hrs of of incubation at inducing temperature, cells were harvested by centrifugation and resuspended in 50 ml of ice-cold 20 mM Tris-HCl pH 8.0 containing 0.5 M NaCl. The cells were disrupted by sonication and insoluble material including rAsp f4 protein was collected by centrifugation. The insoluble material was washed twice by resuspension in 20 mM Tris-HCl pH 8.0 containing 2 M Urea, 0.5 M NaCl and 2% Triton X-100, followed by centrifugation. Partially purified rAsp f4-containing inclusion bodies were extracted for 45 minutes at room temperature in 70 ml of 20 mM Tris-HCl pH8.0 containing 6 M guanidinium hydrochloride, 0.5 M NaCl, 5 mM Imidazol and 1 mM 2-mercaptoethanol (extraction buffer). The extract was clarified by centrifugation and remaining particulate material removed by passage through a 0.22 .mu.m filter. The clarified extract, containing the overexpressed allergen, was loaded onto an assembly of two serially connected 5 ml HiTrap Chelating columns (Pharmacia Biotech AB, Uppsala, Sweden) previously charged with Ni²⁺ and equilibrated with extraction buffer lacking 2-mercaptoethanol. The column assembly was washed first with 50 ml of extraction buffer, then with 50 ml of 6 M urea in 20 mM Tris-HCl pH 8.0, 0.5 M NaCl, 20 mM Imidazol and 1 mM 2-mercaptoethanol (urea wash buffer). In order to renature the immobilized rAsp f4, a 960 ml linear gradient was applied, from urea wash buffer to 20 mM Tris-HCl pH8.0 containing 0.5 M NaCl, 20 mM Imidazol and 1 mM 2-mercaptoethanol (renaturation buffer). To elute rAsp f4, a 30 ml gradient of 20-1000 mM imidazol in renaturation buffer was applied while 1 ml fractions were collected and analysed by SDS-PAGE. Fractions containing rAsp f4 were pooled and subjected to gel filtration through a Superdex 75 column (Pharmacia Biotech AB, Uppsala, Sweden) equilibrated and eluted with 0.15 M NaCl. Fractions containing rAsp f4 were pooled and concentrated using an Amicon cell fitted with a YM10 membrane. Final yield of purified rAsp f4 from one liter of bacterial culture was 34 mg.

Production has also been carried out with the vector described by Hochli et al (60-63).

Analysis of cDNL inserts

Only inserts coding for peptides/proteins relevant for the diagnosis of ABPA will be discussed.

rAsp f6 (SEQ ID NO 1). A clone containing an insert of 751 base pairs with an open reading frame of 624 base pairs revealed a strong homology with nucleotide sequences encoding superoxide dismutases. The 3'-noncoding region had a polyadenylated tail of 24 base pairs. The deduced amino acid sequence of this cDNA clone (SEQ ID NO 1) was homologous to manganese SOD, showing the highest sequence identity of 48-52% to the human, fruit fly, gum tree, yeast, E. coli, and Mycobacterium leprae enzymes. Apparently the A. fumigatus MnSOD displays a similar high degree of sequence identiy to MnSODs from a wide variety of phylogenetically distant organisms (43). Multiple sequence alignment shows that the A. fumigatus MnSOD (rAsp f6) shares high homology with human MnSOD (51.8% identity, 67.2% homology). IgE raised against A. fumigatus MnSOD is detected predominantly in sera of patients suffering from ABPA. Therefore MnSOD could be a candidate for a serologic differential discrimination between ABPA and A. fumigatus allergy (see below). Notably, both recombinant A. fumigatus and human MnSOD induce proliferation in peripheral blood mononuclear cells of A. fumigatus allergic subjects with detectable levels of specific IgE to A. fumigatus MnSOD. Moreover, both the fungal and human recombinant MnSODs elicited Type I skin reactions in individuals sensitized to the fungal enzyme, providing evidence for auto-reactivity to human MnSOD in allergic individuals sensitized to the environmental A. fumigatus allergen (43).

rAsp f4 (SEQ ID NO 2). This was the second recombinant ABPA-related allergen discovered in our screening system. The clone contained an isert of 1103 base pairs with an open reading frame of 858 base pairs. Its deduced amino acid sequence does not share significant homology to any known protein. The gene product encoded by the used cDNA was only characterized by the function for which it was selected: IgE binding.

In Vivo Tests Utilizing Recombinant Allergens.

These are mainly illustrated by skin prick tests in which a small amount of a solution of an allergen is inserted into the dermis of an individual whereupon a wheal reaction occurs around the place for administration.

One protocol for skin prick tests of the invention implied that a recombinant allergen was dissolved in 0.9% physiological saline as a diluent at an end concentration of 100 .mu.g/ml. 20 .mu.l of these solutions were placed on the patient's forearms. Thereafter the skin was pricked with a sterile needle, which was entered into the epidermis at a degree angle and lifted up to elevate a small portion of the epidermis (38). The needle was discarded after the application of each solution to avoid contamination. The test sites were placed 3 to 4 cm apart to avoid false positive results.

For intradermal tests, an allergen solution (100 .mu.g/ml) were diluted at serial 10-fold dilutions and applied at concentrations starting from 10-4 .mu.g/ml to 10 .mu.g/ml. For testing the solutions (100 .mu.l) were injected on the patients' backs starting from the solution with lowest concentration resulting in a size of the wheal of half the size of the skin reaction induced by the histamine control. The test sites were placed 5 to 8 cm apart to avoid false-positive results. Histamine dihydrochloride was used as a positive control at concentrations of 0.1% in skin prick tests or 0.01% in intradermal tests, respectively. Physiological 0.9% salime was used as a negative control. The reactions were recorded after 15 minutes by measuring the maximal longitudal and transversal diameter of the wheal and evaluated as described (66).

The Use of Recombinant A. fumigatus Allergens for In Vitro Diagnostics.

The binding of recombinant A. fumigatus allergens to antibodies may be used in immunoassays for measuring allergen/antigen specific antibodies of various classes (IgA, IgG, IgD, IgE and IgM), including specific subclasses thereof, for instance in connection with diagnoses of allergy and ABPA. Among IgG subclasses may be mentioned IgG1, IgG2, IgG3 and IgG4. The methodology for the assays is the same as that used in the prior art for conventional antigens/allergens. Suitable immunoassay protocols thus contemplate formation of a ternary immune complex:

[allergen]-[anti-allergen antibody]-[anti-antibody]

where allergen and anti-antibody are added reagents and anti-allergen antibody derives from the sample to be assayed. The complex is formed in an insoluble or insolubilizable form. Insoluble forms are accomplished by having either the allergen or the anti-antibody bound to a solid phase before, after or during formation of the complex. Well known solid phases in the field are walls of tubes and wells, particulate and monolithic more or less porous materials used as adsorbents in chromatography and heterogeneous imunoassays etc. In order to measure the amount of complex, either the allergen or the anti-antibody is labelled with an analytically detectable group, with the provision that the reagent linked to a solid phase or causing post-insolubilization is not labelled. Well known detectable groups are enzymes (ELISA), fluorophors, chromophors, chemiluminescent groups, radioactive isotopes, metal atoms, biotin, haptens etc. In order to measure class/subclass specific antigen/allergen specific antibodies the anti-antibody has to be class/subclass specific. Normally this type of immunoassay is run with sequential incubation, i.e.

step 1: sample with allergen followed by

step 2: incubation of the complex formed in step 1, i.e. [allergen]-[anti-allergen antibody] with anti-antibody

or vice versa. In case the reagent used in step 1 is bound to a solid phase, separation and washing after each step should be carried out in order to remove unspecific interference.

For ABPA diagnosis, IgE and certain IgG subclasses are the most relevant Igs to measure. It is believed that the recombinant allergens to be used should be derived from A fumigatus proteins not being exposed on the cell surface or secreted. This may indicate that the most relevant A. fumigatus allergens relevant for ABPA may be cell-bound, for instance as intracellular peptides/proteins.

Relevant antibodies can be found in blood (including plasma and serum), saliva, cerebrospinal fluid (CSF), bronchioalveolar fluid, tear drops (lacrymal fluid) etc.

Claim 1 of 17 Claims

What is claimed is:

1. A method for the diagnosis of ABPA in a human individual, comprising determining if the individual carries antibodies reactive with one or more ABPA-related recombinant allergens, which one or more ABPA-related recombinant allergens discriminate between ABPA and allergic sensitization to A. fumigatus and wherein the one or more allergens are selected from the group consisting of rAsp f4 and rAsp f6, and ABPA-related fragments thereof which bind with IgE or IgG antibody.

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