WO1995019437A1 - Allergene a reactivite croisee - Google Patents

Allergene a reactivite croisee Download PDF

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Publication number
WO1995019437A1
WO1995019437A1 PCT/CA1995/000021 CA9500021W WO9519437A1 WO 1995019437 A1 WO1995019437 A1 WO 1995019437A1 CA 9500021 W CA9500021 W CA 9500021W WO 9519437 A1 WO9519437 A1 WO 9519437A1
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composition
protein
nucleic acid
allergen
pollen
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PCT/CA1995/000021
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English (en)
Inventor
Shyam S. Mohapatra
Alec H. Sehon
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University Of Manitoba
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Priority to AU14109/95A priority Critical patent/AU1410995A/en
Publication of WO1995019437A1 publication Critical patent/WO1995019437A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is related to the field of molecular genetics and is particularly concerned with the cloning of a cross-reactive allergen.
  • Pollen allergens are multideterminant proteins or glycoproteins capable of eliciting IgE-mediated allergic diseases such as hayfever and asthma in approximately 17% of the population who are genetically predisposed to develop allergies (ref. 1 - Throughout this specification, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the disclosure. The disclosure of these references are hereby incorporated by reference into the present disclosure) . In contrast to some other allergens (e.g., cat or house dust mite allergens) , the global distribution of pollens of a large variety of monocotyledonous (grasses) and dicotyledonous plants
  • hayfever consists primarily in symptomatic relief.
  • Sufferers take drugs, such as anti-histamines and steroids, which do not suppress the formation of IgE antibodies and often have harmful side effects.
  • Attempts to downregulate the IgE immune responses of allergic subjects by the "time-honoured" immunotherapy consist of a series of injections of increasing amounts of the allergenic extracts of the appropriate pollen or pollen-mixtures over prolonged periods lasting usually 3 to 5 years.
  • pollen extracts used therapeutically are crude mixtures of a multiplicity of chemical constituents, some of these components bearing no relation whatsoever to the few allergenic components which are actually responsible for a given patient's hypersensitivity. Because some of the proteins present in these extracts may not be allergens, standardization of allergenic extracts based on total protein content is an unreliable guide for determining the potency of an extract. Moreover, large (up to lOOx) variations in allergen content occur in the preparations used for immunotherapy because of the different methods used for (i) pollen collection and storage, which lead to variations in raw materials from lot to lot and from year to year, and (ii) the extraction procedures.
  • rDNA recombinant DNA
  • allergenic constituents Upon initial exposure to allergenic constituents, they are internalized by antigen-presenting cells (APC) , which include nonantigen-specific phagocytic cells or specific B cells, and are "processed" by these cells. The net effect of this processing is the breakdown of the antigens into peptidic determinants which, in turn, are re-expressed in association with class I or class II molecules of the major histocompatibility complex (MHC) on the surface of the APC.
  • APC antigen-presenting cells
  • MHC major histocompatibility complex
  • the binary peptide-MHC complexes interact with the corresponding specific receptors (TCR) of, respectively, (i) Th cells or (ii) CTLs or Ts cells, and the resulting triads determine the up- or down-regulation of the appropriate B cells (ref. 13) .
  • TCR specific receptors
  • the Th cell subpopulation may be further subdivided into three subsets, i.e., ThO, Thl and Th2 cells (ref. 14).
  • ThO Th2 cells
  • Th2 cells have been shown to produce IL-4, IL-5 and IL-6, and IL-4 has been shown to activate B cells leading to the production of IgE antibodies.
  • Thl cells produce IFN ⁇ , which blocks the production of IL-4.
  • the mechanism(s) involved in the suppression of antibody responses by Ts cells is still not fully understood. It has been suggested that the suppression of antibody production is due to inactivation of specific Th cells as a result of non-professional APC, i.e. a T-cell (ref. 15) .
  • the primary reaction of the IgE antibodies secreted from the Be cells is their binding to specific IgE receptors on the surface of mast cells, basophils and eosinophils.
  • the cell-fixed IgE antibodies react with and are crosslinked by the allergenic molecules. This process leads to the release from these cells of chemical mediators of anaphylaxis. In turn, these mediators act rapidly on the smooth muscles of different target organs, resulting in the inflammatory manifestations characteristic of immediate type hypersensitivity.
  • the development of therapeutic strategies (ref.
  • IgE antibodies 16 that may influence the formation of IgE antibodies requires detailed knowledge of (i) the structures of individual allergens and, in particular, of their B cell epitopes (which are recognized by IgE/IgG antibodies) , (ii) structures recognized by MHC molecule (i.e., la epitope) , and (iii) the structures recognized by T cell receptors of Th or Ts cells, known as T cell epitopes. Therefore, determination of primary sequences of individual pollen allergens of a complex repertoire of allergens of a given pollen by immunochemical and physiochemical methods is of central importance, and has been a major objective in allergy research for a long time. However, using these classical methods, the progress in isolation and characterization of allergens has been slow.
  • the epitopes of allergenic proteins are either continuous or discontinuous.
  • continuous antigenic epitopes can be localized to segments composed of amino acid residues in a linear sequence, whereas discontinuous
  • epitopes comprise residues which appear adjacent to one another on the protein surface, but are widely separated in their primary sequence. The latter epitopes depend on the native conformation of the protein.
  • Conventional methods for identification of B cell epitopes consist of probing a polyspecific antiserum or a set of monoclonal antibodies produced against the intact antigen with cleavage fragments of antigen or synthetic peptides, which may yield information on continuous epitopes (refs. 26 to 27) .
  • Atassi and his associates synthesized ten overlapping pentadecapeptides which represented the entire Amb a III molecule (ref. 28) .
  • These peptides served to localize four antigenic sites in Amb a III that were recognized by IgG antibodies in human, rabbit and murine antisera. It is noted that the same regions were also recognized by human IgE antibodies (ref. 29).
  • allergens present in dust mite refs. 31 to 33
  • hornet venom ref. 34
  • birch pollen ref. 35
  • grass pollens ref. 36
  • the grass allergens cloned to-date may be classified to two main groups, one group includes allergens of 11 and 35 kDa in size (ref. 13) and the other group includes allergens of 28 to 34 kDa in size.
  • Published Canadian patent application No. 2,066,801 in which we are named as coinventors (corresponding to copending United States patent applications Serial No. filed , the disclosure of which is incorporated herein by reference) discloses the cloning of the cDNAs of three isoallergens of Kentucky Blue Grass Poa pratensis (KBG) pollen and identified immunologically important regions within these latter group of allergens. These allergens are present in some other grasses and are thus useful for diagnosis of and desensitizing for grass-specific allergies.
  • KBG Grass Poa pratensis
  • the present invention is directed toward improvements in allergen-specific immunotherapy by providing the means for overcoming the lack of pure allergens or peptides corresponding to portions thereof, as referred to above, by the determination of DNA sequences coding for a novel group of proteins of plant pollens. This group of allergens, though constituting major allergenic components of plant pollens, remained uncloned prior to this invention.
  • the present invention provides, in one aspect, a purified and isolated nucleic acid molecule comprising at least a portion coding for an allergenic protein which is present in pollens from both monocotyledonous and dicotyledonous plants.
  • the monocotyledonous plants are generally those selected from the family Gramineae, particularly grasses including Bermuda grass, Kentucky Blue grass, Red Top grass, Reed Canary grass, Rye grass, Timothy grass, Brome grass, Orchard grass and cultivated corn.
  • the dicotyledonous plants include trees, for example, birch, and weeds, for example, ragweed, particularly short ragweed, and parietaria.
  • the portion of the nucleic acid molecule coding for the allergenic protein provided herein generally is selected from:
  • the DNA sequences defined in (c) have at least about 75% sequence identity with the sequence defined in (a) or (b) .
  • the present invention provides a recombinant plasmid adopted for transformation of a host, comprising a plasmid vector into which has been inserted a DNA segment comprising at least a 15 bp fragment of a DNA molecule as provided herein.
  • the present invention also includes an expression vector adapted for transformation of a host, comprising at least a DNA segment comprising at least a 15 bp fragment of a DNA molecule as provided herein and expression means operatively coupled to the DNA segment for expression thereof in a host.
  • the DNA segment encodes an allergenic protein including the amino acid sequence of Figure 2.
  • the DNA segment contained in the expression vector may further comprise a nucleic acid sequence encoding a carrier protein, for example, glutathione-S-transferase
  • GST ⁇ -galactosidase or protein A, for expression of a carrier-allergen fusion.
  • plasmid pNHl deposited with the American Type Culture Collection, Rockville, Maryland, U.S.A., on January 10, 1994 and having ATCC accession number 75,634.
  • An additional aspect of the invention provides a recombinant protein produced by expression in the host of the DNA fragment contained in the expression vector provided herein or a functional analog of the protein.
  • a first protein is a "functional analog" of a second protein if the first protein is immunologically related to and/or has the same function as the second protein.
  • the functional analog may be, for example, a fragment of the protein or a substitution addition or deletion mutant thereof.
  • the present invention also includes synthetic allergenic or antigenic peptides corresponding in amino acid sequence to portions of the recombinant protein or allergen.
  • the present invention provides, in a further aspect, a composition for protecting allergic individuals from developing an allergic reaction, comprising at least one active component selected from at least one nucleic acid molecule and at least one recombinant protein, provided in accordance with aspects of the invention, and a pharmaceutically-acceptable carrier therefor, particularly formulated for in vivo administration.
  • the composition may comprise at least one recombinant protein conjugated to a non-immunogenic substrate, which may be a polymeric material, for example, a carboxymethyl cellulose, a monomethoxypolyethylene glycol and a polyvinyl alcohol.
  • the non-immunogenic substrate may comprise beads for targeted uptake of the at least one recombinant protein by antigen-presenting cells.
  • composition may be formulated as a microparticle, capsule or liposome preparation and may be provided a combination with a targeting molecule for delivery to specific cells of the immune system or to ucosal surfaces.
  • composition provided in this aspect of the invention may be combined with at least one additional desensitizing agent, which may be selected from Poa p_ IX allergen, Lol E - ⁇ allergen, Bet v I allergen, Amb a I allergen and Amb a II allergen.
  • additional desensitizing agent which may be selected from Poa p_ IX allergen, Lol E - ⁇ allergen, Bet v I allergen, Amb a I allergen and Amb a II allergen.
  • the composition may comprise at least one compound having anti-histamine activity and/or at least one compound having anti-inflammatory activity and/or at least one compound which is immunosuppressive.
  • the composition also may comprise an adjuvant.
  • the present invention provides a method for desensitizing an allergic individual, particularly a human, by administering an effective amount of the composition provided herein, particularly one containing the at least one additional desensitizing agent.
  • the present invention provides, in a further aspect, a method of depleting allergen-specific antibodies from an individual, particularly a human, by contacting the antibodies with the composition provided herein to form a complex, and removing the complex from the individual.
  • the present invention provides a method of anergizing allergen-specific antibody-producing cells, particular in a human, by contacting the cells with the composition provided herein.
  • the present invention also provides, in an additional aspect, an antiserum specific for a recombinant protein as provided herein.
  • Figure 1 shows the nucleotide sequence (SEQ ID NO: 1) and the deduced amino acid sequence (SEQ ID NO: 2) of a portion of a cDNA clone (KBG 51) obtained by screening a Kentucky Bluegrass pollen - ⁇ gtll library;
  • Figure 2 contains Northern blot analysis of KBG pollen mRNA using KBG 30 as lane 1, KBG 51 as lane 2 and KBG 60 as lane 3 insert DNAs as probes;
  • Figure 3 illustrates detection of homology in mRNA in a number of pollen by RT-PCR followed by Southern analysis using KBG 51 cDNA as the probe;
  • Figure 4 illustrates vector plasmid pNHl and the cloning site for KBG 51
  • Figure 5 illustrates high-level expression of KBG 51 in the pGEX-2T-l system
  • Figure 6 contains Western blots of a variety of pollen extracts utilizing antiserum to GST-KBG 51 fusion protein.
  • the various embodiments of the present invention have many applications in the fields of diagnosis and therapy of allergic diseases, such as allergic rhinitis, asthma, food allergies and atopic eczema.
  • allergic diseases such as allergic rhinitis, asthma, food allergies and atopic eczema.
  • the demonstrated cross-reactivity of the allergen is particularly useful.
  • the usual method of determining to which aeroallergens an individual is allergic involves an intra-dermal exposure to many crude extracts of allergen extracts (for example, mixtures of grasses, weeds and trees) . This procedure involves the use and administration of many allergens.
  • the cross-reactive allergen of the present invention (specifically CRAL51) now allows for the demonstration of IgE antibodies against a whole range of allergens.
  • An allergic individual who has CRAL51 specific IgE/antibodies can now be immediately excluded from being allergic to this whole range of allergens.
  • a further non-limiting discussion of such uses is further presented below. Preparation and Use of Composition for Protecting Allergic Individuals for Developing an Allergic Reaction
  • compositions suitable to be used for protecting allergic individuals from developing an allergic reaction, may be prepared from CRAL51 allergen, analogs, fragments and/or peptides as disclosed herein. Compositions may be prepared as injectables, as liquid solutions or emulsions.
  • the CRAL51 allergen fragment analogs or peptides may be mixed with pharmaceutically- acceptable excipients which are compatible with the allergen proteins, fragment analogs or peptides. Excipients may include, water, saline, dextrose, glycerol, ethanol, and combinations thereof.
  • the composition may further contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, or adjuvants to enhance the effectiveness thereof.
  • compositions may be administered parenterally, by injection subcutaneously or intramuscularly. Alternatively, other modes of administration including suppositories and oral formulations may be desirable.
  • binders and carriers may include, for example, polyalkylene glycols or triglycerides.
  • Oral formulations may include normally employed incipients, such as, for example, pharmaceutical grades of saccharine, cellulose, magnesium carbonate and the like.
  • compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 10 to about 95% of the allergen fragment analogs and/or peptides.
  • the compositions are administered in a manner compatible with the dosage formulation, and in such amount as is therapeutically effective to protect allergic individuals from developing an allergic reaction.
  • the quantity to be administered depends on the subject to be treated, including, for example, the capacity of the individual's immune system to synthesize antibodies. Precise amounts of allergen required to be administered depends on the judgement of the practitioner. However, suitable dosage ranges are readily determinable by one skilled in the art and may be of the order of nanograms to micrograms of the allergen, analog fragment and/or peptides.
  • Suitable regimens for initial administration and booster does are also variable, but may include an initial administration followed by subsequent administrations.
  • the dosage of the composition may also depend on the route of administration and will vary according to the size of the host.
  • the cross-reactive allergen may be conjugated to a non-immunogenic substrate including polymeric materials, such as carboxymethyl celluloses, monomethoxypolyethylene glycols (mPEG) and polyvinyl alcohols, to render it non-immunogenic and non-allergenic for protecting allergic individuals from developing an allergic reaction.
  • polymeric materials such as carboxymethyl celluloses, monomethoxypolyethylene glycols (mPEG) and polyvinyl alcohols
  • the nucleic acid molecules encoding the allergen of the present invention or portions thereof may also be used directly for immunization by adminstration of the DNA directly, for example, by injection for genetic immunization or by constructing a live vector, such as Salmonella, BCG, adenovirus, poxvirus, vaccinia or poliovirus.
  • a live vector such as Salmonella, BCG, adenovirus, poxvirus, vaccinia or poliovirus.
  • peptide analogs extends to any functional chemical equivalent of a peptide characterized by its increased stability and/or efficacy in vivo or in vitro in respect of the practice of the invention.
  • peptide analog is also used herein to extend to any amino acid derivative of the peptides as described herein.
  • Peptide analogs contemplated herein are produced by procedures that include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide synthesis and the use of cross-linkers and other methods which impose conformational constraint on the peptides or their analogs.
  • side chain modifications contemplated by the present invention include modification of amino groups, such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidation with methylacetimidate; acetylation with acetic anhydride; carbamylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6, trinitrobenzene sulfonic acid (TNBS) ; alkylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxa-5' - phosphate followed by reduction with NaBH 4 .
  • the guanidino group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents, such as 2, 3-butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via o-acylisourea formation followed by subsequent derivatisation, for example, to a corresponding amide.
  • Sulfhydryl groups may be modified by methods, such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of mixed disulphides with other thiol compounds; reaction with maleimide; maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4- chloromercuriphenylsulfonicacid, phenylmercurychloride, 2-chloromercuric-4-nitrophenol and other mercurials; carbamylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides. Tryosine residues may be altered by nitration with tetranitromethane to form a 3- nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
  • Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4- amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid-, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
  • the purified and isolated DNA molecules comprising at least a portion coding for an allergenic protein which is present in pollens from both monocotyledonous and dicotyledonous plants typified by the embodiments described herein are advantageous as: nucleic acid probes for the specific identification of other allergens that contain a DNA sequence comprising at least a portion coding for the cross-reactive allergen CRAL51 or analogs thereof;
  • the products encoded by the DNA molecules are useful as diagnostic reagents to identify CRAL51 specific IgE antibodies in an individual to demonstrate or exclude allergy to a whole range of allergens;
  • - peptides corresponding to portions of the allergen as typified by the embodiments described herein are advantageous as diagnostic reagents, antigens for the production of allergen-specific antisera, for example, for the demonstration of an allergic reaction to a whole range of allergens.
  • the cross-reactive allergen, analog, fragment and/or peptide of the present invention are useful as immunogens, as antigens in immunoassays including enzyme- linked immunosorbent assays (ELISA) , RIAs and other non- enzyme linked antibody binding assays or procedures known in the art for the detection of allergen specific IgE antibodies.
  • ELISA assays the allergen, analog, fragment and/or peptide corresponding to portions of the allergen are immobilized onto a selected surface, for example, a surface exhibiting a protein affinity, such as the wells of a polystyrene microtiter plate.
  • a nonspecific protein such as bovine serum albumin (BSA) or casein that is known to be antigenically neutral with regard to the test sample, may be bound to the selected surface.
  • BSA bovine serum albumin
  • the peptides are at least 12 residues in length and preferably 14 to 30 residues. It is understood however, that a mixture of peptides may be used either as an immunogen in a composition or as a diagnostic agent.
  • the immobilizing surface is then contacted with a sample, such as clinical or biological materials to be tested in a manner conducive to immune complex (antigen/antibody) formation.
  • a sample such as clinical or biological materials to be tested in a manner conducive to immune complex (antigen/antibody) formation.
  • This may include diluting the sample with diluents, such as solutions of BSA, bovine gamma globulin (BGG) and/or phosphate buffered saline (PBS)/Tween.
  • BGG bovine gamma globulin
  • PBS phosphate buffered saline
  • the sample is then allowed to incubate for from about 2 to about 4 hours, at temperatures, such as of the order of about 25° to about 37°C.
  • the sample-contacted surface is washed to remove non-immunocomplexed material.
  • the washing procedure may include washing with a solution, such as PBS/Tween, or a borate buffer
  • the occurrence, and even amount, of immunocomplex formation may be determined by subjecting the immunocomplex to a second antibody having specificity for the first antibody.
  • the second antibody would be an antibody having specificity for human IgE or IgG antibodies.
  • the second antibody may have an associated activity, such as an enzymatic activity that will generate, for example, a color development upon incubating with an appropriate chromogenic substrate. Quantification may then achieved by measuring the degree of color generation using, for example, a visible spectra spectrophotometer.
  • nucleotide sequences of the present invention comprising the sequence of the cross-reactive allergenic protein, now allow for the identification and cloning of the allergenic protein genes from other sources.
  • the nucleotide sequences comprising the sequence of the allergenic protein of the present invention are useful for their ability to selectively form duplex molecules with complementary stretches of other allergenic protein genes.
  • hybridization conditions may be employed to achieve varying degrees of selectivity of the probe toward the other allergenic protein genes.
  • relatively stringent conditions are used to form the duplexes, such as low salt and/or high temperature conditions, such as provided by 0.02 M to 0.15 M NaCl at temperatures of between about 50°C to 70°C.
  • less stringent hybridization conditions are required such as 0.15 M to 0.9 M salt, at temperatures ranging from between about 20°C to 55°C.
  • Hybridization conditions can also be rendered more stringent by the addition of increasing amounts of formamide, to destabilize the hybrid duplex.
  • particular hybridization conditions can be readily manipulated, and will generally be a method of choice depending on the desired results.
  • indicator means are known in the art for determining hybridization, including radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of providing a detectable signal.
  • an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of a radioactive tag may be used.
  • colorimetric indicator substrates are known which can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with samples containing allergenic protein gene sequences.
  • the nucleic acid sequences of allergenic protein genes of the present invention are useful as hybridization probes in solution hybridizations and in embodiments employing solid-phase, procedures.
  • the test DNA (or RNA) from samples is adsorbed or otherwise affixed to a selected matrix or surface.
  • the fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes comprising the nucleic acid sequences of the allergenic protein genes or fragments thereof of the present invention under desired conditions.
  • the selected conditions will depend on the particular circumstances based on the particular criteria required depending on, for example, on the G+C contents, type of target nucleic acid, source of nucleic acid, size of hybridization probe etc.
  • Plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell may be used for the expression of the allergenic protein genes in expression systems.
  • the vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells.
  • E. coli may be transformed using pBR322 which contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
  • the pBR322 plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of its own proteins.
  • phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as a transforming vector in connection with these hosts.
  • the phage in lambda GEMTM-11 may be utilized in making recombinant phage vectors which can be used to transform host cells, such as E. coli LE392.
  • Promoters commonly used in recombinant DNA construction include the 3-lactamase (penicillinase) and lactose promoter systems (Chang et al. , 1978 (ref. 48) : Itakura et al . , 1977 (ref. 49); Goeddel et al. , 1979 (ref.
  • microbial promoters such as the T7 promoter system. Details concerning the nucleotide sequences of promoters are known, enabling a skilled worker to ligate them functionally with plasmid vectors. The particular promoter used will generally be a matter of choice depending upon the desired results.
  • Hosts that are appropriate for expression of the allergen genes, fragments, analogs or variants thereof include E. coli , Bacillus species, fungi, yeast, higher eukaryotic cells, such as CHO cells, or the baculovirus expression system may be used.
  • the allergenic protein, fragment or analog by recombinant methods.
  • Particularly desirable hosts for expression include Gram positive bacteria which do not have LPS and are therefore endotoxin free.
  • Such hosts include species of Bacillus and may be particularly useful for the production of allergenic protein, fragments or analogs thereof.
  • the allergenic protein may also be produced as a fusion protein with, for example, glutathione-S- transferase, /3-galactosidase and protein A.
  • a plasmid pNHl (pUM 94.1) that contains at least a portion coding for an allergenic protein that is described and referred to herein has been deposited with the American Type Culture Collection (ATCC) located at Rockville, Maryland USA pursuant to the Budapest Treaty on January 10, 1994 and has been accorded accession number 75,634.
  • a further plasmid pUM 94.2 (KBG 15) also containing at least a portion coding for an allergenic protein has been deposited with ATCC on and has been accorded accession number . Samples of the deposited plasmids will become available to the public upon grant of a patent based upon this United States patent application.
  • This Example describes the cloning and sequencing of a cDNA encoding a portion of the cross- reactive allergen (CRAL 51) . Certain aspects of the cloning of allergen-encoding cDNAs is described in the aforementioned Canadian patent application No. 2,066,801 (and corresponding USA
  • Plaques (250,000) from a ⁇ gtll library (ref. 38) were screened immunologically for the expression of pollen allergens using pooled sera of eight patients allergic to KBG pollen. Patients were selected on the basis of case history, positive skin prick and radioallerosorbant tests but had not undergone any hyposensitization treatments. Plaques were lifted onto nitrocellulose filters (Bio-Rad, Toronto) and washed twice for 5 and 30 minute intervals in 30 mis of buffer
  • the filters were re-washed as previously described and developed using 5-bromo-4-chloro-3-indolylphosphate p- toluidine and nitroblue tetrazolium chloride. Positive clones were picked and re-screened, one of these clones, (KBG51) was further characterized.
  • DNA was prepared from plaque purified phage using the liquid lysate method (ref. 39) . Inserts recovered from Eco RI digestion were ligated into the multiple cloning site (MCS) of pBluescript and used to transform E. coli DH5 ⁇ -.
  • MCS multiple cloning site
  • cDNA KBG51 is a partial cDNA clone with an open reading frame.
  • the deduced polypeptide comprises 145 amino acids with an estimated Mr of 16.636 kDa and pi of 12.02. Comparison of the nucleotide and deduced amino acid sequences with the GenBank DNA and PIR protein sequence Data Banks, respectively indicated that the sequences were unique and not homologous to any other known allergen or non-allergen DNA or proteins in these Data Banks.
  • the cloned cDNA encodes a novel allergen which has not been described previously.
  • This Example describes the demonstration of mRNA species in KBG pollen corresponding to the KBG51 cDNA.
  • Total pollen and leaf RNA was electrophoresed in 1.5% agarose gels under dewatering conditions (ref. 40) and blotted onto nylon membrane.
  • This Example demonstrates the presence of transcripts corresponding to KBG51 in a variety of other pollens.
  • RNA prepared from the pollens indicated in Figure 3 as described in Example 1 was subjected to reverse transcription reaction.
  • the first strand cDNA was synthesized in a 50 ⁇ l final volume at 42°C for 1 h using a 5 ⁇ g of total RNA, reverse transcriptase and oligo dT primer.
  • the double strand cDNA was synthesized using DNA polymerase and dNTP according to the manufacturers instructions (BRL, Mississauga, ON) .
  • the products obtained were heat denatured and an aliquot of 5 ⁇ l was utilized in amplification reaction with specific primers (DNA Synthesis Laboratory, Univ. of Manitoba) for the KBG51 cDNA.
  • the primers are shown on the DNA sequence of Figure 1 and had the sequences KBG, forward 5' - TCT TGG CTT GAC CGA AGC (SEQ ID NO: 3) and KBG, reverse 5' - GAT ACA GCC CAT CAC CGC (SEQ ID NO: 4) .
  • the PCR amplification (ref. 41) was carried out in a buffer containing 50 mM KCl, 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl 2 , 0.1% (w/v) gelatin, 200 ⁇ M dNTP, 2.5 Taq polymerase (Cetus Perkin Elmer) and 50 pmol of each primer.
  • the reaction consisted of 30 cycles of amplification followed by a 15 min. final extension at 12° C Each cycle included denaturation at 95°C for 30 sec, annealing at 65 ° C for 60 sec. and extension at 72°C for 2 min.
  • This Example describes the production of a GST-KBG51 fusion protein.
  • the insert was cloned in the pGEX 2T-1 expression vector (ref. 42) to produce plasmid pNHl ( Figure 4) which permitted high-level expression of the GST-KBG51 fusion protein ( Figure 6) .
  • the fusion protein was expressed as a soluble fusion protein of approximately 43 kDa in conjunction with the glutathione- S-transferase (GST) , which upon cleavage yielded the GST of about 26 kDa.
  • GST glutathione- S-transferase
  • the protein expression was estimated to be about 20% of the total cell extract or about 2 mg/g wet wt of cells.
  • This Example describes the generation of GST-KBG51 fusion protein specific antiserum and its cross reactivity to proteins in other plant pollens.
  • the GST-KBG51 fusion protein with dextran sulfate as adjuvant was used as immunogen to immunize 6 to 8 week old female BDF1 mice (Jackson Laboratories, Bar Harbour Michigan) .
  • the mice were immunized subcutaneously and after 23 days the mice were boosted with the same antigen preparation. Blood was collected at day 30 after primary immunization and serum was prepared. Proteins from various pollens (as shown in Figure 6) were isolated as previously described (ref. 17) and electrophoresed on a 15% polyacrylamide gel.
  • Proteins were then stained with Coomassie Blue or electroblotted onto a nitrocellulose membrane.
  • Membranes were blocked with 1% gelatin in TBS (10 mM Tris-HCl pH7.5, 150 mM NaCl) and probed with the antiserum raised against GST- KBG51. Immunoreactive bands were detected using an alkaline phosphatase conjugated goat anti-mouse second antibody.
  • a Western blot analysis (ref. 43) employing the polyclonal murine antiserum to the GST-KBG51 fusion protein led to the identification of two components in the KBG extract, a major component having an approximate Mr of 59 kDa and a minor component having an approximate Mr- of 30 kDa ( Figure 6 lane 3 and Table 1 (the Tables appear at the end of the disclosure) ) .
  • a Western blot analysis of a number of pollen extracts showed that a number of polypeptides in these extracts reacted with the antiserum indicating the presence of cross-reactive allergens in these pollens.
  • Table 1 summarizes the polypeptides recognized by the antiserum in protein extracts prepared from a number of different grasses, weeds and tree pollens. This antibody recognized two high molecular weight polypeptides in the ryegrass pollen extract which has not been reported previously. From these studies it is concluded that the allergen encoded by the KBG51 cDNA comprises a very highly cross-reactive group of allergens in plant pollens.
  • This Example describes the percentage of the allergic population that have KBG51 specific IgE antibodies.
  • GST-KBG51 protein (10 ⁇ g/ml) was coated to wells of microtiter plates (Nunc, Immunoplate, Denmark) overnight. The wells were further blocked with phosphate buffer containing 1% gelatin for 8 hrs. Subsequent to washing 5 times with phosphate buffer containing 0.05% Tween 20, the wells were incubated with human serum diluted with the washing buffer containing 0.1% gelatin overnight. After washing, alkaline phosphatase-conjugated secondary antibodies (goat anti-human IgE (TAGO) , diluted 300-fold) were added and the wells were incubated overnight.
  • TAGO alkaline phosphatase-conjugated secondary antibodies
  • the color reaction was developed by adding the substrate para-nitro phenyl phosphate (1 mg/ml) in 0.1 M Tris-HCl buffer containing NaCl and MgCl 2 and the OD was measured at 405 nm in a Titertek Mul iscan spectrophotometer (Flow Laboratories, McLean, VA) .
  • the present invention provides certain novel nucleic acid molecules which code for allergenic proteins present in a wide spectrum of plant pollens. Modifications are possible within the scope of this invention. Table 1
  • CMS Update Desensitizing vaccines Brit. Med. J. 293: 948 (1986) .

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Abstract

Le criblage d'une banque de pollen-μgt11 de pâturin des prés avec des sérums provenant de patients allergiques au pollen des graminées a permis d'identifier un clone d'ADNc partiel, le KBG51. L'analyse de la séquence nucléotidique de KBG51 a révélé que le polypeptide codé par cet ADNC est différent de celui des allergènes du pollen des graminées recombinés connus. On a détecté deux polypeptides d'environ 30 et 59 kDa dans l'analyse par immunotransfert SDS-PAGE des protéines de pollen de KBG en utilisant un antisérum murin dirigé contre la protéine de fusion de GST-KBG51 produite à l'aide du système d'expression pGEX-2T-1. La réactivité de cet antisérum, avec un certain nombre de polypeptides de 29,5 à 115 kDa d'extraits de pollen de plusieurs graminées, du bouleau, de l'ambrosia et de la pariétaria, ainsi que l'hybridation des produits d'amplification RT-PCR de divers pollens avec de l'ADNc-KBG51 radiomarqué a démontré la réactivité croisée de cet AL avec d'autres Als de pollen. De par sa (leur) réactivité croisée importante, la(les) protéine(s) correspondant à KBG51 est (sont) désignée(s) par CRAL51. La technique ELISA dans laquelle des sérums d'environ 1000 personnes allergiques aux pollens dans le monde entier a démontré que les individus provenant de plusieurs zones géographiques possèdent des anticorps IgE reconnaissant la protéine de fusion GST-KBG51. Ces découvertes ont permis de conclure que CRAL51 représente une membre de la famille d'ALs à réactivité croisée élevée dans les pollens de plantes.
PCT/CA1995/000021 1994-01-14 1995-01-16 Allergene a reactivite croisee WO1995019437A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1356826A1 (fr) * 2002-04-22 2003-10-29 BIOMAY Produktions- und Handels- Aktiengesellschaft Microparticules à perles de hydrates de carbone auquels sont liés des allergènes de facon covalente
WO2007030748A2 (fr) * 2005-09-09 2007-03-15 The University Of Chicago Methodes et compositions utiles pour le diagnostic et l'immunotherapie contre l'allergie aux pollens

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992003551A1 (fr) * 1990-08-13 1992-03-05 Biomay Biotechnik Produktions- Und Handelsgesellschaft M.B.H. Allergene p14 au pollen de bouleau servant au diagnostic et a la therapie des maladies allergiques

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Publication number Priority date Publication date Assignee Title
WO1992003551A1 (fr) * 1990-08-13 1992-03-05 Biomay Biotechnik Produktions- Und Handelsgesellschaft M.B.H. Allergene p14 au pollen de bouleau servant au diagnostic et a la therapie des maladies allergiques

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Title
DONOVAN AND BALDO: "Crossreactivity of IgE antibodies from sera of subjects allergic to both ryegrass pollen and wheat endosperm proteins: evidence for common allergenic determinants", CLINICAL AND EXPERIMENTAL ALLERGY, vol. 20, no. 5, OXFORD, GB, pages 501 - 509 *
LUO ET AL.: "Identification of a novel highly crossreactive allergen in Kentucky Bluegrass pollen", THE JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY, vol. 93, no. 1, par, pages 231 *
SILVANOVICH ET AL.: "Nucleotide sequence analysis of three cDNAs coding for Poa pIX isoallergens of Kemtucky Bluegrass pollen", JOURNAL OF BIOLOGICAL CHEMISTRY (MICROFILMS), vol. 266, no. 2, 15 January 1991 (1991-01-15), MD US, pages 1204 - 1210 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1356826A1 (fr) * 2002-04-22 2003-10-29 BIOMAY Produktions- und Handels- Aktiengesellschaft Microparticules à perles de hydrates de carbone auquels sont liés des allergènes de facon covalente
WO2003089009A1 (fr) * 2002-04-22 2003-10-30 Biomay Produktions- Und Handels-Aktiengesell Schaft Microparticules comprenant des billes d'hydrates de carbone liees par covalence a un allergene
CN100460019C (zh) * 2002-04-22 2009-02-11 比奥梅产品及贸易股份公司 包含与过敏原共价连接的糖类珠粒的微粒
US8454964B2 (en) 2002-04-22 2013-06-04 Biomay Ag Microparticles comprising carbohydrate beads covalently linked with allergen
WO2007030748A2 (fr) * 2005-09-09 2007-03-15 The University Of Chicago Methodes et compositions utiles pour le diagnostic et l'immunotherapie contre l'allergie aux pollens
WO2007030748A3 (fr) * 2005-09-09 2007-06-07 Univ Chicago Methodes et compositions utiles pour le diagnostic et l'immunotherapie contre l'allergie aux pollens

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