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WO1998004274A1 - Allergenes salivaires du moustique - Google Patents

Allergenes salivaires du moustique Download PDF

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Publication number
WO1998004274A1
WO1998004274A1 PCT/US1997/013573 US9713573W WO9804274A1 WO 1998004274 A1 WO1998004274 A1 WO 1998004274A1 US 9713573 W US9713573 W US 9713573W WO 9804274 A1 WO9804274 A1 WO 9804274A1
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mosquito
allergen
recombinant
set forth
raed
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WO1998004274A9 (fr
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Zhikang Peng
F. Estelle R. Simons
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University of Manitoba
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University of Manitoba
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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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the field of the invention relates to the use of recombinant mosquito salivary protein allergens in skin tests, i munoassays and allergen immunotherapy.
  • Mosquito bites are a global problem not only because they facilitate transmission of potentially fatal diseases such as malaria and yellow fever, but also because they cause local skin reactions and, rarely, systemic reactions including urticaria, angioedema, and even anaphylactic shock [Frazier, 1973; McCormack et al . , 1995] .
  • Skin reactions to mosquito bites are caused by the proteins in the mosquito saliva that enter the skin when mosquitoes take a blood meal [Hudson et al . , 1960] .
  • Mosquito saliva proteins elicit both IgE-mediated immediate hypersensitivity and lymphocyte-mediated delayed hypersensitivity [Oka 1989; Peng et al . , 1996] .
  • Mosquito salivary proteins are also involved with many aspects of the process of hematophagy which provide new perspectives for evaluating the transmission dynamics of pathogens [James, 1994] .
  • Mosquito saliva contains a complex of proteins. Protein visualization techniques using gel electrophoresis and silver staining have revealed as many as 20 peptides in adult mosquito Aedes aegypti saliva [Racioppi and Spielman, 1987] , which include ⁇ - amylase, anticoagulants, anti-TNF, apyrase, esterase, D7, ⁇ -glucosidase, and sialokinins [James, 1994].
  • each of the saliva proteins Purification or isolation of each of the saliva proteins is required to improve studies of the diagnosis and immunotherapy of mosquito allergy, and studies of mechanisms of mosquito-transmitted diseases.
  • purification of each salivary protein from mosquito whole body extract is an extremely laborious task and may result in the potential loss of important allergens or their biologic activity during the necessary multiple purification procedures. Collection of mosquito saliva is time-consuming and labor- intensive and therefore is also impractical . Utilization of molecular techniques to clone and express mosquito salivary proteins would be useful in developing allergens for skin testing and immunotherapy.
  • a recombinant mosquito salivary allergen for use in skin tests, immunoassays and immunotherapy for allergic reactions to mosquito bites.
  • the recombinant allergen is produced by a cDNA encoding an IgE-binding protein or fragment thereof or analogue thereof which is found in mosquito saliva.
  • the recombinant allergen is rAed a 1, a 68 kDa recombinant allergen, or rAed a 2, a 37 kDa recombinant allergen or rAed a 3, a 30 kDa recombinant allergen.
  • the recombinant allergen may share common allergenicity among at least two species of mosquitos or may be species-specific .
  • the present invention also provides a method of skin testing and undertaking immunotherapy utilizing the recombinant salivary allergen and a kit for practicing the method of the invention.
  • the recombinant allergens are selected to share common allergenicity among the mosquito species common to the geographic area for which testing or treatment is required.
  • the recombinant allergens included in the kit are selected so as to be species- specific for each mosquito species common to the geographic area for which testing is required.
  • a combination of allergens with common specificities among species and specificities that are species-specific are used so as to effectively represent the mosquito species distribution of the geographic area for which testing and/or immunotherapy is needed.
  • the present invention also provides an immunoassay for measurement of mosquito salivary allergen-specific IgE and IgG using recombinant mosquito salivary allergen as the substrate to which the allergen- specific IgE and IgG binds.
  • the present invention also provides a kit for the immunoassay including the appropriate recombinant allergen, antibody directed to the allergen and may also contain reference sera.
  • the present invention further provides antibodies directed against the recombinant salivary allergens, wherein the recombinant allergen is produced by a cDNA encoding an IgE-binding protein or fragment thereof or analogue thereof which is found in mosquito saliva.
  • the antibody is directed against rAed a 1, a 68 kDa recombinant allergen, or rAed a 2, a 37 kDa recombinant allergen or rAed a 3, a 30 kDa recombinant allergen.
  • the antibody may be polyclonal or monoclonal.
  • the antibodies are used for immunoassays, purification and antigen standardization.
  • FIGURE 1 is a scan of an immunoblot showing fusion protein-selected mouse IgG recognizes a 30 kDa salivary protein.
  • Aedes aegypti saliva proteins were separated by 12% SDS-PAGE and immunoblotted with mouse anti- saliva serum (lane 1), fusion protein-selected mouse antibodies (lane 2) , or PBST containing 1% BSA (lane 3) .
  • the reaction was detected by incubation of the filters with peroxidase-conjugated goat anti-mouse IgG followed by ECL detecting reagents.
  • FIGURE 2 is a scan of a fusion protein-selected human IgE reacts with a 30 kDa salivary protein. Aedes aegypti salivary proteins were separated by 12% SDS- PAGE and immunoblotted with a pooled human mosquito- allergic serum (lane 1) , fusion protein-selected human antibodies (lane 2) , or PBST containing 1% BSA (lane 3) . The reaction was developed by incubation of the filters with monoclonal anti-human IgE followed by incubation with peroxidase-conjugated goat anti-mouse IgG. The bound human IgE is indicated by an arrow.
  • FIGURE 3 is a scan of an immunoblot showing patterns of IgE and IgG responses to Ae. vexans in five subjects with or without skin reactions to mosquito bites. SDS-PAGE and immunoblot using the sera of three subjects with severe skin reactions to the bites (strips #1-3), two subjects without skin reactions to the bites (strips #4 and #5) , and a cord serum (strip #6) .
  • Strip #7 is PBS control.
  • the 2 IgE binding antigens indicated by arrows show different antibody responses in individuals.
  • FIGURE 4 is a scan of an immunoblot showing comparison of IgE and IgG responses to Ae. vexans by SDS-PAGE and immunoblot in three subjects with severe skin reactions to mosquito bites.
  • FIGURE 5A-B are graphs showing the rate of skin reactions to rAed a 1 in subjects with positive reactions in mosquito bite tests. Skin prick tests were performed with the purified rAed a 1 and a commercial mosquito Ae. aegypti whole-body extract in subjects with or without skin reactions in the mosquito bite tests. Immediate wheal and flare reactions were measured 20 minutes after the pricking and delayed indurated reactions were measured 24 hours later.
  • FIGURE 6 is a graph showing correlation of immediate flare sizes of rAed a 1 and Aedes aegypti bite in subjects with positive skin reaction to both rAed a 1 and an Ae. aegypti bite.
  • FIGURE 7 is a scan of the identification of Aed a 2 by Western blot.
  • Aedes aegypti saliva and rAed a 2 expressed in the culture medium were separated from other proteins using SDS-PAGE. Proteins were then transferred to nitrocellulose membranes, which were immunoblotted with either mosquito-allergic human serum (left panel) or rabbit anti-Aed a 2 (right panel) and bands visualized.
  • rAed a 2 possesses identical antigenic properties and the same molecular weight as the native Aed a 2 present in the saliva.
  • FIGURE 8 is a scan of an immunoblot analysis of Aed a 2 allergens in 11 mosquito species.
  • FIGURE 9 is a scan of an immunoblot showing salivary allergens of 10 mosquito species. Proteins in the saliva or salivary gland extracts of 10 species were separated by SDS-PAGE and then transferred to nitrocellulose membranes. The membranes were sequentially incubated with a pooled mosquito-allergic serum, monoclonal anti-human IgE, and enzyme-conjugated goat anti-mouse IgG as described.
  • FIGURE 10 is a scan of an immunoblot showing analysis of shared Aed a 1 and Aed a 2 allergens in different mosquito species. Proteins in the 11 mosquito saliva or salivary gland extracts were separated by SDS-PAGE and then transferred to nitrocellulose membranes. The membranes were incubated with rabbit anti-Aed a 1 (left) or rabbit anti-Aed a 2 (right) followed by incubation with enzyme-conjugated goat anti-mouse IgG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
  • the present invention provides recombinant mosquito salivary allergens for use in skin tests, immunoassays and immunotherapy.
  • the recombinant allergen is identified and produced by a cDNA encoding an IgE-binding protein or fragment thereof or analogue thereof which is found in mosquito saliva as is shown in the Examples herein.
  • the allergen is generally a protein or protein fragment or analogue thereof found in the saliva of mosquitos as described herein above.
  • the allergen elicits an IgE response and may also elicit an IgG response.
  • the fragment contains some or all of the epitopes on the whole allergen. It elicits an IgE response and may also elicit an IgG response.
  • the recombinant allergen expresses the same epitopes, either sequence based or conformational, as the native allergen and shares the same antigenic functions as the native allergen.
  • the antigenic functions essentially mean the possession of an epitope or antigenic site that is capable of cross-reacting with antibodies raised against a naturally occurring salivary protein. Further, the recombinant antigen must also have the same biologic activity, that is it must elicit an immune response in vivo .
  • Analogue as used herein is defined as a variant (alternatively the terms alteration, amino acid sequence alteration, amino acid sequence variant can be used) with some differences in their amino acid sequences as compared to the native sequence of salivary proteins, but functionally equivalent.
  • the analogue will be generally at least 70% homologous over any portion that is functionally relevant for eliciting an immune response.
  • the homology will be at least 80% and can approach 95% homology to the mosquito salivary protein sequence. The homology will extend over a region of at least nine contiguous amino acids.
  • amino acid sequence of an analog may differ from that of the native protein when at least one residue is deleted, inserted or substituted, but the protein retains its antigenic competence and biological activity in vivo in relation to eliciting an immune response. Differences in glycosylation can provide analogues.
  • Functionally equivalent refers to the biological property of the molecule and in this context means an in vivo eliciting of an immune response by a naturally occurring (native) salivary proteins.
  • the antigenic functions essentially mean the possession of an epitope or antigenic site that is capable of cross-reacting with antibodies raised against a naturally occurring salivary protein and eliciting skin reactions as do the native salivary allergens.
  • Biologically active in vivo activi ty means that the analogues share an antigenic function and elicits an immune response in vivo .
  • any expression system may be used as is known in the art that will provide recombinant allergens or allergen fragments that express the eliciting epitopes and have activity in vivo.
  • the allergen is produced in a baculovirus expression vector system.
  • recombinant allergens rAed a 1, a 68 kDa recombinant allergen, and/or rAed a 2, a 37 kDa recombinant allergen and/or rAed a 3 (SEQ ID No:l), a 30 kDa are used.
  • the allergens are named using the rules as set forth in Larson and Lowenstein [1996] .
  • the recombinant allergens can be selected such that they share common allergenicity among at least two species of mosquitos as shown in the Examples. Alternatively, the allergen is species specific.
  • the allergens used for skin testing are selected on the basis of the mosquito species distribution in the geographic area in which the patient to be tested is exposed or will be exposed. It is contemplated by the present invention that in one embodiment patients moving into new geographic areas or planning to vacation in a new geographic area will be skin tested to determine their sensitivity to the predominant mosquito species population of that area and can then be desensitized as described herein below.
  • the allergens may be cross-reactive across several species or may be species-specific.
  • the skin testing will utilize the recombinant allergens that effectively represent the mosquito species distribution for the geographic area in which the patient is exposed or will be exposed.
  • concentration of each recombinant allergen used in skin testing will be determined using in vivo and/or in vitro standardization techniques. The standardization techniques have been described in detail previously [Ipsen et al . 1993] and are summarized herein below. Each recombinant allergen extract will be standardized against a standard reference to assure lot-to-lot consistency and relative potency of allergenic recombinant extracts.
  • the response to the recombinant mosquito allergens can be determined by an immunoassay as is known in the art and described herein below.
  • an ELISA for IgE is preferred but Western Blotting can also be used.
  • the present invention also provides a kit including the appropriate recombinant allergen, antibody directed to the allergen and may contain reference sera for the practice of the immunoassay.
  • the present invention also provides an allergy immunotherapy medicament which contains the recombinant mosquito allergens for use in immunotherapy
  • the recombinant allergens are suspended in pharmaceutically acceptable carriers, diluents, adjuvants and/or vehicles as is known in the art of immunotherapy. These pharmaceutically acceptable carriers and the like are selected such that they do not react with the active ingredients of the invention and that the allergens retain their immunologically eliciting conformation and biological activity.
  • an alternative term for the immunotherapy medicament is "extract" which is generally used to indicate allergens (immunogens) that have been isolated or prepared from a native or natural source and not produced recombinantly. An example of an extract would be mosquito whole body preparations.
  • the allergy immunotherapy medicament of the present invention can be referred to as a "recombinant extract” .
  • the allergy immunotherapy medicament will contain at least one recombinant mosquito allergen.
  • the recombinant mosquito allergens are selected on the basis of the skin test results.
  • the starting dose of the allergen can be determined by skin test endpoint-titration using a dose that is equal to 0.1 ml of the end-point dilution that initiates a skin reaction or other methods known in the art.
  • the medicament will contain a combination of recombinant allergens that effectively represents the mosquito species distribution for the geographic area to which the patient is allergic and for which they need desensitization.
  • the present invention provides a kit for skin testing for allergy to mosquito bites and a kit for providing immunotherapy including recombinant mosquito salivary allergens.
  • the kit includes recombinant allergens that are selected to share common allergenicity among the mosquito species common to the geographic area for which testing or immunotherapy is required.
  • the kit may also include species-specific recombinant allergens for each mosquito species common to the geographic area for which testing and/or immunotherapy is required.
  • the kit includes recombinant allergens rAed al, a 68 kDa recombinant allergen, rAed a2 , a 37 kDa recombinant allergen and rAed a3, a 30 kDa recombinant allergen. Since 1865, skin tests have been used to provide helpful confirmatory evidence for diagnosis of specific allergy.
  • Skin tests include epicutaneous and intradermal tests. Detailed techniques have been previously described [Bousquet and Michel 1993] . Briefly, in the epicutaneous tests, drops of recombinant allergen extracts are placed approximately 2 cm apart on the volar surface of the forearm. The point of a disposable needle is passed through the drop, inserted into the epidermal surface, and then gently lifted without inducing bleeding. The immediate wheal and flare reactions are traced and recorded 20 minutes after the test, and delayed indurated papules are traced 24 hours later. In the intradermal tests, a volume of approximately 0.01 to 0.05 ml of the recombinant allergen is injected into the skin to produce a small superficial bleb approximately 2-3 mm in diameter. Results are traced and recorded in the same manner as for the epicutaneous tests.
  • a starting dose will contain 0.5 allergy units/ml or can be chosen by skin test end-point titration using a dose that is equal to 0.1 ml of the end-point dilution that initiate skin reaction. When the highest tolerated dose is reached, this dose is used to maintain allergen-specific immunity, that is, injected every 2 - 4 weeks for a period of time, as determined by clinical history and monitored immunologic tests.
  • Diagnosis of mosquito allergy can be also made by measurement of serum recombinant allergen-specific IgE and IgG antibodies using immunoassays .
  • immunoassays are the preferred immunoassays employed to assess the amount of IgE and IgG in a specimen.
  • ELISA assays are well known to those skilled in the art. Both polyclonal and monoclonal antibodies can be used in the assays. Where appropriate, other immunoassays, such as radioimmunoassays (RIA) can be used as are known to those in the art. Available immunoassays are extensively described in the patent and scientific literature.
  • any expression system as is known in the art that will provide recombinant allergens or allergen fragments that express the eliciting epitopes and have activity in vivo can be used in the practice of the present invention.
  • the baculovirus insect cell expression system which performs many of the post-translational modifications found in mammalian cells, is an excellent system for the production of large amounts of biologically active proteins (see generally O'Reilly et al, 1994.
  • Baculovirus Expression Vectors A Laboratory Manual . Oxford University Press
  • the efficiency of expression of baculovirus system differs from gene to gene by approximately 1000-fold.
  • this system provides for glycosylation such that proper carbohydrate expression is provided which plays a role in the immunological response and in vivo activity.
  • Vectors can be constructed containing the cDNA of the present invention by those skilled in the art and should contain all expression elements necessary to achieve the desired transcription of the sequences in the selected expression system. Other beneficial characteristics can also be contained within the vectors such as mechanisms for recovery of the nucleic acids in a different form.
  • the vectors can also contain elements for use in either procaryotic or eucaryotic host systems. One of ordinary skill in the art will know which host systems are compatible with a particular vector.
  • the vectors can be introduced into cells or tissues by any one of a variety of known methods within the art (calcium phosphate transfection; electroporation; lipofection; protoplast fusion; polybrene transfection) .
  • the host cell can be any eucaryotic and procaryotic cells, which can be transformed with the vector and which will support the production of the allergen with proper glycosylation and conformation.
  • pBlueBacHis vectors are designed for efficient expression and purification of recombinant proteins [Chen et al, 1993; Reddy et al, 1994; Rotrosen et al ,
  • expression vectors are selected for each recombinant allergen as is known in the art such that yield of allergen is maximized
  • Expressed recombinant allergens in the cell culture media will be purified according to the physio- biochemical characteristics of each recombinant allergen as is known in the art. For example, histidine tagging [Xu et al . , 1996] for purification of recombinant proteins by immobilized metal affinity chromatography can be used for a number of proteins both in prokaryotic [Dudler et al . , 1992.] and eukaryotic [Janssen et al . , 1995; Reddy et al, 1994] expression systems. Other purification systems as are known in the art can be used including affinity chromatography utilizing the monoclonal antibodies to recombinant allergens of the present invention.
  • the present invention provides antibodies directed against the recombinant salivary allergens. These antibodies can be used in immunoassays and for purification and standardization of allergens. Applicants are producing mAb directed against recombinant allergens in BALB/c mice.
  • Antibodies may be either monoclonal or polyclonal and are raised against the immunogen.
  • the antibodies may be prepared against the immunogen or part of the immunogen for example a synthetic peptide based on the sequence, or prepared recombinantly by cloning techniques or the natural gene product and/or portions thereof may be isolated and used as the immunogen.
  • Such immunogens can be used to produce antibodies by standard antibody production technology well known to those skilled in the art as described generally in Harlow and Lane, Antibodies : A Labora tory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988 and Borrebaeck, Antibody Engineering - A Practical Guide, W.H. Freeman and Co., 1992.
  • Antibody fragments may also be prepared from the antibodies and include Fab, F(ab') 2 , and Fv by methods known to those skilled in the art .
  • a host such as a rabbit or goat
  • the immunogen generally with an adjuvant and, if necessary, coupled to a carrier; antibodies to the protein are collected from the sera.
  • the polyclonal antibody can be absorbed such that it is monospecific . That is, the sera can be absorbed against related immunogens so that no cross-reactive antibodies remain in the sera rendering it monospecific .
  • the technique involves hyperimmunization of an appropriate donor with the immunogen or immunogen fragment, generally a mouse, and isolation of splenic antibody producing cells. These cells are fused to a cell having immortality, such as a myeloma cell, to provide a fused cell hybrid which has immortality and secretes the required antibody. The cells are then cultured, in bulk, and the monoclonal antibodies harvested from the culture media for use.
  • an appropriate donor with the immunogen or immunogen fragment, generally a mouse
  • splenic antibody producing cells are fused to a cell having immortality, such as a myeloma cell, to provide a fused cell hybrid which has immortality and secretes the required antibody.
  • the cells are then cultured, in bulk, and the monoclonal antibodies harvested from the culture media for use.
  • RNAs from antibody producing B-lymphocytes of animals, or hybridoma are reverse-transcribed to obtain complimentary DNAs (cDNAs) .
  • Antibody cDNA which can be full or partial length, is amplified and cloned into a phage or a plasmid.
  • the cDNA can be a partial length of heavy and light chain cDNA, separated or connected by a linker.
  • the antibody, or antibody fragment is expressed using a suitable expression system to obtain recombinant antibody.
  • Antibody cDNA can also be obtained by screening pertinent expression libraries.
  • the antibody or antibody fragment can be bound to a solid support substrate or conjugated with a detectable moiety or be both bound and conjugated as is well known in the art.
  • a solid support substrate or conjugated with a detectable moiety or be both bound and conjugated as is well known in the art.
  • the binding of antibodies to a solid support substrate is also well known in the art. (see for a general discussion Harlow & Lane Antibodi es : A Labora tory
  • the detectable moieties contemplated with the present invention can include, but are not limited to, fluorescent, metallic, enzymatic and radioactive markers such as biotin, gold, ferritin, alkaline phosphatase, /S-galactosidase , peroxidase, urease, fluorescein, rhodamine, tritium, 14 C and iodination.
  • fluorescent, metallic, enzymatic and radioactive markers such as biotin, gold, ferritin, alkaline phosphatase, /S-galactosidase , peroxidase, urease, fluorescein, rhodamine, tritium, 14 C and iodination.
  • a rnimetope can be used as the antigenic source. That is a molecule having an epitope the same or similar to the antigenic determinant (epitopes) of interest may be used as the source of the eliciting antigen.
  • An Aedes aegypti mosquito colony was obtained from the Department of Entomology, University of Manitoba, and maintained in applicants' laboratory.
  • Female mosquitoes were used in the preparation of mosquito whole body, head and thorax, salivary gland, and saliva extracts.
  • Saliva was collected from 3-15 day old adult mosquitoes by placing the proboscis of each mosquito into a capillary tube filled with water. Salivation was induced by applying 0.5 ⁇ l of 0.5% malathion in acetone to the thorax [Boor an, 1987] .
  • the contents of capillary tubes containing saliva were collected, pooled, and lyophilized.
  • the saliva was reconstituted by dissolving the lyophilized proteins in 0.02 M phosphate buffered saline.
  • the protein concentration was 0.6 mg/ml for Ae . vexans extract, 0.4 mg/ml for Cx. quinquefaciatus extract, and 0.3 mg/ml for Ae . aegypti extract as measured by a Bio- Rad Protein Assay kit (Bio-Rad Labs, Richmond, CA) .
  • Skin bite tests and blood samples Skin bite tests were performed with one Ae. vexans mosquito and one Ae. aegypti mosquito on the volar aspect of each subject's forearm as previously described [Peng et al . , 1996] .
  • the wheal and flare circumferences were traced at 20 minutes and 24 hours after the bite, using a felt- tipped pen. All wheal and flare tracings were transferred to transparent paper.
  • the area of the wheal, flare or induration was measured using an IBM- PC (IBM Instruments, Inc., Danbury, Conn.) digitizer and stereometric measurement software [Simons, et al, 1990] .
  • a wheal of less than 0.3 cm 2 with no flare and no itch was considered to be a negative immediate reaction.
  • An induration less than 0.3 cm 2 was considered to be a negative delayed reaction.
  • Mosquitos (Aedes vexans) , collected and identified in the Department of Entomology, University of Manitoba, were anaesthetized by chilling them at 4°C in a refrigerator.
  • Salivary glands were dissected from female mosquitos in 0.02 M phosphate buffered saline (PBS), pH 7.2 under a binocular microscope and immediately transferred to 1 ml of PBS on ice.
  • PBS phosphate buffered saline
  • a total of 370 salivary glands were gathered in 1 ml of PBS, ultrasonicated for 30 seconds, and centrifuged at 8820 g for 15 minutes. The supernatant was collected, aliquoted, and stored at -70°C.
  • the protein concentration of the antigen preparation was 0.6 mg/ml as determined by the Lowry method.
  • Standardization of ELISA results between assays and the estimation of relative amount of mosquito-specific IgE or IgG in each sample was accomplished by using reference IgE and IgG sera. These reference sera were obtained from one subject with a high value of mosquito-specific IgE and another subject with a high value of mosquito-specific IgG and were defined as 1,000 U/ml for mosquito-specific IgE and IgG antibodies, respectively.
  • Polystyrene immunoplates (Nunc-Immuno Plate Maxisorp, Denmark) were coated with mosquito salivary gland extract (0.05 ⁇ g/well) diluted in 0.05 M carbonate buffer (pH 9.6) and incubated overnight at 4°C.
  • the reaction was stopped by addition of 0.1 ml of 1 N NaOH.
  • Optical absorbance at 410 nm was read, using the THERMOmax microplate reader (Molecular Devices, CA, USA) .
  • the value of mosquito-specific antibodies was calculated by interpolation from the dilution curve of the reference serum.
  • IgE and IgG determinations were performed on two dilutions of serum, with each dilution duplicated. 50 samples were assayed twice for mosquito-specific IgE and IgG.
  • Inhibition tests were conducted [Peng et al, 1995] .
  • the mosquito salivary gland extract was 10- fold sequentially diluted in ELISA buffer. Each dilution of the extract was incubated with a diluted serum with high mosquito-specific IgE or high mosquito- specific IgG for 2 hours at 37°C and then followed by 4°C overnight. The final serum dilution was 1 in 20 for the inhibition of IgE and 1 in 200 for that of IgG.
  • Incubation of the serum dilution with ELISA buffer served as a control.
  • Incubations of the diluted serum with 10-fold serial dilutions of grass pollen Hollister-Stier, Miles Canada Inc., Ont .
  • the proteins in the mosquito extracts were separated by SDS-PAGE in a discontinuous system according to Laemmli [1970] using a Bio-Rad slab gel apparatus. For each mosquito extract, fifteen micrograms of proteins prepared in a reducing buffer were loaded onto each well and separated by electrophoresis in 12% SDS-PAGE. These proteins were then electro-transferred onto nitrocellulose membranes. Free binding sites on the membranes were blocked by incubation with 3% bovine serum albumin (Sigma, St. Louis, MO) dissolved in 0.02 M PBS for 2 hours.
  • bovine serum albumin Sigma, St. Louis, MO
  • Immunoblot was completed by incubation of the membranes with a pooled serum exhibiting high mosquito-specific IgE and IgG (1:10 dilution for IgE and 1:50 for IgG) over night. This was followed by incubation with monoclonal anti-human IgE (1:15,000) (ascites, clone No. 7.12, a gift from Dr. A. Saxon, Univ. of California) or monoclonal anti-human IgG (1:15,000) (PharMingen, CA) for 1.5 hours.
  • the membranes were incubated with horseradish peroxidase conjugated goat anti-mouse IgG (1:5,000 dilution for IgE and 1:10,000 for IgG) (Calbiochem Corporation, CA) for 1.5 hours. After washing, the membranes were incubated in ECL detecting reagents (Amersham Life
  • PBS and umbilical cord serum were used respectively to replace the pooled human serum as controls.
  • Pre- stained SDS-PAGE standards Bio-Rad, Richmond, CA) were used to determine the relative molecular weights of the electrophoresed components.
  • Epicutaneous tests and mosqui to bi te tests Epicutaneous tests with the 7 commercial mosquito extracts and the 4 laboratory mosquito preparations were performed on 2 subjects with severe skin reactions to Aedes aegypti bites and on 2 subjects with no skin reaction to the bites. Histamine phosphate (1 mg/ml), saline and 50% glycerin in saline were used as positive and negative controls, respectively.
  • Mosquito bite tests were performed using female mosquitoes (Aedes aegypti) reared in the laboratory. Skin immediate wheal and flare reactions were measured 30 minutes after the tests and skin delayed papule reactions were measured 24 hours later using the largest and the orthogonal diameters of the wheal or the papule, respectively. The area of the wheal or papule was calculated after subtracting the area of the wheal or papule produced by the relevant negative control (if present) .
  • Protein assay Protein concentration of each commercial mosquito extract and laboratory mosquito preparations was determined by a Bio-Rad Protein Assay kit (Bio-Rad Labs, Richmond, CA) .
  • the pooled serum used in immunoblotting was obtained from 6 subjects exhibiting severe skin reactions to mosquito bites and high mosquito-specific IgE and IgG as measured by ELISA. These subjects, including the 2 skin and bite test positive subjects, lived in Manitoba or Texas where the Aedes and/or Culex mosquito species are abundant . In the comparison of commercial mosquito extracts, 10 ⁇ l of each commercial extract, 6 ⁇ l of Aedes aegypti saliva and 10 ⁇ l of Culex quinquefacia tus salivary gland extract were loaded onto different wells.
  • Proteins separated by 12% SDS-PAGE were electrophoretically transferred onto nitrocellulose membranes. Free binding sites on the membranes were blocked by incubation with 3% bovine serum albumin in 0.05 M PBS Tween 20 for 2 hours. Immunoblotting was completed by incubation of the membranes with the pooled serum (1:10 dilution for IgE binding antigens, 1:50 for IgG binding antigens). After washing, this was followed by sequential incubations of the membranes with monoclonal anti -human IgE or monoclonal anti -human IgG (PharMingen, CA) , and HRP-conjugated goat anti- mouse IgG (Calbiochem Corporation, CA) .
  • Epicutaneous tests and protein concentra tions Immediate and delayed reactions were found at the sites of skin tests and mosquito bites in the 2 subjects allergic to mosquito bites.
  • the immediate reaction was a pruritic wheal with a surrounding flare appearing within a few minutes, peaking at 30 minutes and then subsiding.
  • the delayed papules were found several hours later, reaching a peak 24 hours after the epicutaneous and the bite tests. In contrast, there was little or no skin reaction to either the epicutaneous sites or the bite site in the 2 control subjects .
  • the ratio of skin reactions/protein concentration was calculated (Table 2) .
  • the skin reactivity per gram of protein varied from 0.3 to 14.9 in the 7 commercial extracts.
  • the skin reactivity per gram of protein increased significantly .
  • the skin reactivity per gram of protein in saliva (78.0) was less than that found in salivary gland (128.6) because the lyophilization required in the preparation of saliva extract reduced some biological activity of saliva.
  • SDS-PAGE and sil ver stain In the 4 laboratory preparations, rank ordered from whole body, head and thorax, salivary gland to saliva extracts, the amount of salivary antigens significantly increased, while non-salivary proteins and antigens significantly decreased. There were 24 visible protein bands in the head and thorax extract, 16 in the salivary gland extract, but only 9 in the saliva extract. When commercial extracts containing the same amount of protein were used, the protein bands were obscure and the background was very dark.
  • the salivary gland extract of Culex quinquefaciatus was used to compare antigens with the Culex pipiens whole body extracts.
  • IgE and IgG binding antigens varied greatly both in the number of antigen bands and the amount of each antigen. Multiple antigens were found in the commercial extracts. Most of these antigens were not present in the saliva or salivary gland extract.
  • Cl, C2 , C3 contained small amounts of saliva antigens.
  • the IgE and IgG binding antigens in one commercial mosquito whole body extract and 4 laboratory-made mosquito preparations were observed.
  • Whole body and head and thorax extracts contained multiple antigens which were not present in saliva, but they contained few of the antigens present in saliva, although more micrograms of proteins were loaded for these extracts. As the purity of the preparation increased from whole body extract to saliva, the number of proteins and antigens decreased dramatically.
  • mosquito extracts contained multiple proteins and antigens, many of which are unrelated to the antigens in mosquito saliva.
  • the antibodies observed in the human subjects directed against these non-saliva antigens in the commercial extracts may have been induced by inhalation of insect particles or by being bitten by other insects whose antigens cross- reacted with mosquito body components leading to the formation of IgE and IgG antibodies against mosquito body antigens.
  • An in-house reference mosquito extract and a pooled serum for each mosquito genus should be used to evaluate the relative biological activity and the lot-to-lot variation of different batches of mosquito extracts including species specific and nonspecific components.
  • This procedure is not efficient and better means of standardized extracts are required.
  • the present invention provides the use of recombinant salivary allergens to be used as a "recombinant extract" to simplify the need for standardization and to provide greater safety in immunotherapy .
  • mosquito saliva antigens In order to improve diagnosis and immunotherapy of mosquito allergy, purified or recombinant mosquito saliva antigens should be used as shown in Example 1. In order to do so, it should be determined if there are any cross-reactive skin and IgE responses and species- shared antigens among various mosquito species, especially those with world-wide distribution.
  • Ae . vexans, Ae . aegypti , and Cx. quinquefascia tus are the three most important mosquito species distributed globally.
  • Ae . aegypti see generally Christophers, 1960.
  • Aedes aegypti the yellow fever mosquito: its life history, Bionomics and Structure. London: Cambridge University Press
  • Cx. quinquefascia tus see generally Knight and Stone, 1977.
  • a Catalog of the Mosquitoes of the World. 2nd ed . Washington: Ent Soc Am (Thomas Say Found.)) are found throughout the tropical regions of the world within 20°C isotherms, and Ae .
  • vexans is found in North America, Eurasia, Asia, and Africa [Wood et al , 1979] . Comparison of the human immunological response to the three species has never been made. Because of climate, Ae . aegypti and Cx. quinquefascia tus are not present in Canada, while Ae . vexans is the major pest in Canada representing up to 80% of the local mosquito population. In order to determine whether the three mosquito species have cross-reactive immunological responses and species-shared antigens, skin bite tests were performed and serum mosquito specific IgE was evaluated for the three mosquito species in 41 Manitobans who had been exposed to Ae . vexans bites, but not to Ae.
  • Mosquitoes, mosquito saliva and salivary gland extracts Female Ae . vexans mosquitoes were collected in local fields and identified by scientists in the
  • ELISA Serum mosquito-specific IgE to Ae.
  • vexans, Ae . aegypti , and Cx . quinquefaciatus were measured by an indirect ELISA as described herein above.
  • Optimal conditions for dilutions of the 3 mosquito extracts, serum samples, goat anti-human IgE, and conjugated rabbit anti -goat IgG were chosen by checkerboard titration. Standardization of ELISA results between assays and estimation of the relative amount of mosquito-specific IgE in each sample was accomplished by using reference sera as described.
  • the reference serum used to measure Ae. aegypti -IgE and Cx . quinquefaciatus- IgE was obtained from a subject with systemic reactions to mosquito bites (kindly provided by Dr.
  • Another reference serum used to measure Ae . vexans- IgE came from a Manitoban with severe skin reactions to mosquito bites (immediate wheal 1.5 cm 2 and flare 11.6 cm 2 ) . Both reference sera were defined as 1000 U/ml for mosquito-specific IgE.
  • Microplates coated with mosquito saliva or salivary gland extract (0.02 - 0.05 ⁇ g/well) were sequentially incubated with serum samples (1:20) or reference serum (2-fold dilutions from 1:20 to 1:10,240), 1:1,000 goat anti-human IgE (P.S. myeloma-affinity purified, a gift from Dr. N.F.
  • vexans-specific IgE levels (> 1,000 U/ml) were pooled and used for immunoblotting. SDS-PAGE and immunoblotting were performed as described herein above and in Peng et al . , 1996. In the analysis of IgE and IgG antigens, two ⁇ g of the proteins from each mosquito extract were loaded and electrophoresed in 12% acrylamide SDS-PAGE under reducing conditions. Proteins separated by SDS-PAGE were electrophoretically transferred onto nitrocellulose membranes.
  • Mosquito-specific IgE levels The geometric mean Ae . vexans- IgE, Ae . aegypti -IgE, and Cx . quinquefaciatus- IgE were all significantly higher in the subjects with immediate skin reactions to Ae . vexans bites than in those with no immediate skin reaction to the bites (p's
  • the immunologic basis for the reactive skin and IgE responses among different mosquito species is the existence of species-shared antigens which are based on their identical protein sequences. Salivary secretions have been demonstrated to be directly responsible for skin reactions to mosquito bites. In the present Example, immunoblot analysis using saliva or salivary gland extracts, a number of species-shared antigens and several Ae. vexans- specific antigens were found.
  • Reactions to mosquito bites are generally caused by immunologic mechanisms, with both type I (IgE- mediated) and type IV (cell-mediated) hypersensitivities being involved [Oka K, 1989; Peng et al, 1996; Reunala et al, 1994a; 1994b].
  • Serum mosquito- specific IgE has been demonstrated to correlate with cutaneous mosquito bite reactions [Oka K, 1989; Peng et al, 1996] .
  • Mosquito-specific IgG has also been found to correlate with skin mosquito bite reactions, suggesting that IgG may also be involved in the development of mosquito allergy [Peng et al, 1996] .
  • Mosquito antigens have been identified by immunoblot analysis. A number of mosquito antigens with molecular masses ranging from 14 to 126 kDa have been reported in various mosquito species [Penneys et al, 1989; Shen et al . , 1989; Wu and Lan, 1989; Brum er-Korvenkontio, 1990, 1994] . In this Example antigens are analyzed using immunoblot techniques on three mosquito species not previously examined; Aedes (Ae . ) vexans, a globally distributed species (and the major pest species in Manitoba) , and two North American species Culex (Cx. ) tarsalis and Culiseta (Cs . ) inorna ta .
  • a pooled serum from reactive subjects was used in the analysis of antigens in the three mosquito species.
  • Mosquito head and thorax extracts Extracts prepared from mosquito heads and thoraxes were used in the study.
  • Female mosquitoes of the three species (Ae. vexans, Cx. tarsalis , Cs . inornata) were collected and identified by the Insect Control Branch, Parks and Adventure Department, City of Winnipeg, and then stored at -70°C.
  • Inhibition test To examine the specificity of the immunoblot analysis, an antigen inhibition test was performed. Prior to immunoblotting, the pooled serum with high mosquito-specific IgE and IgG was incubated with Ae . vexans head and thorax extract with a final dilution of 1:2, 1:20 or 1:200 at 4°C overnight. Incubation of the serum with PBS served as a positive control. SDS-PAGE and immunoblotting for IgE and IgG binding antigens was then performed with these preincubated serum samples. After incubation with mosquito extract, both IgE and IgG binding bands were significantly reduced compared to the positive control. This inhibition exhibited dose-dependency demonstrates that the immunoblot analysis of IgE and IgG binding antigens is specific to mosquito antigens.
  • Antigens in the three mosqui to species IgE and IgG antibodies bound to various mosquito antigens in the three species. Twelve antigens in Ae . vexans, 16 antigens in Cx. tarsalis , and 14 antigens in Cs . inornata, with molecular masses ranging from 18.5 to 160 kDa, were found by immunoblot analysis (Table 4) . Most of the antigens bound to both IgE and IgG, and were shared by species. Nine antigens (24, 32.5, 40, 46, 50, 62, 65, 110, 160 kDa) were shared by 3 species, especially the 40 kDa antigen.
  • extracts of one globally distributed mosquito species (Aedes vexans) and two North American species ( Culex tarsalis and Culiseta inorna ta) were prepared from heads and thoraxes. Proteins of the 3 extracts were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes for immunoblotting. Immunoblotting was completed by sequential incubations of the membranes with a pooled human serum from subjects allergic to mosquito bites, monoclonal anti- bodies to human IgE or IgG, and goat anti-mouse IgG conjugate. Twelve to sixteen antigens with molecular masses ranging from 18.5 to 160 kDa were found in each extract.
  • IgE and IgG antibodies against Aedes vexans were studied by immunoblotting using individual serum from subjects with or without skin reactions to Aedes vexans bites. All 3 subjects with severe skin reactions had strong IgE and IgG antibodies to 32.5, 40, and 50 kDa proteins. The patterns and magnitudes of IgE and IgG antibodies to the antigens varied among individuals. Very faint IgE antibodies to these antigens were found in the 2 subjects with no skin reactions, suggesting that IgE plays a role in the development of mosquito allergy.
  • ⁇ -galactosidase part of the fusion protein is 114 kDa
  • the cDNA coding part ranged from 10 to 60 kDa.
  • Three clones with different size of cDNA inserts were subcloned into pBluescript II SK vector (Stratagene, La Jolla, CA, USA) and sequenced by the Sanger method [1977] using a US Biochemicals sequencing kit (Cleveland, Ohio, USA) .
  • PCR was designed to clone the 5' terminal fragment from the cDNA library using the lambda gtll forward primer (5' GACTCCTGGAGCCCG 3', Clontech; SEQ ID No:2) and a synthesized 3' primer (3 'ATATCTGTCCACCAACG 5 ' ; SEQ ID No: 3) complementary to a sequence of the 3' terminal fragment.
  • PCR was performed in 100 ⁇ l of the sample containing 0.5 ⁇ g of library DNA, 10 ⁇ l of lOx buffer, 2 ⁇ l of 25 mM dNTP's, 2 ⁇ l of 100 ng/ ⁇ l each primer, 2 U of Taq polymerase supplied by the PCR kit (Boehringer Mannheim Canada, Quebec) .
  • the reaction was subjected to 25 cycles of amplification consisting of 1 minute at 94°C, 1 minute at 55°C and 1 minute at 72°C, with a final 72 °C extension for 7 minute.
  • the PCR product was cloned into the TA vector (Invitrogen, San Diego, CA, USA) . Three PCR clones were obtained and sequenced. The sequences of the 3 clones were found to be identical and overlapped AA22 cDNA.
  • AA22 cDNA In the search for an initiation codon in the same open reading frame as AA22 cDNA, an ATG was found, which was characterized with an adenosine at the crucial -3 position of the Kozak consensus sequence, A/GXXXATG, for initiation of translation by eukaryotic ribosomes [Kozak, 1987] .
  • the sequence flanking the putative translational start sites, GAAAATG is very similar to the consensus sequence, C/AAAA/CATG, for initiation of Drosophila , an insect gene (Cavener, 1987] .
  • the full-length cDNA is 0.85 kb, coding for a protein of 253 amino acid residues, approximately 30 kDa.
  • Fig. 1 many bands were revealed in the immunoblotting with mouse anti-saliva serum (lane 1) .
  • lane 1 mouse anti-saliva serum
  • a 30 kDa protein was specifically revealed (Fig. 1, lane 2).
  • the fusion protein-selected mouse antibodies recognize the 30 kDa saliva protein, indicating that this cDNA clone encodes the 30 kDa saliva protein.
  • the 30 kDa saliva protein of mosquito Aedes aegypti elicited an IgE response in 42% of mosquito-allergic subjects and in none of the subjects without skin reactions to the bites.
  • the fusion protein-selected human IgE strongly bound to the 30 kDa native protein (lane 2) , suggesting that the cDNA isolated codes the 30 kDa salivary allergen which induces a specific IgE response in mosquito-allergic humans.
  • This IgE-binding protein is the third salivary allergen of Aedes aegypti whose cDNA has been cloned and sequenced.
  • the protein is rich in glutamic acid residues (16.5% of amino acid residues), and has a hydrophobic amino terminal region characteristic of a secretory signal peptide [Hopp et al . , 1981; Kyte et al . , 1982].
  • Aed a 1 a 68 kDa mosquito Aedes aegypti salivary protein, is an allergen which binds to the IgE of mosquito-allergic subjects.
  • an expressed, purified recombinant Aed a 1 a 1
  • rAed a 1 was characterized to determine if it bound to antibodies directed to the native protein. Additionally, responses to it in mosquito-allergic subjects was investigated to determine its biologic activity, that is activity in vivo to elicit an immune response .
  • Two cDNA segments were ligated together forming the full-length Aed a 1 gene, which was inserted into the baculovirus expression vector pBlueBacHis C.
  • Recombinant baculoviruses were generated by co- infection of Sf9 insect cells with wild-type baculovirus AcMNPV DNA and the recombinant vector.
  • This Example demonstrates that the recombinant Aed a 1 expressed by the baculovirus system has the same antigenicity and biological activity as the native Aed a 1 present in mosquito saliva and is a major salivary allergen of Ae. aegypti .
  • Mosquito salivary proteins cause allergic reactions in humans.
  • Aed a 2 a 37 kDa mosquito Aedes aegypti salivary protein, is an allergen which binds to the IgE of mosquito-allergic subjects.
  • an expressed, purified recombinant Aed a 2 (rAed a 2) , was characterized to determine if it bound to antibodies directed to the native protein. Additionally, responses to it in mosquito-allergic subjects was investigated to determine its biologic activity, that is activity in vivo to elicit an immune response.
  • Sf9 insect cells were co-infected with the transfer vector pVL1392/Aed a 2 DNA and wild- type baculovirus.
  • Aed a 2 was also shown to be a species-shared allergen, being present in the saliva or salivary gland extracts of 6 Aedes and one Cul ex species among the 12 species studied (Fig. 8) .
  • Aed a 2 expressed by the baculovirus system has identical antigenicity and biological activity with native Aed a 2 present in mosquito saliva and that Aed a 2 is a common allergen shared by Aedes genus and other species.
  • saliva or salivary gland extracts were prepared as described herein above from ten mosquito species including seven species with worldwide distribution. These species are Ae . aegypti , Ae . vexans, Ae . albopi ctus , Ae . togoi , Ae . triseria tus, Cx. quinquefasciatus , Cx. pipiens, Cx. tarsalis , An . sinensis and Cs . inorna ta . Proteins from the mosquito preparations were separated by SDS-PAGE and then transferred to nitrocellulose membranes.
  • the membranes were immunoblotted by sequential incubations of the membranes with human serum, monoclonal anti -human IgE, and enzyme-conjugated goat anti-mouse IgG.
  • Salivary allergens were analyzed using a pooled serum from mosquito-allergic subjects.
  • Fig. 9 Three to 16 salivary allergens with molecular masses ranging from 16 to 95 kDa were found in each species (Fig. 9). Both species-shared and species- specific allergens were identified by molecular masses, binding to the 2 rabbit antibodies, and the individual IgE responses to species which were not indigenous to the areas where the subjects lived. As shown in Fig. 10, the existence of species- shared allergens was confirmed by immunoblot using rabbit anti-Aed a 1 and Aed a 2 antibodies, respectively. A 68 kDa allergen which was recognized by rabbit anti-Aed a 1 was found not only in the Ae . aegypti extract (Fig. 10A strip #1) but also in the extracts of Ae .
  • Species-specific allergens also existed as evidenced by a 23 kDa Ae . vexans allergen which reacted only with the sera from Canada where Ae . vexans is abundant .
  • Salivary allergens elicited higher IgE responses in mosquito-allergic subjects than in non-allergic subjects.
  • Three major Aedes species (Ae . aegypti , Ae . vexans, Ae . albopictus) had a higher number of allergens which also elicited stronger IgE responses, suggesting that they are major biting species.
  • GGC AAA GAG GAG AAT ACA GGA CAT GAG GAT GCT GGT GAG GAA 251 G K E E N T G H E D A G E E
  • Peng et al 1996b. Allergens and antigens in extracts of Aedes vexans, Culiseta inornata, and Culex tarsalis . Int Arch Allergy Immunol 110:46-51. Penneys et al , 1989. Mosquito salivary gland antigens identified by circulating human antibodies. Arch Dermatol 125:219-222.
  • ORGANISM Aedes aegypti

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Abstract

L'invention concerne un allergène salivaire recombiné de moustique, à utiliser dans des tests dermatologiques, des dosages immunologiques et un traitement antiallergique des réactions allergiques aux piqûres de moustiques. L'allergène de recombinaison est produit par un ADNc codant pour une protéine de fixation d'IgE, un fragment ou un analogue de celle-ci que l'on trouve dans la salive du moustique. L'allergène recombiné partager une allergénicité commune avec au moins deux espèces de moustique ou peut être spécifique à une espèce. L'invention concerne également un procédé d'essai dermatologique, un dosage immunologique et une immunothérapie dans laquelle l'allergène salivaire du moustique est utilisé ainsi que des kits permettant la mise en oeuvre des procédés selon l'invention. Ladite invention se rapporte également à des anticorps dirigés contre les allergènes salivaires recombinés.
PCT/US1997/013573 1996-07-31 1997-07-31 Allergenes salivaires du moustique Ceased WO1998004274A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999025826A1 (fr) * 1997-11-13 1999-05-27 University Of Manitoba Methode permettant de determiner de violentes reactions inflammatoire aux piqures de moustiques et necessaire correspondant
WO2001077353A3 (fr) * 2000-04-05 2002-04-11 Syngenta Participations Ag Proteines therapeutiquement actives dans des vegetaux
WO2009009493A3 (fr) * 2007-07-09 2009-02-26 Us Gov Health & Human Serv Aégyptine et utilisations de celle-ci
WO2022093899A1 (fr) * 2020-10-28 2022-05-05 BioVaxys Inc. Procédé et kit pour la détection d'une réponse immunitaire à médiation cellulaire

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
INT. ARCH. ALLERGY APPL. IMMUNOL., 1990, Vol. 93, BRUMMER-KORVENKONTIO et al., "Immunization of Rabbits with Mosquito Bites: Immunoblot Analysis of IgG Antimosquito Antibodies in Rabbits and Man", pages 14-18. *
INT. ARCH. ALLERGY IMMUNOL., 1994, Vol. 104, REUNALA et al., "Frequent Occurence of IgE and IgG4 Antibodies Against Saliva of Aedes Communis and Aedes Aegypti Mosquitoes in Children", pages 366-371. *
THE JOURNAL OF DERMATOLOGY, 1994, Vol. 21, OHTAKI et al., "Quantitative Study of Specific Immunoglobulins to Mosquito Salivary Gland Antigen in Hypersensitive and Common Types of Mosquito Bite Reaction", pages 639-644. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999025826A1 (fr) * 1997-11-13 1999-05-27 University Of Manitoba Methode permettant de determiner de violentes reactions inflammatoire aux piqures de moustiques et necessaire correspondant
WO2001077353A3 (fr) * 2000-04-05 2002-04-11 Syngenta Participations Ag Proteines therapeutiquement actives dans des vegetaux
WO2009009493A3 (fr) * 2007-07-09 2009-02-26 Us Gov Health & Human Serv Aégyptine et utilisations de celle-ci
US8383589B2 (en) 2007-07-09 2013-02-26 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Aegyptin and uses thereof
US8980859B2 (en) 2007-07-09 2015-03-17 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Aegyptin and uses thereof
US9441022B2 (en) 2007-07-09 2016-09-13 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Aegyptin and uses thereof
WO2022093899A1 (fr) * 2020-10-28 2022-05-05 BioVaxys Inc. Procédé et kit pour la détection d'une réponse immunitaire à médiation cellulaire

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