GB2455108A - T-Cell dependent method for detecting non-allergic or intrinsic disorders - Google Patents

T-Cell dependent method for detecting non-allergic or intrinsic disorders Download PDF

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Publication number
GB2455108A
GB2455108A GB0723338A GB0723338A GB2455108A GB 2455108 A GB2455108 A GB 2455108A GB 0723338 A GB0723338 A GB 0723338A GB 0723338 A GB0723338 A GB 0723338A GB 2455108 A GB2455108 A GB 2455108A
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Prior art keywords
allergen
individual
peptide
condition
cells
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GB0723338D0 (en
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Rod Hafner
Mark Larche
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Circassia Ltd
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Circassia Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases

Abstract

An in vitro method of determining whether an individual has or is at risk of a condition classified as a non-allergic or intrinsic disorder is disclosed, the method comprising testing whether the individual has T cells which respond to an allergen, thereby determining whether the individual has or is at risk of the condition, and additionally identifying the allergen as a trigger for the condition. The condition may be intrinsic (non-allergic) asthma or intrinsic (non-allergic) rhinitis, and the allergen may be Fel d1, Der p1, Der p2, Der p7, Amb a 1, Lol p1, or Lol p5, among others. Methods of treatment of individuals suffering from the aforementioned conditions are also claimed.

Description

-2455108
PROGNOSTIC ASSAY
Field of the Invention
The invention relates to a prognostic method.
Background to the Invention
Diagnosis of allergic disease typically focuses on detecting the presence of allergen-specific IgE antibodies in a patient, and thereby identifying the causative allergen and enabling appropriate measures to treat the disease. A number of methods are commonly used to detect allergen-specific IgE, including skin prick testing with solutions of allergen extracts, or radioallergosorbent test (RAST) following challenge with inhaled allergen.
However, various disorders such as intrinsic asthma and intrinsic rhinitis are defined by the absence of allergen-specific IgE. For example, upto 40% of adult (late-onset) asthmatics in the USA have instrinsic or so-called non-allergic asthma.
The symptoms of these patients ase equivalent to those of allergic asthmatics, and the biochemical and histological effects of the disorders are highly similar.
As with equivalent allergic disorders, management of intrinsic or non-allergic disorders which are provoked by environmental agents requires accurate trigger identification to control symptoms. However, as described above, the main method available for the identification of triggers of allergy symptoms is not applicable to intrinsic disorders since no allergen-specific IgE is found.
Accordingly, there is a need for a diagnostic method which can determine the provocation agent responsible for intrinsic or non-allergic disorders.
Summary of the Invention
The pathogenesis of intrinsic or non-allergic disorders. is poorly understood.
Whilst the absence of allergen-specific IgE argues for a non-immunological cause, the infiltration of tissues with eosinophils and the upregulation of cytokines such as IL-4 and IL-5 suggests that a non-IgE mediated immune response is taking place.
Accordingly it is necessary to determine whether patients are responding to environmental proteins, for example allergens, in an IgE independent manner. The
S
present inventors have found that subjects exhibit T cell responses to particular peptide epitopes derived from various allergens. Accordingly, by determining T cell responses to such peptide epitopes, there is provided a method to identify individuals at risk of a reaction to allergen which does not rely on the detection of allergen-specific IgE.
Thus, the present invention provides an in Vitro method of determining whether an individual has or is at risk of a condition wherein the condition is classified as a non-allergic or intrinsic disorder, the method comprising testing whether the individual has I cells which respond to an allergen, thereby determining whether the individual has or is at risk of the condition, and additionally identifying the allergen as a trigger for the condition.
Description of the Figures
Figure 1 -Sequence comparison of Der p 1 versus Der f 1 (Fig IA), Der p 2 versus Der f 2 (Fig I B) and Der p 7 versus Der f 7 (Fig 1 C). Regions containing epitopes are highlighted in grey. Locations of specific peptides of the invention are indicated by lines above or below the sequence.
Figure 2 -Peptides derived from Fel dl chains I and 2 were tested for ability to bind to multiple DR types in MHC class II binding assays. Peptides that showed promiscuous binding characteristics were selected and combined to generate a mixture of peptides that bind to a broad population of MHC class II types.
Figure 3 -Percentage of responders by cytokine. Figure 3 summarises the percentage of individuals who mounted a detectable response to each of the peptides/antigens by production of the three cytokines measured. The positive control antigen PPD elicited a cytokine production in almost all individuals (IFN-y: 91%, IL-13: 97% and IL-lO: 96%). Whole cat allergen and the mixture of 7 peptides elicited a cytokine response in approximately 80% or more of subjects. Individual peptides elicited responses of differing frequency. In general cytokine production appeared to be a more sensitive method of detecting responses with larger percentages of individuals giving positive cytokine responses than proliferative responses. In most cases, IL-I 0 secretion was detected in the largest number of subjects and IFN-'y detected least frequently. O -3-
Figure 4 -Percentage of individuals producing IFN-'y and strength of response following cell culture with peptide/antigen. [FN-1 responses were detected in 26-44% of subjects in response to individual peptides. These responses were predominantly very low to low to moderate. Complex antigens induced more frequent responses (peptide mixture 80%, cat dander 79%, PPD 9 1%). These responses were low to moderate to high. PPD responses were particularly high (89 of PPD responses were above 1 OOpg/ml).
Figure 5 -Percentage of individuals producing IL-13 and strength of response following cell culture with peptide/antigen. IL-13 responses were detected in between 33-68% of subjects in response to individual peptides. These responses were predominantly very low to low, although a significant number of moderate responses were detected. This may reflect the Th2 nature of allergic sensitisation in these subjects. Complex antigens induced more frequent responses (jeptide mixture 85%, cat dander 93%, PPD 97%). These responses were low to moderate to high.
Figure 6 -Percentage of individuals producing IL-10 and strength of response following cell culture with peptide/antigen. IL-lO responses were detected in between 46-75% of subjects in response to individual peptides. These responses were predominantly very low to low. Complex antigens induced more frequent responses (peptide mixture 93%, cat dander 96%, PPD 96%). These responses were low to moderate. Very few "high" IL- 10 responses were observed.
Description of the Sequences
SEQ ID NOS: 1 to 65 show the peptides of the invention
Detailed Description of the Invention
The invention provides a method of detecting whether an individual has or is at risk of developing a disorder, wherein the disorder is classified as intrinsic or non-allergic.
The individual is typically a mammal, preferably a human. The individual to be tested in the method is preferably between the ages of 1 year and 80 years, more S -4-preferably between the ages of 1 year and 60, 50, 40, 30 or 20 years, and most preferably between the ages of 1 year and 16 years.
The individual may have been diagnosed or may be suspected of suffering from a disorder which is classified as intrinsic or non-allergic, for example, intrinsic or non-allergic asthma, or intrinsic or non-allergic (perennial) rhinitis. The individual typically lacks a detectable antibody response to an allergen, in particular an IgE response to allergen. Suitable assays to detect IgE include the PharmaciaTM CAP system. Using this system, the individual typically scores 0 or 0/1.
The individual may be a patient suffering from or diagnosed as suffering from symptoms which are typically associated with allergy such as itchy eyes, runny nose, breathing difficulties, red itchy skin or rash, in the absence of an identifiable trigger. The first occurrence or diagnosis of these symptoms may occur when the individual is older than 15 years of age. For example, the individual may be at least 15, 16, 17, 18, 20, 22, 24, 26, 28 or 30 years of age at the first occurrence or diagnosis of symptoms of allergy which are typically associated with allergy.
The method of the invention concerns determining whether an individual has a T cell response to particular allergens. Without being bound by any hypothesis, the inventors consider that the intrinsic or non-allergic disorders are in fact caused by a T cell-driven, IgE independent, immune response. Accordingly these disorders do have an allergen trigger, but it does not give rise to allergen-specific IgE. Rather, it gives rise to a T cell response which can be characterised by the release of cytokines.
For example, the cytokines released may include IL-5, which is involved in the recruitment of eosinophils. Accordingly, the T cell response can drive the induction of eosinophilic reactions in an individual.
Whether an individual has a T cell response to a particular allergen may be determined by any suitable method, typically a method which can be used to detect the presence of cytokine released by allergen-experienced T cells. A positive response by the patient's T cells to the allergens indicates that the patient has or is more likely to develop allergy-like symptoms in response to the allergen. A negative response indicates that the patient has allergy-like symptoms which are not caused by the allergen, or is less likely to develop allergy-like symptoms in response to the allergen. *
The I cells which respond to the allergen in the method are generally T cells which have been pre-sensitised in vivo to allergen. These allergen-experienced I cells are generally present in the peripheral blood of a individual, i.e. within the population of peripheral blood mononuclear cells (PBMCs) in the individual. The T cells may be CD4 and/or CD8 T cells.
in the method the T cells can be contacted with the allergen in vitro or in vivo, preferably in vitro in a sample from the individual.
Generally the T cells which are contacted in the method are taken from the individual in a blood sample, although other types of samples which contain T cells can be used. The sample may be added directly to the assay or may be processed first. Typically the processing may comprise standard techniques such as gradient centrifugation to separate the T cells, with resuspension in any suitable volume.
Alternatively, the processing may comprise diluting of the sample, for example with water, buffer or media. The sample may be diluted from 1.5 to 100 fold, for example 2to50or5tolOfold.
The processing may comprise separation of components of the sample.
Typically mononuclear cells (MCs) are separated from the samples. The MCs will comprise the T cells and antigen presenting cells (APCs). Thus in the method the APCs present in the separated MCs can present the peptide to the T cells. In another embodiment only T cells, such as only CD4 T cells, can be purified from the sample.
PBMCs, MCs and T cells can be separated from the sample using techniques known in the art, such as those described in Lalvani eta! (1997) J.Exp. Med. .i.., p859-865.
Preferably the T cells used in the assay are in the form of unprocessed or diluted samples, are freshly isolated T cells (such as in the form of freshly isolated MCs or PBMCs) which are used directly ex vivo, i.e. they are not cultured before being used in the method or are thawed cells (which were previously frozen).
However the T cells can be cultured before use, for example in the presence of the allergen, and generally also exogenous growth promoting cytokines. During culturing the allergen is typically present on the surface of APCs, such as the APC used in the method. Pre-culturing of the T cells may lead to an increase in the sensitivity of the method. Thus the T cells can be converted into cell lines, such as short term cell lines (for example as described in Ota eta! (1990) Nature p183-187).
The APC which is typically present in the method may come from the same individual as the T cell or from a different individual. The APC may be a naturally occurring APC or an artificial APC. The APC is a cell which is capable of presenting the antigen to a T cell. It is typically a B-cell, dendritic cell or macrophage. It is typically separated from the same sample as the T cell and is typically co-purified with the T cell. Thus the APC may be present in MCs or PBMCs. The APC is typically a freshly isolated ex vivo cell or a cultured cell. It may be in the form of a cell line, such as a short term or immortalised cell line. The APC may express empty MHC class II molecules on its surface.
In one embodiment the allegen is added directly to an assay comprising T cells and APCs. As discussed above the T cells and APCs in such an assay could be in the form of MCs. When an allergen which can be recognised by the T cell without the need for presentation by APCs is used then APCs are not required. Analogues which mimic the original allergen bound to a MHC molecule are an example of such an antigen.
In one embodiment the allergen is provided to the APC in the absence of the T cell. The APC is then provided to the T cell, typically after being allowed to present the allergen on its surface. The allergen may have been taken up inside the APC and presented, or simply be taken up onto the surface without entering inside the APC.
Typically iO to i07, preferably 2.5x i� to 106 PBMCs are added to each assay. In the case where allergen is added directly to the assay it is typically added as a peptide with a concentration from 10' to 103tgIml, preferably 0.5 to 50j.tg/ml or I to l0.tg/ml.
Typically the length of time for which the T cells are incubated with the allergen is from 4 to 24 hours (preferably 5 to 18 hours) for effector T cells or for more than 24 hours for central memory cells. When using ex vivo PBMCs it has been found that 5.0 x106 PBMCs can be incubated in l0tg/ml of peptide for 5 hours at 3 7°C.
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The allergen may be in any suitable form. The allergen generally comprises one or more T cell epitopes from an allergen, or a variant thereof. Classes of suitable allergens include, but are not limited to, pollens, animal dander (in particular cat dander), grasses, molds, dusts, antibiotics, stinging insect venoms, and a variety of environmental (including chemicals and metals), drug and food allergens. Common tree allergens include pollens from cottonwood, popular, ash, birch, maple, oak, elm, hickory, and pecan trees; common plant allergens include those from mugwort, ragweed, English plantain, sorrel-dock and pigweed; plant contact allergens include those from poison oak, poison ivy and nettles; common grass allergens include rye grass, Timothy, Johnson, Bermuda, fescue and bluegrass allergens; common allergens can also be obtained from molds or fungi such as Alternaria, Fusarium, Hormodendrum, Aspergillus, Micropolyspora, Mucor and thermophilic actinomycetes; epidermal allergens can be obtained from house or organic dusts (typically fungal in origin), from arthropods such as house mites (Dermatophagoides pteronyssinus), or from animal sources such as feathers, and dog dander; common food allergens include milk and cheese (diary), egg, wheat, nut (e.g., peanut), seafood (e.g., shellfish), pea, bean and gluten allergens; common environmental allergens include metals (nickel and gold), chemicals (formaldehyde, trinitrophenol and turpentine), Latex, rubber, fiber (cotton or wool), burlap, hair dye, cosmetic, detergent and perfume allergens; common drug allergens include local anesthetic and salicylate allergens; antibiotic allergens include penicillin, tetracycline and sulfonamide allergens; and common insect allergens include bee, wasp and ant venom, and cockroach calyx allergens. Particularly well characterized allergens include, but are not limited to, the major allergen produced by the domestic cat Fe/is catus (Fe/is domesticus) glycoprotein Fel dl, the major and cryptic epitopes of the Der p I allergen (Hoyne et al. (1994) Immunology 83190-195), bee venom phospholipase A2 (PLA) (Akdis et al. (1996) J. Clin. Invest. :1676-l683), birch pollen allergen Bet v I (Bauer et al. (1997) C/in. Exp. Immunol. j2:536-541), and the multi-epitopic recombinant grass allergen rKBG8.3 (Cao et al. (1997) Immunology Q:46-5 1). These and other suitable allergens are commercially available and/or can be readily prepared as extracts following known techniques.
Preferably, the allergen is selected from the list of allergen sequences and database accession numbers (NCBI Entrez accession numbers) below. NCBI is the National Center for Biotechnology information and is a division of the US National Institutes of Health. The NCBI web site, from which access to the database may be sought, is www.ncbi.nlm.nih.gov/. Allergen sequences and database accession numbers (NCBI Entrez accession numbers): House dust mite Dermatophagoides pteronyssinus Derp 1
MKIVLAIASLLALSAVYARPSSIKTFEEYKKAFNKSYATFEDEEAARKNFLES
VKYVQSNGGAJNHLSDLSLDEFKNRFLMSAEAFEHLKTQFDLNAETNACSIN
is GNAPAEIDLRQMRTVTPIRMQGGCGSCWAFSGVAATESAYLAYRNQSLDLA
EQELVDCASQHGCHGDTIPRGIEYIQHNGVVQESYYRYVAREQSCRRPNAQ
RFGISNYCQIYPPNVNKIREALAQTHSAIAVJIGIKDLDAFRHYDGRTIIQRDN
GYQPNYHAVNIVGYSNAQGVDYWJVRNSWDTNWGDNGYGYFAANIDLM
MIEEYPYVVIL Derp2
MMYKJLCLSLLVAAVARDQVDVKDCANHEIKKVLVPGCHGSEPCIIHRGKPF
QLEAVFEANQNTKTAKIEIIKASIDGLEVDVPGIDPNACHYMKCPLVKGQQYD
IKYTWNVPKIAPKSENVVVTVKVMGDDGVLACAIATHAKIRD
Derp 3
MIIYNILIVLLLAINTLANPILPASPNATIVGGEKALAGECPYQISLQSSSHFCG
GTILDEYWILTAAHCVAGQTASKLSIRYNSLKHSLGGEKJSVAKIFAHEKYDS
YQIDNDIALrKLKSPMKLNQKNAKAVGLPAKGSDVKVGDQVRVSGWGYLE
EGSYSLPSELRRVDIAVVSRKECNELYSKANAEVTDNMJCGGDVANGGKDS
CQGDSGGPVVDVKNNQVVGIVSWGYGCARKGYPGVYTRVGNFIDWIESKR
SQ
S
Der p4
KYXNPHFIGXRSVITXLME Derp5
MKFHAFFVATLAVMTVSGEDKKHDYQNEFDFLLMERIIHEQII(KGELALFYL QEQINHFEEKPTKEMKDKWAEMDTIIAMJDGVRGVLDRLMQRJ(DLDIFEQY
NLEMAKKSGDILERDLKKEEARVKKIEV Derp6
AIGXQPAAEAEAPFQISLMK
Derp 7
MMKLLLIAAAAFVAVSADPIHYDKITEEJNKAVDEAVAAIEKSETFDPMKVP
DHSDKFERHIGIIDLKGELDMRNIQVRGLKQMKRVGDANVKSEDGVVKAHL
LVGVHDDVVSMEYDLAYK.LGDLHPNTHVISDIQDFVVELSLEVSEEGNMTL TSFEVRQFANVVNHIGGLSILDPIFAVLSDVLTA[FQDTVRAEMTKVLAPAFK
KELERNNQ Derp9
IIVGGSNASPGDAVYQIAL
Dermatophagoides farinae Derfl MKFVLAIASLLVLTVYARPASIKTFEFKKAFNKNYATVEEEEVARI(NFLESL KYVEANKGAINHLSDLSLDEFKNRYLMSAEAFEQLKTQFDLNAETSACRmJS
VNVPSELDLRSLRTVTPIRMQGGCGSCWAFSGVAATESAYLAYRNTSLDLSE
QELVDCASQHGCHGDTllRGIEYIQQNGVVEERSYPYVAREQRCRRPNSQHY
GISNYCQIYPPJDVKQIREALTQTHTAIAVIIGIKDLRAFQHYDGRTIIQHDNGY
QPNYHAVNIVGYGSTQGDDYWIVRNSWDTTWGDSGYGYFQAGNNLMMIE
QYPYV VIM
S
Der f2
MISKILCLSLLVAAVVADQVDVKDCANNEIKKVMVDGCHGSDPCIIHRGKPF
TLEALFDANQNTKTAJUEH(ASLDGLEIDVPGIDTNACHFMKCPLVKGQQYD1
KYTWNVPKIAPKSENVVVTVKLIGDNGVLACAIATHGKIRD
Der f 3
MMILTIVVLLAANILATPILPSSPNATIVGGVKAQAGDCPYQISLQSSSHFCGG
SILDEYWILTAAHCVNGQSAKKLSIRYNTLKHASGGEKIQVAEIYQHENYDS
MTIDNDVALIKLKTPMTLDQTNAKPVPLPAQGSDVKVGDK1RVSGWGYLQE
GSYSLPSELQRVDIDVVSREQCDQLYSKAGADVSENMICGGDVANGGVDSC
QGDSGGPVVDVATKQIVGIVSWGYGCARKGYPGVYTRVGNFVDWIESKRS
Q Derf4
AVGGQDADLAEAPFQISLLK
Der f 7
MMKFLLIAAVAFVAVSADPIHYDKJTEEINKAIDDAIAAIEQSETIDPMKVPD
HADKFERHVGIVDFKGELAMRNIEARGLKQMKRQGDANVKGEEGIVKAHL
LIGVHDDJVSMEYDLAYKLGDLHPTTHVISDIQDFVVALSLEISDEGNITMTSF
EVRQFANVVNHIGGLSILDPIFGVLSDVLTAIFQDTVRKEMTKVLAPAFKREL
EKN
Additional mite allergen sequences (NCBI entrez accession): 1170095; 1359436; 2440053; 666007; 487661; 1545803; 84702; 84699; 625532; 404370; 1091577; 1460058; 7413; 9072; 387592. Cat
Felis sequences (NCBI entrez accession):
S
539716; 539715; 423193; 423192; 423191; 423190; 1364213; 1364212; 395407; 163827; 163823; 163825; 1169665; 232086; 1169666. Latex
Hevea sequences: Hey b 1
MAEDEDNQQGQGEGLKYLGFVQDAATYAVTTFSNVYLFAKDKSGPLQPGV
DIIEGPVKNVAVPLYNRFSYIPNGALKFVDSTVVASVTIIDRSLPPIVKDASIQV
VSAIRAAPEAARSLAS SLPGQTKILAKVFYGEN
Hey b 3
MAEEVEEERLKYLDFVRAAGVYAVDSFSTLYLYAKDISGPLKPGVDTIENVV
KTVVTPVYY1PLEAVKFVDKTVDVSVTSLDGVVPPVIKQVSAQTYSVAQDAP
RIVLDVASSVFNTGVQEGAKALYANLEPKAEQYAVITWRALNKLPLVPQVA
NV V VPTA VYFSEKYND V VRGTTEQGYRVSSYLPLLPTEKITK VFGDEAS
Hey b 5 (Q39967)
MASVEVESAATALPKNETPEVTKAEETKTEEPAAPPASEQETADATPEKEEP
TAAPAEPEAPAPETEKAEEVEKIEKTEEPAPEADQTTPEEKPAEPEPVAEEEPK
HETKETETEAPAAPAEGEKPAEEEKPITEAAETATTEVPVEKTEE
Hey b 6 (P02877)
MNIFIVVLLCLTGVAIAEQCGRQAGGKLCPNNLCCSQWGWCGSTDEYCSPD
HNCQSNCKDSGEGVGGGSASNVLATYHLYNSQDHGWDLNAASAYCSTWD
ANKPYSWRSKYGWTAFCGPVGAHGQSSCGKCLSVTNTGTGAKTTVRIVDQ
CSNGGLDLDVNVFRQLDTDGKGYERGHITVNYQFVDCGDSFNPLFSVMKSS
VIN
Additional Hevea sequences (NCBI entrez accession):
S
3319923; 3319921; 3087805; 1493836; 1480457; 1223884; 3452147; 3451147; 1916805; 232267; 123335; 2501578; 3319662; 3288200; 1942537; 2392631; 2392630; 1421554; 1311006; 494093; 3183706; 3172534; 283243; 1170248; 1708278; 1706547; 464775; 266892; 231586; 123337; 116359; 123062; 2213877; 542013; 2144920; 1070656; 2129914; 2129913; 2129912; 100135; 82026; 1076559; 82028; 82027; 282933; 280399; 100138; 1086972; 108697; 1086976; 1086978; 1086978; 1086976; 1086974; 1086972; 913758; 913757; 913756; 234388; 1092500; 228691; 1177405; 18839; 18837; 18835; 18833; 18831; 1209317; 1184668; 168217; 168215; 168213; 168211; 168209; 348137.
Rye grass Lolium sequences: 126385 Lol p 1
MASSSSVLLVVALFAVFLGSAHGIAKVPPGPNITAEYGDKWLDAKSTWYGK
PTGAGPKDNGGACGYKNVDKAPFNGMTGCGNTPIFKDGRGCGSCFE1KCTK
PESCSGEAVTVTITDDNEEPIAPYHFDLSGHAFGSMAKKGEEQNVRSAGELE
LQFRRVKCKYPDDTKPTFHVEKASNPNYLAILVKYVDGDGDVVAVDKEKG
KDKWIELKESWGAVWRIDTPDKLTGPFTVRYTTEGGTKSEFEDVIPEGWKA
DTSYSAK
126386 Lol p 2a
AAPVEFTVEKGSDEKNLALSIKYNKEGDSMAEVELKEHGSNEWLALKKNG
DGVWEIKSDKPLKGPFNFRFVSEKGMRNVFDDVVPADFKVGTTYKPE
126387 Lol p3 TKVDLTVEKGSDAKTLVLNIKYTRPGDTLAEVELRQHGSEEWEPMTKJ(GNL
WEVKSAKPLTGPMNFRFLSKGGMKNVFDEVIPTAFTVGKTYTPEYN
2498581 Lol p 5a
MAVQKYTVALFLRRGPRGGPGRSYAADAGYTPAAAATPATPAATPAGGWR
EGDDRRAEAAGGRQRLASRQPWPPLPTPLRRTSSRSSRPPSPSPPRASSPTSA
S
AKAPGLIPKLDTAYDVAYKAAEAHPRGQVRRLRHCPHRSLRVIAGALEVHA
VKPATEEVLAAKIPTGELQIIVDKIDAAFKJAATAANAAPTNDKFTVFESAFNK
ALNECTGGAM1PTSSSPPSRPRSSRPTPPPSPAAPEVKYAVFEAALTKAITAM
TQAQKAGKPAAAAATAAATVATAAATAAAVLPPPLLVVQSLISLLIYY
2498582 Lot p Sb
MAVQKHTVALFLAVALVAGPAASYAADAGYAPATPATPAAPATAATPATP
ATPATPAAVPSGKATTEEQKL1EKINAGFKAAVAAAAVVPPADKYKTFVETF
GTATNKAFVEGLASGYADQSKNQLTSKLDAALKLAYEAAQGATPEAKYDA
YVATLTEALRVIAGTLEVHAVKPAAEEVKVGAIPAAEVQLIDKVDAAYRTA
ATAANAAPANDKFTVFENTFNNAIKVSLGAAYDSYKFIPTLVAAVKQAYAA
KQATAPEVKYTVSETALKKAVTAMSEAEKEATPAAAATATPTPAAATATAT
PAAAYATATPAAATATATPAAATATPAAAGGYKV
455288 Lot p isoform 9
MAVQKHTVALFLAVALVAGPAASYADAGYAPATPATPAAPATAATPATP
ATPATPAAVPSGKATTEEQKLIEKINAGFKAAVAAAAVVPPADKYKTFVETF
GTATNKAFVEGLASGYADQSKNQLTSKLDAALKLAYEAAQGATPEAKYDA
YVATLTEALRVIAGTLEVHAVKPAAEEVKVGAIPAAEVQLIDKVDAAYRTA
ATAANAAPANDKFTVFENTFNNAIIKVSLGAAYDSYKNPTLVAAVKQAYAA
KQATAPEVKYTVSETALKKAVTAMSEAEKEATPA&ATATPTPAAATATAT
PAAAYATATPAAATATATPAAATATPAAAGGYKV
1582249 LoIp 11
DKGPGFVVTGRVYCDPCRAGFETNVSHNVEGATVAVDCRPFDGGESKLKA
EATTDKDGWYKIEIDQDHQEEICEVVLAKSPDKSCSEIEEFRDRARVPLTSNX
GIKQQGIRYANPJAFFRKEPLKECGGILQAY
Additional Lotium sequences (NCBI entrez accession): 135480; 417103; 687261;687259; 1771355; 2388662; 631955; 542131;542130; 542129; 100636; 626029; 542132; 320616; 320615; 320614; 100638; 100634; 82450; 626028; 100639; 283345; 542133; 1771353; 1763163; 1040877; 1040875; 250525; 551047; 515377; 510911; 939932; 439950; 2718; 168316; 168314; 485371; 2388664; 2832717; 2828273; 548867.
Olive tree Olive sequences 416610 Olee 1
EDIPQPPVSQFHIQGQVYCDTCRAGFITELSEFIPGASLRLQCKDKENGDVTFT
EVGYTRAEGLYSM LVERDHKNEFCEITLISSGRKDCNEIPTEGWAKPSLKFKL
NTVNGTTRTVNPLGFFKKEALPKCAQVYNKLGMYPPNM
Parietaria Parietaria sequences: 2497750 Parj P2
MRTVSMAALVVIAAALAWTSSAEPAPAPAPGEEACGKVVQDIMPCLHFVKG
EEKEPSKECCSGTKKLSEEVKTTEQKREACKC1VRATKGISGIKNELVAEVPK
KCDIKTTLPPITADFDCSKIQSTIFRGYY
1352506 Parj PS
MVRALMPCLPFVQGKEKEPSKGCCSGAKRLDGETKTGPQRVHACECIQTAM
KTYSDIDGKLVSEVPKHCGIVDSKLPPIDVNMDCKTVGVVPRQPQLPVSLRH
GPVTGPSDPAHKARLERPQIRVPPPAPEKA
1532056 Parj P8
MRTVSMAALVVIAAALAWTSSAELASAPAPGEGPCGKVVHHIMPCLKFVKG
EEKEPSKSCCSGTKKLSEEVKTTEQKREACKCIVAATKGISGIKNELVAEVPK
KCGJTTTLPPITADFDCSKIESTIFRGYY
1532058 Parj P9
MRTVSAPSAVALVVIVAAGLAWTSLASVAPPAPAPGSEETCGTVVRALMPC
O
LPFVQGKEKEPSKGCCSGAKRLDGETKTGLQRVHACECIQTAMKTYSDIDG
KLVSEVPKHCGIVDSKLPPIDVNMDCKTLGVVPRQPQLPVSLRHGPVTGPSD
PAHKARLERPQIRVPPPAPEKA
2497749ParjP9
MRTVSARSSVALVVIVAAVLVWTSSASVAPAPAPGSEETCGTVVGALMPCL
PFVQGKEKEPSKGCCSGAKRLDGETKTGPQRVHACECIQTAMKTYSDIDGK
LVSEVPKHCGIVDSKLPPIDVNMDCKTLGVLHYKGN
1086003 Parj I
MVRALMPCLPFVQGKEKEPSKGCCSGAKRLDGETKTGPQRVHACECIQTAM
KTYSDIIDGKLVSEVPKHCGIVDSKLPPIDVNMDCKTVGVVPRQPQLPVSLRH
GPVTGPSRSRPPTKHGWRDPRLEFRPPHRKKPNPAFSTLG
Additional Panetaria sequences (NCBI entrez accession): 543659; 1836011; 1836010; 1311513; 1311512; 1311511; 1311510; 1311509; 240971.
Timothy grass Phleum sequences: Phi p 1
MASSSSVLLVVVLFAVFLGSAYGIPKVPPGPNITATYGDKWLDAKSTWYGK
PTGAGPKDNGGACGYKDVDKPPFSGMTGCGNTPIFKSGRGCGSCFEIKCTKP
EACSGEPVVVHITDDNEEPL&PYHFDLSGHAFGAMAKKGIDEQKLRSAGELEL
QFRRVKCKYPEGTKVTFHVEKGSNPNYLALLVKYVNGDGDVVAVDIIKEKG
KDKWIELKESWGAIWR1DTPDKLTGPFTVRYTTEGGTKTEAEDVLpEGwKj
DTSYESK
PhIp 1
O
MASSSSVLLVVALFAVFLGSAHGIPKVPPGPNITATYGDKWLDAKSTWYGK
PTAAGPKDNGGACGYKDVDKPPFSGMTGCGNTPWKSGRGCGSCFEIKCTKP
EACSGEPVVVHITDDNEEPIAAYHFDLSGIAFGSMAKKGDEQKLRSAGEVEI
QFRRVKCKYPEGTKVTFHVEKGSNPNYLALLVKFSGDGDVVAVDIKEKGKD
KWIALKESWGAIWRIDTPEVLKGPFTVRYTTEGGTKARAKDVIPEGWKADT
AYESK
PhIp 2
MSMASSSSSSLLAMAVLAALFAGAWCVPKVTFTVEKGSNIEKHLAVLVKYE
GDTMAEVELREHGSDEWVAMTKGEGGVWTFDSEEPLQGPFNFRFLTEKGM
KNVFDDVVPEKY'TIGATYAPEE Phi p 5
ADLGYGGPATPAAPAEAAPAGKATTEEQKLIEKINDGFKAALAAAAGVPPA
DKYKTFVATFGAASNKAFAEGLSAPKGAAESSSKAALTSKLDAAYKLAYK
TAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVTE
KVDSAFKVAATAANAAPANDKFTVFEAAFNNAIKASTGGAYESYKFIPALE
AAVKQAYAATVATAPEVKYTVFETALKKAFTAMSEAQKAAKPATEATATA
TAAVGAATGAATAATGGYKV
Phi p 5
ADLGYGGPATPAAPAEAAPAGKATTEEQKLIEKINDGFKAALAAAAGVPPA
DKYKTFVATFGAASNKAFAEGLSAEPKGAAESSSKAALTSKLDAAYKLAYK
TAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIE
KVDSAFKVAATAANAAPANDKFTVFEAAFNNAIKASTGGAYESYKFIPALE
AAVKQAYAATVATAPEVKYTVFETALKKAJTAMSEAQKAAKPATEATATA
TAAVGAATGAATAATGGYKV
O
Phi p Sb
AAAAVPRRGPRGGPGRSYTADAGYAPATPAAAGAAAGKATTEEQKLIEDIN
VGFKAAVAAAASVPAADKFKTFEAAFTSSSKAAAAKAPGLVPKLDAAYSV
AYKAAVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVTEEPGMAKIPA
GELQHDK1DAAFKVAATAAATAPADDKFTVFEAAFNKAKESTGGAYDTYK
CIPSLEAAVKQAYAATVAAAPQVKYAVFEAALTKAITAMSEVQKVSQPATG
AATVAAGAATTAAGAASGAATVAAGGYKV
Phlp5a
ADLGYGPATPAAPAAGYTPATPAAPAGADAAGKATTEEQKLIEKINAGFKA
ALAGAGVQPADKYRTFVATFGPASNKAFAEGLSGEPKGAAESSSKAALTSK
LDAAYKLAYKTAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEV
KVIPAGELQVIEKVDAAFKVAATAANAAPANDKFTVFEAAFNDEII(ASTGG
AYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAITAMSEAQKA
AKPAAAATATATAAVGAATGAATAATGGYKV
Phi p 5
MAVQKYTVALFLAVALVAGPAASYAADAGYAPATPAAAGAEAGKATTEE
QKLIEDINVGFKAAVAAAASVPAADKFKTFEAAFTSSSKAATAKAPGLVPKL
DAAYSVSYKAAVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVTEEPG
MAKIPAGELQIIIDKIDAAFKVAATAAATAPADTVFEAAFNKAIKESTGGAYD
TYKCIPSLEAAVKQAYAATVAAAPQVKYAVFEAALTKAITAMSEVQKVSQP
ATGAATVAAGAATTAAGAASGAATVAAGGYKV Phlp5
MAVQKYTVALFLAVALVAGPAASYAADAGYAPATPAAAGAEAGKATTEE
QKLIEDINVGFKAAVAAAASVPAADKFKTFEAAFTSSSKAATAKAPGLVPKL
DAAYSVAYKAAVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVTEDPA
WPKIPAGELQIIDKJDAAFKVAATAAATAPADDKFTVFEAAFNKAIKESTGG
AYDTYKCIPSLEAAVKQAYAATVAAAPQVKYAVFEAALTKAITAMSEVQK
VSQPATGAATVAAGAATTATGAASGAATVAAGGYKV
O
Phi p 5
ADAGYAPATPAAAGAEAGKATTEEQKLIEDINVGFKAAVAAAASVPAADKF
KTFEAAFTSSSKAATAKAPGLVPKLDAAYSVAYKAAVGATPEAKFDSFVAS
LTEALRVIAGALEVHAVKPVTEEPGMAKIIPAGELQIIDKJIDAAFKVAATAAA
TAPADDKFTVFEAAFNKALKESTGGAYDTYKCIPSLEAAVKQAYAATVAAA
PQVKYAVFEAALTKAITAMSEVQKVSQPATGAATVAAGAATTAAGAASGA
AT VAAGGYK V
Phi p 5
SVKRSNGSAEVHRGAVPRRGPRGGPGRSYAADAGYAPATPAAAGAEAGKA
TTEEQKLIEDINVGFKAAVAAAASVPAADKFKTFEAAFTSSSKAATAKAPGL
VPKLDAAYSVAYKAAVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVT
EEPGMAKJIPAGELQIIDKIDAAFKVAATAAATAPADDKFTVFEAAFNKAIKES
TGGAYDTYKCIPSLEAAVKQAYAATVAAAPQVKYAVFEAALTKAITAMSEV
QKVSQPATGAATVAAGAATTAAGAASGAATVAAGGYKV
Phi p 5
MAVHQYTVALFLAVALVAGPAGSYAADLGYGPATPAAPAAGYTPATPAAP
AGAEPAGKATTEEQKLIEKINAGFKAALAAAAGVPPADKYRTFVATFGAAS
NKAFAEGLSGEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAY
VATVSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIEKVDAAFKVAATAA
NAAPANDKFTVFEAAFNDAIKASTGGAYESYKFIPALEAAVKQAYAATVAT
APEVKYTVFETALKKAITAMSEAQKAAKPAAAATATATAAVGAATGAATA
ATGGYKV
Phi p 5
ADLGYGGPATPAAPAEAAPAGKATTEEQKLIEKINDGFKAALAAAAGVPPA
DKYKTFVATFGAASNKAFAEGLSAEPKGAAESSSKAALTSKLDAAYKLAYK
TAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIE
KVDSAFKVAATAANAAPANDKFTVFEAAFNNAIKASTGGAYESYKFIPALE
AAVKQAYAATVATAPEVKYTVFETALKKAFTAMSEAQKAAKPATEATATA
TAAVGAATGAATAATGGYKV
S
Phi p5b
AAAAVPRRGPRGGPGRSYTADAGYAPATPAAAGAAAGKATTEEQKLIEDIN
VGFKAAVAAAASVPAADKFKTFEAAFTSSSKAAAAKAPGLVPKLDAAYSV
AYKAAVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVTEEPGMAKIPA
GELQIIDKIDAAFKVAATAAATAPADDKFTVFEAAFNKAIIKESTGGAYDTYK
CIPSLEAAVKQAYAATVAAAPQVKYAVFEAALTKAJTAMSEVQKVSQPATG
AATVAAGAATTAAGAASGAATVAAGGYKV
Phlp5a
DLGYGPATPAAPAAGYTPATPAAPAGADAAGKATTEEQKLIEKINAGFKA
ALAGAGVQPADKYRTFVATFGPASNXAFAEGLSGEPKGAAESSSKAALTSK
LDAAYKLAYKTAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEV
KVIPAGELQVIEKVDAAFKVAATAANAAPANDKFTVFEAAFNDE[KASTGG
AYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAITAMSEAQKA
AKPAAAATATATAAVGAATGAATAATGGYKV
Phi p 5
AVPRRGPRGGPGRSYAADAGYAPATPAAAGAEAGKATTEEQKUEDINVGF
KAAVAAAASVPAGDKFKTFEAAFTSS SKAATAKAPGLVPKLDAAYSVAYK
AAVGATPEAJ(FDSFVASLTEALRVIAGALEVHAVKPVTEEPGMAKIPAGELQ
IIDKIDAAFKVAATAAATAPADDKFTVFEAAFNKAIKESTGGAYDTYKCIPSL
EAAVKQAYAATVAAAPQVKYAVFEAALTKAITAMSEVQKVSQPATGAATV
AAGAATTATGAASGAATVAAGGYKV
Phi p Sb MAVPRRGPRGGPGRSYTADAGYAPATPAAAGAAAGKATTEEQKLffiDINVG
FKAAVAARQRPAADKFKTFEAASPRHPRPLRQGAGLVPKLDAAYSVAYKA
AVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVTEEPGMAKIPAGELQII
DKTDAAFKVAATAAATAPADDKFTVFEAAFNKAIKESTGGAYDTYKCIPSLE
AAVKQAYAATVAAAAEVKYAVFEAALTKAITAMSEVQKVSQPATGAATVA
AGAATTAAGAASGAATVAAGGYKV
O
Phi p 5
MAVHQYTVALFLAVALVAGPAASYAADLGYGPATPAAPAAGYTPATPAAP
AEAAPAGKATTEEQKLIEKINAGFKAALAAAAGVQPADKYRTFVATFGAAS
NKAFAEGLSGEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAY
VATLSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIEKVDAAFKVAATAA
NAAPANDKFT VFEAAFNDAII(ASTGGAYESYKFIPALEAAVKQAYAAT VAT
APEVKYTVFETALKKAITAMSEAQKAAKPAATATATAAVGAATGAATA
ATGGYKV
Phi p 5
EAPAGKATTEEQKLIEKJINAGFKAALARRLQPADKYRTFVATFGPASNKAFA
EGLSGEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAYVATLS
EALRIIAGTLEVHAVKPAAEEVKVIPAAELQVIEKVDAAFKVAATAANAAPA
NDKFTVFEAAFNDEIKASTGGAYESYKFIPALEAAVKQAYAATVATAPEVK
YTVFETALKKAITAM SEAQKAAKPPPLPPPPQPPPLAATGAATAATGGYKV
Phi p 5
MAVHQYTVALFLAVALVAGPAASYAADLGYGPATPAAJAAGYTPATPAAP
AEAAPAGKATTEEQKLIEKINAGFKAALAAAAGVQPADKYRTFVATFGAAS
NKAFAEGLSGEPKGAAES SSKAALTSKLDAAYKLAYKTAEGATPEAKYDAY
VATLSEALRIIAGTLEVHAVKPAAEEVKVIIPAGELQVIEKVDAAFKVAATAA
NAAPANDKFTVFEAAFNDAHASTGGAYESYKFIPALEAAVKQAYAATVAT
APEVKYTVFETALKKAITAMSEAQKAAKPAWTATATAAVGAATGAATA
ATGGYKV
Phi p Sb MAVPRRGPRGGPGRSYTADAGYAPATPAAAGAAAGKATTEEQKLIEDINvG FKAAVARQRPAADKFKTFEAASPRJiPRPLRQGAGLVPKLDAAYSVAYKA
AVGATPEAKFDSFVASLTEALRVIAGALEVHAVKPVTEEPGMAKJPAGELQH
DKDAAFKVAATAAATAPADDKFTVFEAAFNKAIKESTGGAYDTYKCIpSLE *
AAVKQAYAATVAAAAEVKYAVFEAALTKAITAMSEVQKVSQPATGAATVA
AGAATTAAGAASGAATVAAGGYKV
Phi p 5a
ADLGYGPATPAAPAAGYTPATPAAPAGADAAGKATTEEQKLIEKINAGFKA
ALAGAGVQPADKYRTFVATFGPASNKAFAEGLSGEPKGAAESSSKAALTSK
LDAAYKLAYKTAEGATPEAKYDAYVATLSEALRIIAGTLEVHAVKPAAEEV
KVIPAGELQVIEKVDAAFKVAATAANAAPANDKFTVFEAAFNDE[KASTGG
AYESYKFIPALEAAVKQAYAATVATAPEVKYTVFETALKKAITAMSEAQKA
AKPPPLPPPPQPPPLAATGAATAATGGYKV
Phi p 5
MAVHQYTVALFLAVALVAGPAASYAADLGYGPATPAAPAAGYTPATPAAP
AEAAPAGKATTEEQKLIEKINAGFKAALAAAAGVQPADKYRTFVATFGAAS
NKAFAEGLSGEPKGAAESSSKAALTSKLDAAYKLAYKTAEGATPEAKYDAY
VATLSEALRIIAGTLEVHAVKPAAEEVKVIPAGELQVIEKVDAAFKVAATAA
NAAPANDKFTVFEAAFNDAIKASTGGAYESYKFLPALEAAVKQAYAATVAT
APEVKYTVFETALKKAITAMSEAQKAAKPAAAATATATAAVGAATGAATA
ATGGYKV
Phi p 6
MAAHKFMVAMFLAVAVVLGLATSPTAEGGKATTEEQKLIEDVNASFRAAM
ATTANVPPADKYKTFEAAFTVSSKRNLADAVSKAPQLVPKLDEVYNAAYN
AADHAAPEDKYEAFVLHFSEALRIIAGTPEVHAVKPGA
Phi p 6
SKAPQLVPKLDEVYNAAYNAADHAAPEDKYEAFVLHFSEALHIJAGTPEVH
AVKPGA PhJp6
ADKYKTFEAAFTVSSKRNLADAVSKAPQLVPKLDEVYNAAYNAADHAAPE
DKYEAFVLHFSEALHIIAGTPEVHAVKPGA *
Phi p 6
TEEQKLIEDVNASFRAAMATTANVPPADKYKTLEAAFTVS SKRNLADAVSK
APQLVPKLDEVYNAAYNAADHAAPEDKYEAFVLHFSEALRIJAGTPEVHAV
KPGA
Phi p 6 MAAI{KFMVAMFLAVAVVLGLATSPTAEGGKATTEEQKLIEDINASFRAAM ATTANVPPADKYKTFEAAYTVSSKRJ'LADAVSKAYQLVPKLDEVYNAAYN
AADHAAPEDKYEAFVLHFSEALHHAGTPEVHAVKPGA
Phi p 6
MVAMFLAVAVVLGLATSPTAEGGKATTEEQKLIEDVNASFRAAMATTANV
PPADKYKTFEAAFTVSSKRNLADAVSKAPQLVPKLDEVYNAAYNAADHAA
PEDKYEAFVLHFSEALRIIAGTPEVHAVKPGA
Phi p 7
MADDMERIFKRFDTNGDGKISLSELTDALRTLGSTSADEVQRMMAEIDTDG
DGFIDFNEFISFCNANPGLMKDVAKVF
Phi p 11 MSWQTYVDEHLMCEIEGHHLASAAILGHDGTVWAQSA1FPQFKpEEITGrM KDFDEPGHLAPTGMFVAGAKYMVIQGEPGRVIRGKKGAGGITIKKTGQALv
VGIYDEPMTPGQCNMVVERLGDYLVEQGM
Additional Phieum sequences (NCBI entrez accession): 458878; 548863; 2529314; 2529308; 2415702; 2415700; 2415698; 542168; 542167; 626037; 542169; 541814; 542171; 253337; 253336; 453976; 439960.
Wasp (and related) Vespula sequences:
S
465054 ALLERGEN VES V 5
MEISGLVYLIIIVTIIDLPYGKANNYCKIKCLKGGVHTACKYGSLKPNCGNKV
VVSYGLTKQEKQDILKEHNDFRQKIARGLETRGNPGPQPPAKNMKNLVWN
DELAYVAQVWANQCQYGHDTCRDVAKYQVGQNVALTGSTAAKYDDPVK
LVKMWEDEVKDYNPKXKFSGNDFLKTGHYTQMVWANTKEVGCGSIKYIQE
KWHKHYLVCNYGPSGNFMNEELYQTK
1709545 ALLERGEN VES M 1 GPKCPFNSDTVSIIIETRENRNRDLYTLQTLQN}[PEFKKKTITRPVVFITHGFTS
SASEKNFINLAKALVDKDNYMVISIDWQTAACTNEYPGLKYAYYPTAASNT
RLVGQYIATITQKLVKDYKJSMANIRLIGHSLGAHVSGFAGKRVQELKLGKY
SEHGLDPARPSFDSNHCSERLCETDAEYVQIJHTSNYLGTEKILGTVDFYMNN
GKNNPGCGRFFSEVCSHTRAVIYMAECIKHECCLIGIPRSKSSQPISRCTKQEC
VCVGLNAKKYPSRGSFYVPVESTAPFCNNKGKII
1352699 ALLERGEN VES V 1
MEENMNLKYLLLFVYFVQVLNCCYGHGDPLSYELDRGPKCPFNSDTVSIIIE
TRENRNRDLYTLQTLQNHPEFKKKTITRPVVFITHGFTSSASETNFINLAKAL
VDKDNYMVISIDWQTAACTNEAAGLKYLYYPTAARNTRLVGQYIATITQKL
VKHYKISMANIRLIGHSLGAHASGFAGKKVQELKLGKYSEIIGLDPARPSFDS
NHCSERLCETDAEYVQJIHTSNYLGTEKTLGTVDFYMNNGKNQPGCGRFFSE
VCSHSRAVIYMAECIKHECCLIGIPKSKSSQPISSCTKQECVCVGLNAKKYPSR
GSFYVPVESTAPFCNNKGKII
1346323 ALLERGEN VES V 2
SERPKRVFNIYWNVPTFMCHQYDLYFDEVTNFNIIKRNSKDDFQGDKJAIFYD
PGEFPALLSLKDGKYKKRNGGVPQEGNITIHLQKFIENLDK1YPNRNFSGIGVI
DFERWRPIFRQNWGNMKJHKNFSJDLVRNEHPTWNKKMIELEASKRFEKYA
RFFMEETLKLAKKTRKQADWGYYGYPYCFNMSPNNLVPECDVTAMHEND
SWLFNNQNVLLPSVYVRQELTPDQRIGLVQGRVKEAVRJSNNLKHSPKV
LSYWWYVYQDETNTFLTETDVKKTFQEIVINGGDGIIIWGSSSDVNSLSKCK
RLQDYLLTVLGPIAINVTEAVN
549194 ALLERGEN VES VI 5KVNYCKIKCLKGGVHTACKYGTSTKPNCGKMVVKAYGLTEAEKQEILKV F[NDFRQKVAKGLETRGNPGPQPPAKNIv1I'Th4LVWNDELANIAQVWASQCNY
GHDTCKDTEKYPVGQNIAKRSTTAALFDSPGKLVKMWENEVKDFNPNIEWS
KNNLKKTGHYTQMVWAKTKEIGCGSVKYVKDEWYTHYLVCNYGPSGNFR
NEKLYEKK
Additional vespula sequences (NCBT entrez accession): 549193; 549192; 549191; 549190; 549189; 117414; 126761; 69576; 625255; 627189; 627188; 627187; 482382; 112561; 627186; 627185; 1923233; 897645; 897647; 745570; 225764; 162551.
Tree allergen sequences (mainly birch) sequences: 114922 Betv 1
MGVFNYETETTSVIPAARLFKAFJLDGDNLFPKVAPQAISSVENIEGNGGPGTI
KKJSFPEGFPFKYVKDRVDEVDHTNFKYNYSVIEGGPIGDTLEKISNEIKIVAT
PDGGSILKISNKYHTKGDHEVKAEQVKASKEMGETLLRAVESYLLAHSDAY
N
130975 Bet v 2
MSWQTYVDEHLMCDIDGQASNSLASAIVGHDGSVWAQSSSFPQFKPQEITGI
MKDFEEPGHLAPTGLHLGGIKYMVIQGEAGAVIRGKKGSGGIT[KKTGQALV
FGIYEEPVTPGQCNMVVERLGDYLIDQGL
1168696 Betv3
MPCSTEAMEKAGHGHASTPRKRSLSNSSFRLRSESLNTLRLRRIFDLFDKNSD
GIlT VDELSRALNLLGLETDLSELEST VKSFTREGNIGLQFEDFISLHQSLNDSY *
FAYGGEDEDDNEEDMRKSILSQEEADSFGGFKVFDEDGDGYISARELQMVL
GKLGFSEGSE[DRVEKMIVSVDSNRDGRVDFFEFKDMMRSVLVRSS 809536Betv4 MADDHPQDKAERER[FKRFDANGDGKISAAELGEALKTLGSITPDEVKHMM
AEIDTDGDGFIS FQEFTDFGRANRGLLKDVAKJF
543675 Que a I -Quercus alba=oak trees (fragment)
GVFTXESQETSVIAPAXLFKALFL
543509 Car b I -Carpinus betulus=hombeam trees (fragment)
GVFNYEAETPSVIPAARLFKSYVLDGDKLIPKVAPQAIXK
543491 Am g I -Alnus glutinosa=alder trees (fragment)
GVFNYEAETPSVIPAARLFKAFILDGDKLLPKVAPEAVSSVENI
1204056 Rubisco
VQCMQVWPPLGLKKFETLSYLPPLSSEQLAKEVDYLLRKNILIPCLEFELEHG
FVYREHNRSPGYYDGRYWTMWKLPMFGCNDS SQVLKELEECKKAYPSAFI
RIIGFDDK
Additional tree allergen sequences (NCBI entrez accession number): 131919; 128193; 585564; 1942360; 2554672; 2392209; 2414158; 1321728; 1321726; 1321724; 1321722; 1321720; 1321718; 1321716; 1321714; 1321712; 3015520; 2935416; 464576; 1705843; 1168701; 1168710; 1168709; 1168708; 1168707; 1168706; 1168705; 1168704; 1168703; 1168702; 1842188;2564228; 2564226; 2564224; 2564222; 2564220; 2051993; 1813891; 1536889; 534910; 534900; 534898; 1340000; 1339998; 2149808; 66207; 2129477; 1076249; 1076247; 629480; 481805; 81443; 1361968; 1361967; 1361966; 1361965; 1361964; 1361963; 1361962; 1361961; 1361960; 1361959; 320546; 629483; 629482; 629481; 541804; 320545; 81444; 541814:; 629484; 474911; 452742; 1834387; * 298737; 298736; 1584322; 1584321; 584320; 1542873; 1542871; 1542869; 1542867; 1542865; 1542863; 1542861; 1542859; 1542857; 1483232; 1483230; 1483228; 558561; 551640; 488605; 452746; 452744; 452740; 452738; 452736; 452734; 452732; 452730; 452728; 450885; 17938; 17927; 17925; 17921; 297538; 510951; 289331; 289329; 166953 Peanut Peanut sequences 1168391 Arah 1
MRGRVSPLMLLLGILVLASVSATHAKSSPYQKKTENPCAQRCLQSCQQEPD
DLKQKACESRCTKLEYDPRCVYDPRGHTGTTNQRSPPGERTRGRQPGDYDD
DRRQPRREEGGRWGPAGPREREREEDWRQPREDWRRPSHQQPRKLRPEGRE
GEQEWGTPGSHVREETSRNNPFYFPSRRFSTRYGNQNGRIRVLQRFDQRSRQ
FQNLQNHRWQIEAKPNTLVLPKI-IADADNTLVIQQGQATVTVANGNNRKSFN
LDEGHALRIPSGFISYILNRHDNQNLRVAKISMPVNTPGQFEDFFPASSRDQSS
YLQGFSRNTLEAAFNAEFNEIRRVLLEENAGGEQEERGQRRWSTRSSENNEG
VIVKVSKEHVEELTKHAKSVSKKGSEEEGDITNPINLREGEPDLSNI'WGKLFE VKPDKKNPQLQDLDMMLTCVEJKEGALMLPI{FNSKAMVIVVVNKGTGNLE
LVAVRKEQQQRGRREEEEDEDEEEEGSNREVRRYTARLKEGDVFIMPAAHP
VAINAS SELHLLGFGINAENNHRIFLAGDKDNVIDQIEKQAKDLAFPGSGEQV
EKLIKNQKESHFVSARPQSQSQSPSSPEKESPEKEDQEEENQGGKGPLLSILKA
FN
Peanut Ara h 2 (Q6PSU2)
MAKLTILVALALFLLAAHASARQQWELQGDRRCQSQLERANLRPCEQHLM
QKIQRDEDSYGRDPYSPSQDPYSPSQDPDRRDPYSPSPYDRRGAGSSQHQER
CCNELNEFENNQRCMCEALQQIMENQSDRLQGRQQEQQFKRELRNLPQQCG
LRAPQRCDLEVESGGRDRY
Peanut Ara h 3 (AAC63045) *
RQQPEENACQFQRLNAQRPDNRIESEGGYIETWNPNNQEFECAGVALSRLVL
RRNALRRPFYSNAPQEIF1QQGRGYFGLWPGCPRHYEEPHTQGRRSQSQRPPR
RLQGEDQSQQQRDSHQKVHRFDEGDLIAVPTGVAFWLYNDHDTDVVAVSL
TDTNNNDNQLDQFPRRFNLAGNTEQEFLRYQQQSRQSRRRSLPYSPYSPQSQ
PRQEEREFSPRGQHSRRERAGQEEENEGGNIFSGFTPEFLEQAFQVDDRQIVQ
NLRGETESEEEGAIVTVRGGLRILSPDRKRRADEEEEYDEDEYEYDEEDRR.R
GRGSRGRGNGIEETICTASAKKNIGRNRSPDIYNPQAGSLKTANDLNLLILRW
LGPSAEYGNLYRNALFVAHYNTNAHSIWRLRGRAHVQVVDSNGNRVYDEE
LQEGHVLVVPQNFAVAGKSQSENFEYVAFKTDSRPSIANLAGENSVIDNLPE
EVVANSYGLQREQARQL1USJNNPFKFFVPPSQQSPRAVA Peanut Ara h 5 (Q9SQI9)
MSWQTYVDNHLLCEIEGDHLSSAAILGQDGGVWAQSSHFPQFKPEEITAIMN
DFAEPGSLAPTGLYLGGTKYMVIQGEPGAIIPGKKGPGGVTIEKTNQALIIGIY
DKPMTPGQCNMIVERLGDYLIDTGL
Peanut Ara h 6 (ABQ96216)
MAKSTILVALLALVLVAHASAMRRERGRQGDSSSCERQVDRVNLKPCEQHI
MQRIMGEQEQYDSYDIRSTRSSDQQQRCCDELNEMENTQRCMCEALQQIME
NQCDRLQDRQMVQQFKRELMNLPQQCNFRAPQRCDLDVSGGRC
Ragweed Ambrosia sequences 113478 Amb a I
MGIKHCCYILYFTLALVTLLQPVRSAEDLQQILPSANETRSLTTCGTYNIIDGC
WRGKADWAENRKALADCAQGFAKGTIGGKDGDIYTVTSELDDDVANPKEG
TLRFGAAQNRPLWIIFARDMVIRLDRELAINNDKTIDGRGAKVEIINAGFAIY
NVKNIIIHNIIMHDIVVNPGGLIKSBDGPPVPRKGSDGDAIGISGGSQIWIDHCS
O
LSKAVDGLIDAKHGSTHFTVSNCLFTQHQYLLLFWDFDERGMLCTVAFNKF
TDNVDQRMPNLRHGFVQVVNNNYERWGSYALGGSAGPTILSQGNRFLASDI
KKEVVGRYGESAMSESINWNWRSYMDVFENGAIFVPSGVDPVLTPEQNAG
MIPAEPGEAVLRLTSSAGVLSCQPGAPC
113479Amba2
MGIKHCCYILYFTLALVTLVQAGRLGEEVDILPSPNDTRRSLQGCEAHNIIDK
CWRCKPDWAENRQALGNCAQGFGKATHGGKWGDIYMVTSDQDDDVVN?
KEGTLRFGATQDRPLWTIFQRDMIIYLQQEMVVTSDKTIDGRGAKVELVYGG
ITLMNVNVIIHNID[HDVRVLPGGRII(SNGGPAIPRHQSDGDAIHVTGSSDIW IDHCTLSKSFDGLVDVNWGSTGVIISNCKFTHHEICVLLGASDTHFQDLKJvI HVTLA'{NIFTNTVHERMPRCRFGFFQIVNNFYDRWDKYAIGGSSNPTILSQG
NKFVAPDFIYKKNVCLRTGAQEPEWMTWNWRTQNDVLENGAJFVASGSDP
VLTAEQNAGMMQAEPGDMVPQLTMNAGVLTCSPGAPC
113477 Amba 1.3
MGIKQCCYILYFTLALVALLQPVRSAEGVGEILPSVNETRSLQACEALNIIDK
CWRGKADWENNRQALADCAQGFAKGTYGGKWGDVYTVTSNLDDDVANP
KEGTLRFAAAQNRPLWIIFKNDMVINLNQELVVNSDKTIDGRGVKVEIINGG
LTLMNVKNIIIHNJNIHDVKVLPGGMIKSNDGPPILRQASDGDTINVAGSSQIW
IDHCSLSKSFDGLVDVTLGSTHVTISNCKFTQQSKAILLGADDTHVQDKGML
ATVAFNMFTDNVDQRMPRCRFGFFQVVNNNYDRWGTYAIGGS SAPTILCQG
NRFLAPDDQIKKNVLARTGTGAAESMAWNWRSDKDLLENGAIIFVTSGSDPV
LIPVQSAGMIPAEPGEAAIKLTSSAGVFSCHPGAPC
113476 Amba 1.2 MGIKHCCYILYFTLALVTLLQPVRSAEDVEEFLPSANETRRSLKACEAI-IN1ID
KCWRCKADWANNRQALADCAQGFAKGTYGGIUTGDVYTVTSDKIDDDVAN
PKEGTLRFAAAQNRPLWI1FKR1MVIHLNQELVVNSDKTJDGRGVKVNJVNA GLTLMNVKNIIIHNINIHDJ1KVCPGGMKSNDGPPILRQQSDGDAINVAGSSQI
WIDHCSLSKASDGLLDITLGSSHVTVSNCKFTQHQFVLLLGADDTHYQDKG
MLATVAFNMFTDHVDQRMPRCRFGFFQVVNNNYDRWGTYAIGGSSAPIJLS *
QGNRFFAPDDIIKKNVLARTGTGNAESMSWNWRTDRDLLENGAIFLPSGSDP
VLTPEQKAGMIPAEPGEAVLRLTSSAGVLSCHQGAPC
113475 Amba 1.1
MGIKHCCYJLYFTLALVTLLQPVRSAEDLQEJLPVNETRRLTTSGAYNTIDGC
WRGKADWAENRKALADCAQGFGKGTVGGKDGDIYTVTSELDDDVANPKE
GTLRFGAAQNRPLWIIFERDMVIIRLDKEMVVNSDKTIDGRGAKVEJINAGFT
LNGVKNVIII{NJNMHDVKVNPGGLIKSNDGPAAPRAGSDGDAISISGSSQIWI
DHCSLSKSVDGLVDAKLGTTRLTVSNSLFTQHQFVLLFGAGDENIEDRGML
ATVAFNTFTDNVDQRMPRCRHGFFQV\TNNNYDKWGSyAJGGSASPTILSQG
NRFCAPDERSKKNVLGRHGEAAAESMKWNWRTNKDVLENGAIFVASGVDP
VLTPEQSAGMIPAEPGESALSLTSSAGvLSCQPGAPC Cedar sequences 493634 Cryj lB precursor
MDSPCLVALLVFSFVIGSCFSDNPIDSCWRGDSNWAQNRMKLADCAVGFGS
STMGGKGGDLYTVTNSDDDPVNPPGTLRYGATRDRPLWIIFSGNMNIKLKM
PMYIAGYKTFDGRGAQVYIGNGGPCVFIKRVSNVHHGLYLYGCSTSVLGNV
LINESFGVEPVHPQDGDALTLRTATNIWIDHNSFSNSSDGLVDVTLTSTGvTIS NNLFFN}ll{KVMSLGHDDAYSDDKSMKVTVAFNQFGPNCGQRMPRARYGL
VHVANNNYIDPWTIYAIGGSSNPTILSEGNSFTMNESYKKQVTIRJGCKTSSSC
SNWVWQSTQDVFYNGAYFVSSGKYEGGNIYTKKEAFNVENGNATPHLTQN
AGVLTCSLSKRC
493632 Cryj IA precursor
MDSPCLVALLVLSFVIGSCFSDNPJDSCWRGDSNWAQNRMKLADCAVGFGS
STMGGKGGDLYTVTNSDDDPVNPAPGTLRYGATRDRPLWJIFSGNMNII(LK
MPMYIAGYKTFDGRGAQVYIGNGGPCVFJKRVSNVIIHGLHLYGCSTSVLGN
VLINESFGVEPVHPQDGDALTLRTATNIWIDHNSFSNSSDGLVDVTLSSTGVT
ISNNLFFMHHKVMLLGHDDAYSDDKSMKVTVAJ?NQFGPNCGQRJV1PRARYG LVHVANNNYDPWTIYAIGGSSNPTILSEGNSFTAPNESYKJ(QVTUUGCKTSSS *
CSNWVWQSTQDVFYNGAYFVSSGKYEGGNIYTKKEAFNVENGNATPQLTK
NAG VLTCSLSKRC
1076242 Cry j II precursor -Japanese cedar
MAMKLIAPMAFLAMQLIIMAAAEDQSAQIMLDSVVEKYLRSNRSLRKVEHS
RHDAINIFNVEKYGAVGDGKHDCTEAFSTAWQAACKNPSAMLLVPGSKKF
VVNNLFFNGPCQPHFTFKVDGIIAAYQNPASWKNNRIWLQFAKLTGFTLMG
KGVIDGQGKQWWAGQCKWVNGREICNDRDRPTAIKFDFSTGLIIQGLKLMN
SPEFHLVFGNCEGVKJIGISITAPRDSPNTDGIDIFASKNFHLQKNTIGTGDDCV
AIGTGSSNIVIEDLICGPGHGISIGSLGRENSRAEVSYVHVNGAKFIDTQNGLRI
KTWQGGSGMASHIIYENVEMINSENPILINQFYCTSASACQNQRSAVQIQDVT
YKNIRGTSATAAAIQLKCSDSMPCKDIKLSDISLKLTSGKIASCLNDNANGYF
SGHVIPACKNLSPSAKRKESKSHKHPKTVMVENMRAYDKGNTRILLGSRPP
NCTNKCHGCSPCKAKLVIVHRIMPQEYYPQRWICSCHGKIYHP
1076241 Cry j H protein -Japanese cedar
MAMKFIAPMAFVAMQLIIMAAAEDQSAQIMLDSDIEQYLRSNRSLRKVEHS
RHDAINIFNVEKYGAVGDGK}{DCTEAFSTAWQAACKKPSAMLLVPGNKKF
VVNNLFFNGPCQPHFTFKVDGHAAYQNPASWKNNRJWLQFAKLTGFTLMG
KGVIDGQGKQWWAGQCKWVNGREICNDRDRPTAIKFDFSTGLHQGLKLMN
SPEFHLVFGNCEGVKJIGISITAPRDSPNTDGIDIFASKNFHLQKNTIGTGDDCV
AIGTGSSNIVIEDLICGPGHGISIGSLGRENSRAEVSYVHVNGAKFIDTQNGLRI
KTWQGGSGMASHIJYENVEMINSENPILINQFYCTSASACQNQRSAVQIQDVT
YKNIRGTSATAAAIQLKCSDSMPCKDIKLSDISLKLTSGKIASCLNDNANGYF
SGHVIPACKNLSPSAKRKESKSHKHPKTVMVKNMGAYDKGNRTRILLGSRP
PNCTNKCHGCSPCKAKLVIVHRIMPQEYYPQRWMCSRHGKIYHP
541803 Cryj I precursor -Japanese cedar
MDSPCLVALLVLSFVIGSCFSDNPIDSCWRGDSNWAQNRMKLADCAVGFGS
STMGGKGGDLYTVTNSDDDPVNPPGTLRYGATRDRPLWIIFSGNMNIKLKM
PMYIAGYKTFDGRGAQVYIGNGGPCVFIKRVSNVIIHGLHLYGCSTSVLGNV
UNBSFGVEPVHPQDGDALTLRTATNIWIDHNSFSNSSDGLVDVTLSSTGVTIS
NNLFFNHHKVMLLGHDDAYSDDKSMKVTVAFNQFGPNCGQRMPRARYGL
VHVANNNYDPWTIYAIGGSSNPTILSEGNSFTAPNESYKKQVTIRIGCKTSSSC
SNWVWQSTQDVFYNGAYFVSSGKYEGGNIYTKKEAFNVENGNATPQLTKN
AGVLTCSLSKRC
541802 Cryj I precursor-Japanese cedar
MDSPCLVALLVFSFVIGSCFSDNPIDSCWRGDSNWAQNRMKLADCAVGFGS
STMGGKGGDLYTVTNSDDDPVNPAPGTLRYGATRDRPLWIIFSGNMNIKLK
MPMYIAGYKTFDGRGAQVYIGNGGPCVFIKRVSNVIIHGLYLYGCSTSVLGN
VLINESFGVEPVHPQDGDALTLRTATNIWIDHNSFSNSSDGLVDVTLTSTGVT
ISNNLFFNHHKVMSLGHDDAYSDDKSMKVTVAFNQFGPNCGQRMPRARYG
LVHVANNNYDPWTIYAIGGSSNPTILSEGNSFTAPNESYKKQVTIRIGCKTSSS
CSNWVWQSTQDVFYNGAYFVS SGKYEGGNIYTKKEAFNVENGNATPHLTQ
NAG VLTCSLSKRC Dog
Canis sequences: Can f I
MKTLLLTIGFSLIAILQAQDTPALGKDTVAVSGKWYLKAMTADQEVPEKPDS
VTPMILKAQKGGNLEAKITMLTNGQCQNIT V VLHKTSEPGKYTAYEGQR V V
FIQPSPVRDHYILYCEGELHGRQIRMAKLLGRDPEQSQEALEDFREFSRAKGL
NQEILELAQSETCSPGGQ
Serum albumin fragment
EAYKSEIAHRYNDLGEEHFRGLVL S.32.
Serum albumin fragment
LSSAKERFKCASLQKFGDRAFKAWSVARLSQRFPKADFAEISKVVTDLTKVH
KECCHGDLLECADDRADLAKYMCENQDSISTKLKECCDKPVLEKSQCLAEV
ERDELPGDLPSLAADFVEDKEVCKNYQEAKDVFLGTFLYEYSRRHPEYSVSL
LLRLAXEYEATLEKCCATDDPPTCYAJ(VLDEFKPLVDEPQNLVKTNCELFEK
LGEYGFQNALLVRYTKKAPQVSTPTLVVEVSRKLGKVGTKCCKKPESERMS
CADDFLS
Can f2
MQLLLLTVGLALICGLQAQEGNHEEPQGGLEELSGRWHSVALASNKSDLIKP
WGHFRVFIHSMSAKDGNLHGDILIPQDGQCEKVSLTAFKTATSNKFDLEYW
GJ-[NDLYLAEVDPKSYLILYMINQYNDDTSLVAHLMVRDLSRQQDFLPAFES
VCEDIGLHKDQIVVLSDDDRCQGSRD
Additional dog allergen protein (NCBI entrez accession): Horse Equus sequences: 1575778 Equ ci MKLLLLCLGLILVCAQQEENSDVAIRJ.JFDISKJSGEWySJFLASDVKEIuEENG
SMRVFVDVIRALDNSSLYAEYQTKVNGECTEFPMVFDKTEEDGVYSLNYDG
YNVFR1SEFENDEHIILYLVNFDKDRPFQLFEFYAREPDVSPEIKEEFVKJVQJ(
RGIVKENIIDLTKIDRCFQLRGNGVAQA
3121755 Equ c 2
SQXPQSETDYSQLSGEWNTIYGAASNIXK
Euroglyphus (mite)
S
Euroglyphus sequences: Eur m 1 (variant)
TYACSINSVSLPSELDLRSLRTVTPIRMQGGCGSCWAFSGVASTESAYLAYR
NMSLDLAEQELVDCASQNGCHGDTIPRGIEYIQQNGVVQEHYYPYVAREQS
CHRPNAQRYGLKNYCQISPPDSNKIRQALTQTHTAVAVIIGIKDLNAFRHYD
GRTIMQHDNGYQPNYHAVNIVGYGNTQGVDYWWRNSWDTTWGDNGYGY
FAANINL
Eur m 1 (variant)
TYACSINSVSLPSELDLRSLRTVTPIRMQGGCGSCWAFSGVASTESAYLAYR
NMSLDLAEQELVDCASQNGCHGDTIPRGIEYIQQNGVVQEHYYPYVAREQS
CHRPNAQRYGLKNYCQISPPDSNKIRQALTQTHTAVAVIIGIKDLNAFRHYD
GRTIMQHDNGYQPNYHAVNIVGYGNTQGVDYWIVRNSWDTTWGDNGYGY
FAANINL
Eur m I (variant)
ETNACSINGNAPAEIDLRQMRTVTPIRMQGGCGSCWAFSGVAATESAYLAY
RNQSLDLAEQELVDCASQHGCHGDTIPRGIEYIQHNGVVQESYYRYVAREQS
CRRPNAQRFGISNYCQJYPPNANKIREALAQTHSAIAVllGll(DLDAFREYDJR
TIIQDNGYQPNYHAVNIVGYSNAQGVDYWIVRNSWDTNWGDNGYGYFAA
NIDL
Eur m 1 (variant) ETSACR1NSVNVPSELDLRSLRTVTP1RMQGGCGSCWAFSGVAATESAYLAY
RNTSLDLSEQELVDCASQHGCHGDTIPRGIEYIQQNGVVEERSYPYVAREQQ
CRRPNSQHYGISNYCQIYPPDVKQIREALTQTHTAIAVIIGIKDLRAFQHYDGR
TIIQHDNGYQPNYHAVNIVGYGSTQGVDYWIVRNSWDTTWGDSGYGYFQA
GNNL
Poa (grass) sequences
S
113562 POLLEN ALLERGEN POA P 9
MAVQKYTVALFLVALVVGPAASYAADLSYGAPATPAAPAAGYTPAAPAGA
APKATTDEQKMIEKINVGFKAAVAAAGGVPAANKYKTFVATFGAASNKAF
AEALSTEPKGAVDSSKAALTSKLDAAYKLAYKSAEGATPEAJ(YDDYVATL SEALRIIAGTLEVHGVKPAAEEVKATPAGELQV1DKVDAjFKVAATAANAA
PANDKFTVFEAAFNDAIKASTGGAYQSYKFJPALEAAVKQSYAATVATAPA
VKYTVFETALKXAITAMSQAQKKPAAAATGTATAAVGAATGAATAAA
GGYKV
113561P0AP9
MAVHQYTVALFLAVALVAGPAASYAADVGYGAPATLATPATPAAPAAGYT
PAAPAGAAPKATTDEQKLIEK[NAGFKAAVAAAAGVPAVDKYKTFVATFGT
ASNKAFAEALSTEPKGAAAASSNAVLTSKLDAAYKLAYKSAEGATPEAKYD
AYVATLSEALRAGTLEVHAVKPAGEEVKAIPAGELQVIDKVDAAFKVAAT
AANAAPANDKFTVFEAAFNDAIKASTGGAYQSYKFJPALEAAVKQSYAATV
ATAPAVKYTVFETALKKAITAMSQAQKAAKPAAAVTATATGAVGAATQAV
GAATGAATAAAGGYKTGAATPTAGGYKV
113560P0AP9 MDKANGAYKTALKAASAVAPAEKFPVFQATFDKNLKEGLSGPDAvGFMJ
LDAFIQTSYLSTKAAEPKEKFDLFVLSLTEVLRFMAGAVKAPPASKFPAKYAP
KVAAYTPAAPAGAAPKATTDEQKLIEK1NVGFK&VAAAAGVPAASKYKTF
VATFGAASNKAFAEALSTEPKGAAVASSKAVLTSKLDAAYKLAYKSAEGAT
PEAKYDAYVATLSEALRIIAGTLEVHGVKPAAEEVKAIPAGELQVIDKvDAA
FKVAATAANAAPANDKFTVFEAAFNDAIKASTGGAYQSYKFIPALEAAVKQ
SYAATVATAPAVKYTVFETALKKAITAMSQAQKAAKPAAAvTGTATSAvG
AATGAATAAAGGYKV
Cockroach sequences 2833325 Crpl
I
MKTALVFAAVVAFVAARFPDHXDYKQLADKQFLAKQRDVLRLFHIRVHQH
NILNDQVEVGIPMTSKQTSATTVPPSGEAVHGVLQEGHARPRGEPFSVNYEK
HREQAIMLYDLLYFANDYDTFYKTACWARDRVNEGMFMYSFSIAVFHRDD
MQGVMLPPPYEVYPYLFVDHDVIHMAQKYWMKNAGSGEHHSHVIPVNFTL
RTQDHLLAYFTSDVNLNAFNTYYRYYYPSWYNTTLYGHNIDRRGEQFYYTY
KQIYARYFLERLSNDLPDVYPFYYSKPVKSAYNPNLRYHNGEEMPVRPSNM
YVTNFDLYYIADIKNYEKRVEDAIDFGYAFDEHMIKPHSLYHDVHGMEYLAD
MIEGNMDSPNFYFYGSIYFIMYHSMIGHIVDPYHKMGLAPSLEHPETVLRDPV
FYQLWKRVDHLFQKYKNRLPRYTHDELAFEGVKVENVDVGKLYTYFEQYD
MSLDMAVYVNNVDQISNVDVQLAVRLNHKPFTYNIEVSSDKAQDVYVAVF
LGPKYDYLGREYDLNDRRHYFVEMDRFPYHVGAGKTVIERNSHDSNIIAPER
DSYRTFYKKVQEAYEGKSQYYVDKGHNYCGYPENLLIPKGKKGGQAYTFY
VIVTPYVKQDEHDFEPYNYKAFSYCGVGSERKYPDNKPLGYPFDRKIYSNDF
YTPNMYFKDVIIFHKKYDEVGVQGH
2231297 Crp2
INEIHSIIGLPPFVPPSRRHARRGVGINGLIDDVIAILPVDELKALFQEKLETSPD
FKALYDArRSPEFQSJISTLNAMQRSEHHQNLRDKGVDVDHFIQLIRALFGLS
RAARNLQDDLNDFLHSLEPISPRHRHGLPRQRRRSARVSAYLHADDFHKIJTT
IEALPEFANFYNFLKEHGLDVVDYINEIHSIIGLPPFVPPSRRHARRGVGINGLI
DDVIAILPVDELKALFQEKLETSPDFKALYDAIRSPEFQSIISTLNAMPEYQEL
LQNLRDKGVDVDHFIRVDQGTLRTLSSGQRNLQDDLNDFLAUpTDQILAIA
MDYLANDAEVQELVAYLQSDDFHKIITTIEALPEFANFYNFLKEHGLDVVDY
INEffISIIGLPPFVPPSQRHARRGVGINGLIDDVIAILPVDELKALFQEKLETSpD
FKALYDAIDLRSSRA
1703445 BIa g 2 MIGLKLVTVLFAVATITHAAELQRVPLYKLVHVFINTQYAGJTKJc3NQNFLTV
FDSTSCNVVVASQECVGGACVCPNLQKYEKLKPKYISDGNVQVKFFDTGSA
VGRGIEDSLTISNLTTSQQDIVLADELSQEVCILSADVVVGL&APGCPNALKG KTVLENFVEENLIAPVFSIHHARFQDGEHFGEUFGGSDWKYVDGEFTyVpLV GDDSWKFRLDGVKIGDTTVAPAGTQAIrDTSKAIIVGPKAYVNPrNIEAIGCVV *
EKTTTRRICKLDCSKIPSLPDVTFVIINGRNFNTSSQYYIQQNGNLCYSGFQPCG
HSDHFFIGDFFVDHYYSEFNWENKTMGFGRSVE sv
1705483 Blag4
AVLALCATDTLANEDCFRHESLVPNLDYERFRGSWIIAAGTSEALTQYKCWI
DRFSYDDALVSKYTDSQGKNRTTIRGRTKFEGNKFTEDYNDKGKAFSAPYSV
LATDYENYA[VEGCPAAANGHVIYVQIRFSVRRFHPKLGDKEMIQHYTLDQV
NQHKKAIEEDLKHFNLKYEDLHSTCH
2326190 Bla g 5
YKLTYCPVKALGEPIRFLLSYGEKDFEDYRFQEGDWPNLKPSMPFGKTPVLE
[DGKQTHQSVAISRYLGKQFGLSGKDDWENLEIDMWDTISDFRAAIANYHY DADENSKQKKWDPLKKET[PYYTKKFDEVVKANGGYLAAGKLTWADFYFV
AILDYLNHMAKEDLVANQPNLKALREKVLGLPAIKAWVAKRPPTDL
Additional cockroach sequences (NCBI Entrez accession numbers): 2580504; 1580797; 1580794; 1362590; 544619; 544618; 1531589; 1580792; 1166573; 1176397; 2897849.
Alternaria sequences: Alt a I -AAM90320 Alt a 2 -AAD00097 Alt a 6-Q9HDT3 Birch sequences: Bet vI-L -P43185 Betvl-M/N -P43186 Betvl-K -P43184 Bet vl-J -P43183 Bet vl-G -P43180 Bet vi -F/I -P43179 * -37-Bet vl-E -P43178 Betvl-D/H -P43177 Betvl-C -P43176 Betvl-B -P45431 Betvl-A -P15494 P14 -Seq 3 -AAB44348 Seq 4 -AAB44349 Seq 5 -AAB44350 Seqó -AAB44351 Seq7 -AAB44352 Seq 8 -AAB44353 Seq 11 -AAB44354 Russian Thistle sequences: Salk 1.0102 (AAXI 1261)
QPIPPNPAELESWFQGAVKPVSEQKGLEPSVVQAESGGVETIEVRQDGSGKF
KTISDAVKHVKVGNTKRVJJTIGPGEYREKVKJEGLHPYJTLYGIDPKNRPTITF
AGTAAEFGTVDSATLIVESDYFVGANLIVSNSAPRPAGKRKGAQASALRJSG
DRAAFYNCKFTGFQDTVCDDKGNHLFKDCYIEGTVDLIFGEARSLYLNTELH
VVPGDPMAMITAHARKNADGVGGYSFVHCKVTGTGGTALLGRAWFEjJ.
VVFSYCNLSDAVKPEGWSDNNT(PAAQKTIFFGEyKNTGpG&rK1vpyTK
QLTEADAKTFTSLEYIEAAKWLP PPPKV
Salk 1.0103(AAX11262)
QPIPPNPAELESWFQGAVKPVSEQKGLEPSVVQAESGGVETIEVRQDGSGKF
KTISDAVK}IVKVGNTKRVIITIGPGEYREKVKJERLHPYITLYGIDPKNRPTITF
AGTAAEFGTVDSATLIVESDYFVGANLIVSNSAPRPDGKRKGARASALRJSG
DRAAFYNCKFTGFQDTVCDDKGNHLFKDCYIEGTVDFIFGEARSLyLNTELR VVPGDPMAM1TAHARNADGVGGYSFVHCKVTGTGGTALLGRwFEA1&J.
VVFSYCNLSDAVKPEGWSDNNKAAQKTIFFGEYKNTGPGADKRVPYTK
QLTEADAKTFTSLEYIEAAKWLL PPPKV
O
Salk 1.0101 (AAT99258) MEEHVSMLLVGFVLINIAFTS1AQLIPPNPAELESWFQGAVKPVSEQKGLEPS VVQTESGGVET[EVRQDGSGKFKTISDAVKHVKVGNTKRVIITIGPGEYREKV 5}UERLHPYITLYGIDPK}4RPTITFAGTAAEFGTVDSATVIVESDYSVGAJ{LIVT
NSAPRPDGKRKGAQAGALRISGDRAAFYNCKFTGFQDTVCDDKGNHFFTDC
YTEGTVDFIFGEARSLYLNTELHVVPGDPMAMITAHARKNADGVGGYSFVH
CKVTGTGGTALLGRAWFDAARVVFSYCNLSDAAKPEGWSDNNKPEAQKTI
LFGEYKNTGPGAAPDKRAPYTKQLTEADAKTFTS LEYIEAAKWLLPPPKV
Sal k 2 (AAN05083)
MVVADLERLKCQAAMDASQLRERLVDLEEHKRRIAEQKAARHEDEERRLG
TTQSPISYRVYTLKEIEVGTDYFSSSLKIGEGGYGPVYRAMLQHTPVAJKVLR
PNVSQGLKQFQQEIDVLGRMRHPNMVLLVGACPEYGCLVYEYMENGSLED
RLFRKNNSPPIPWKLRFKIAAEIAIALLFLRDAKPEPMVHRDLKPANILLDGN
YISKIADVGLARLVPPTVANEITQYHMTAAAGTFCYIDPEYQQTGQLGTKSDI
YSFGIILLQLLTARPPMALSYHVEEAIDAGNFEEVLDPSISDWPVQEALSLAQ
LALKCCEG
Allergen (general) sequences: NCBI accession numbers 2739154; 3719257; 3703107; 3687326; 3643813; 3087805; 1864024; 1493836; 1480457; 2598976; 2598974; 1575778; 763532; 746485; 163827; 163823; 3080761; 163825; 3608493; 3581965; 2253610; 2231297; 2897849; 3409499; 3409498; 3409497; 3409496; 3409495; 3409494; 3409493; 3409492; 3409491; 3409490; 3409489; 3409488; 3409487; 3409486; 3409485; 3409484; 3409483; 3409482; 3409481; 3409480; 3409479; 3409478; 3409477; 3409476; 3409475; 3409474; 3409473; 3409472; 3409471; 3409470; 3409469; 3409468; 3409467; 3409466; 3409465; 3409464; 3409463; 3409462; 3409461; 3409460; 3409459; 3409458; 3409457; 3409456; 3318885; 3396070; 3367732; 1916805; 3337403; 2851457; 2851456; 1351295; 549187; 136467; 1173367; 2499810; 2498582; 2498581; * 1346478; 1171009; 126608; 114091;2506771; 1706660; 1169665; 1169531; 232086; 416898; 114922; 2497701; 1703232; 1703233; 1703233; 1703232; 3287877; 3122132; 3182907; 3121758; 3121756; 3121755; 3121746; 3121745; 3319925; 3319923; 3319921; 3319651; 3318789; 3318779; 3309647; 3309047; 3309045; 3309043; 3309041; 3309039; 3288200; 3288068; 2924494; 3256212; 3256210; 3243234; 3210053; 3210052; 3210051; 3210050; 3210049; 3210048; 3210047; 3210046; 3210045; 3210044; 3210043; 3210042; 3210041; 3210040; 3210039; 3210038; 3210037; 3210036; 3210035; 3210034; 3210033; 3210032; 3210031; 3210030; 3210029; 3210028; 3210027; 3210026; 3210025; 3210024; 3210023; 3210022; 3210021; 3210020; 3210019; 3210018; 3210017; 3210016; 3210015; 3210014; 3210013; 3210012; 3210011; 3210010; 3210009; 3210008; 3210007; 3210006; 3210005; 3210004; 3210003; 3210002; 3210001; 3210000; 3209999; 3201547; 2781152; 2392605; 2392604; 2781014; 1942360; 2554672; 2392209;3114481;3114480;2981657;3183706;3152922;3135503;3135501; 3135499; 3135497; 2414158; 1321733; 1321731; 1321728; 1321726; 1321724; 1321722; 1321720; 1321718; 1321716; 1321714; 1321712; 3095075; 3062795; 3062793; 3062791; 2266625; 2266623; 2182106; 3044216; 2154736; 3021324; 3004467; 3005841; 3005839; 3004485; 3004473; 3004471; 3004469; 3004465; 2440053; 1805730; 2970629; 2959898; 2935527; 2935416; 809536; 730091; 585279; 584968; 2498195; 2833325; 2498604; 2498317; 2498299; 2493414; 2498586; 2498585; 2498576; 2497749; 2493446; 2493445; 1513216; 729944; 2498099; 548449; 465054; 465053; 465052; 548671; 548670; 548660; 548658; 548657; 2832430; 232084; 2500822; 2498118; 2498119; 2498119; 2498118; 1708296; 1708793; 416607; 416608; 416608; 416607; 2499791; 2498580; 2498579; 2498578; 2498577; 2497750; 1705483; 1703445; 1709542; 1709545; 1710589; 1352699; 1346568; 1346323; 1346322; 2507248; 11352240; 1352239; 1352237; 1352229; 1351935; 1350779; 1346806; 1346804; 1346803; 1170095; 1168701; 1352506; 1171011; 1171008; 1171005; 1171004; 1171002; 1171001; 1168710; 1168709; 1168708; 1168707; 1168706; 1168705; 1168704; 1168703; 1168702; 1168696; 1168391; 1168390; 1168348; 1173075; 1173074; 1173071; 1169290; 1168970; 1168402; 729764; 729320; 729979; 729970; 729315; 730050; 730049; 730048; 549194; 549193; 549192; 549191; 549190; 549189; 549188; 549185; * 549184; 549183; 549182; 549181; 549180; 549179; 464471; 585290; 416731; 1169666; 113478; 113479; 113477; 113476; 113475; 130975; 119656; 113562; 113561; 113560; 416610; 126387; 126386; 126385; 132270; 416611;416612; 416612; 416611; 730035; 127205; 1352238; 125887; 549186; 137395; 730036; 133174; 114090; 131112; 126949; 129293; 124757; 129501;416636; 2801531; 2796177; 2796175; 2677826; 2735118; 2735116; 2735114; 2735112; 2735110; 2735108; 2735106; 2735104; 2735102; 2735100; 2735098; 2735096; 2707295 2154730; 2154728; 1684720; 2580504; 2465137; 2465135; 2465133; 2465131; 2465129; 2465127; 2564228; 2564226; 2564224; 2564222; 2564220; 2051993; 1313972; 1313970; 1313968; 1313966; 2443824; 2488684; 2488683; 2488682; 2488681; 2488680; 2488679; 2488678; 2326190; 2464905; 2415702; 2415700; 2415698; 2398759; 2398757; 2353266; 2338288; 1167836; 414703; 2276458; 1684718; 2293571; 1580797; 1580794; 2245508; 2245060; 1261972; 2190552; 1881574; 511953; 1532058; 1532056; 1532054; 1359436; 666007; 487661; 217308; 1731859; 217306; 217304; 1545803; 1514943; 577696; 516728; 506858; 493634; 493632; 2154734; 2154732; 543659; 1086046; 1086045; 2147643; 2147642; 1086003; 1086002; 1086001; 543675; 543623; 543509; 543491; 1364099; 2147108; 2147107; 1364001; 1085628; 631913; 631912; 631911; 2147092; 477301; 543482; 345521; 542131; 542130; 542129; 100636; 2146809; 480443; 2114497; 2144915; 72355; 71728; 319828; 1082946; 1082945; 1082944; 539716; 539715; 423193; 423192; 423191; 423190; 1079187; 627190; 627189; 627188; 627187; 482382; 1362656; 627186; 627185; 627182; 482381; 85299; 85298; 2133756; 2133755; 1079186; 627181; 321044; 321043; 112559; 112558; 1362590; 2133564; 1085122; 1078971; 627144; 627143; 627142; 627141; 280576; 102835; 102834; 102833; 102832; 84703; 84702; 84700; 84699; 84698; 84696; 477888; 477505; 102575; 102572; 478272; 2130094; 629813; 629812; 542172; 542168; 542167; 481432; 320620; 280414; 626029; 542132; 320615; 320614; 100638; 100637; 100635; 82449; 320611; 320610; 280409; 320607; 320606; 539051; 539050; 539049; 539048; 322803; 280407; 100501; 100498; 100497; 100496; 1362137; 1362136; 1362135; 1362134; 1362133; 1362132; 1362131; 1362130; 1362129; 1362128; 100478; 2129891; 1076531; 1362049; 1076486; 2129817; 2129816; 2129815; 2129814; 2129813; 2129812; 2129805; 2129804; 2129802; 2129801;
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2129800; 2129799; 479902; 479901; 2129477; 1076247; 629480; 1076242; 1076241; 541803; 541802; 280372; 280371; 1361968; 1361967; 1361966; 1361965; 1361964; 1361963; 1361962; 1361961; 1361960; 1361959; 320546; 2119763; 543622; 541804; 478825; 478824; 478823; 421788; 320545; 81444; 626037; 626028; 539056; 483123; 481398; 481397; 100733; 100732; 100639; 625532; 1083651; 322674; 322673; 81719; 81718; 2118430; 2118429; 2118428; 2118427; 419801; 419800; 419799; 419798; 282991; 100691; 322995; 322994; 101824; 626077; 414553;398830; 1311457; 1916292; 1911819; 1911818; 1911659; 1911582; 467629; 467627; 467619; 467617; 915347; 1871507; 1322185; 1322183; 897645; 897647; 1850544; 1850542; 1850540; 288917; 452742; 1842045; 1839305; 1836011; 1836010; 1829900; 1829899; 1829898; 1829897; 1829896; 1829895; 1829894; 1825459; 1808987; 159653; 1773369; 1769849; 1769847; 608690; 1040877; 1040875; 1438761; 1311513; 1311512; 1311511; 1311510; 1311509; 1311689; 1246120; 1246119; 1246118; 1246117; 1246116; 1478293; is 1478292; 1311642; 1174278; 1174276; 1086972; 1086974; 1086976; 1086978; 1086978; 1086976; 1086974; 1086972; 999009; 999356; 999355; 994866; 994865; 913758; 913757; 913756; 913285; 913283; 926885; 807138; 632782; 601807; 546852; 633938; 544619; 544618; 453094; 451275; 451274; 407610; 407609; 404371; 409328; 299551; 299550; 264742; 261407; 255657; 250902; 250525; 1613674; 1613673; 1613672; 1613671; 1613670; 1613304; 1613303; 1613302; 1613240; 1613239; 1613238; 1612181; 1612180; 1612179; 1612178; 1612177; 1612176; 1612175; 1612174; 1612173; 1612172; 1612171; 1612170; 1612169; 1612168; 1612167; 1612166; 1612165; 1612164; 1612163; 1612162; 1612161; 1612160; 1612159; 1612158; 1612157; 1612156; 1612155; 1612154; 1612153; 1612152; 1612151; 1612150; 1612149; 1612148; 1612147; 1612146; 1612145; 1612144; 1612143; 1612142; 1612141; 1612140; 1612139; 1093120; 447712; 447711;447710; 1587177; 158542; 1582223; 1582222; 1531589; 1580792; 886215; 1545897; 1545895; 1545893; 1545891; 1545889; 1545887; 1545885; 1545883; 1545881; 1545879; 1545877; 1545875; 166486; 1498496; 1460058; 972513; 1009442; 1009440; 1009438; 1009436; 1009434; 7413; 1421808; 551228; 452606; 32905; 1377859; 1364213; 1364212; 395407; 22690; 22688; 22686; 22684; 488605; 17680; 1052817; 1008445; 1008443; 992612; 706811 * 886683; 747852; 939932; 19003; 1247377; 1247375; 1247373; 862307; 312284; 999462; 999460; 999458; 587450; 763064; 886209; 1176397; 1173557; 902012; 997915; 997914; 997913; 997912; 997911; 997910; 99790; 997908; 997907; 997906; 997905; 997904; 997903; 997902; 997901; 997900; 997899; 997898; 997897; 997896; 997895; 997894; 997893; 997892; 910984; 910983; 910982; 910981; 511604; 169631; 169629; 169627; 168316; 168314; 607633; 555616; 293902; 485371; 455288; 166447; 166445; 166443; 166435; 162551 160780; 552080; 156719; 156715; 515957; 515956; 515955; 515954; 515953 459163; 166953; 386678; 169865.
Particularly preferred T cell epitopes are derived from the allergens: cat dander protein Fel dl; House dust mite proteins Der P1, Der P2 and Der P7; Ragweed protein amb a 1; Latex allergens Hey b5 and hey b6; Rye grass proteins lol p1 and lol p5; Timothy grass proteins phi p1 and phl p5; Alternaria alternate proteins Alt a 1, Alt a 2 and Enolase (Alt a 6); Birch protein Bet vi and P14; German Cockroach proteins Bla g 1, Bla g 2, Bla g 3, Bla g 4, Bla g 5 and Bla g 6; Mugwort protein Art v 1; Russian thistle proteins Sal k I and Sal k 2; peanut proteins Ara hi, h2,h3,h5,h6 The allergen may also be in the form of an analogue which mimics the epitope of the naturally occurring protein bound to a MHC molecule.
In one embodiment T cell responses to 2 or more different allergens, or 2 or more different fragments of the same allergen are investigated, for example using combinations of allergens or peptides.
The presence (and magnitude) or absence of a T cell response to an allergen is typically determined by detecting cytokine production by a T cell. Preferred cytokines for detection are IL-4, IL-9, IL-13 and IL-5. Other cytokines include, for example, IFN-y, IL-2, IL-lO.
A cytokine can typically be detected by allowing it to bind to a specific capture agent which may be immobilised on a support such as a plate, bead or the cytokine-secreting cell itself, and then measuring the presence of the specific binding agentlcytokine complex typically with a second binding detection agent. A washing step can be incorporated to remove material which is not specifically bound to the capture agent.
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Typically the second agent binds the cytokine at a site which is different from the site which binds the first agent. The second agent may be directly conjugated to an enzyme such as alkaline phosphatase, or fluorescent label or may comprise a biotin moiety to be detected by a third agent comprising streptavidin, which is directly conjugated to an enzyme or fluorescent label. The conjugated enzyme then changes colour of a reagent. The agent is preferably an antibody, mono-or polyclonal. Antibodies to cytokines are commercially available, or can be made using standard techniques.
The preferred method employed to detect cytokines will be based on sandwich immunoassays detecting the frequency of cytokine-secreting cells such as colour or fluorescent ELISpot, limited dilution assays, intracellular cytokine staining and cytokine secretion assays with or without enrichment of cytokine-secreting cells as pioneered by Miltenyi Biotec. Alternatively, the amount of cytokine secreted can be measured for example by an ELISA based system such as the whole blood Quantiferon� system with the capture antibody immobilised on a plate, and its modifications (for example as available from Cellestis) or Luminex� suspension array technology using Beadlyte� kits with the capture antibody immobilised on a bead. Cytokine mRNA expression can also be measured with assays such as RT-PCR.
In one embodiment the detection system which is used is the ex-vivo ELISpot assay described in WO 98/23960. In that assay IFN-7 secreted from the T cell is bound by a first IFN-y specific antibody which is immobilised on a solid support.
The bound IFN-y is then detected using a second IFN-y specific antibody which is labelled with a detectable label. Such a labelled antibody can be obtained from MABTECH (Stockholm, Sweden). Other detectable labels which can be used are discussed below. Kits
The invention also provides a kit for carrying out the methods of the invention comprising a means for determining whether an individual has a T cell response to an allergen, or a variant thereof. Typically the means to detect recognition allows or aids detection based on the techniques discussed above. Thus,
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the means may allow detection of a cytokine secreted by the I cells after recognition.
The kit may thus additionally include a specific binding agent for the cytokine, such as an antibody. The agent is typically immobilised on a solid support. This means that after binding the agent the cytokine will remain in the vicinity of the T cell that secreted it. Thus spots' of cytokine/agent complex are formed on the support, each spot representing a T cell which is secreting the cytokine. Quantifying the spots, and typically comparing against a control, allows determination of the relative numbers of cells that secrete the cytokine.
The kit may also comprise a means to detect the cytokine/agent complexes.
A detectable change may occur in the agent itself after binding cytokine, such as a colour change. Alternatively a second agent directly or indirectly labelled for detection may be allowed to bind the cytokine /agent complex to allow the determination of the spots. As discussed above the second agent may be specific for the cytokine, but binds a different site on the cytokine than the first agent.
The immobilised support may be a plate with wells, such as a microtitre plate.
Each assay can therefore be carried out in a separate well in the plate.
The kit may additionally comprise medium for the T cells, detection agents or washing buffers to be used in the detection steps. The kit may additionally comprise reagents suitable for the separation from the sample, such as the separation of PBMCs or T cells from the sample. The kit may be designed to allow detection of the T cells directly in the sample without requiring any separation of the components of the sample.
The kit may also comprise controls, such as positive or negative controls.
The positive control may allow the detection system to be tested. Thus the positive control typically mimics recognition of the peptide in any of the above methods.
Typically in the kits designed to determine recognition in vitro the positive control is a cytokine, such as IFN-y andlor IL-2.
The kit may also comprise a means to take a sample containing T cells from the human, such as a blood sample. The kit may comprise a means to separate mononuclear cells or T cells from a sample from the individual.
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Peptides As discussed above, the allergen is typically a present as a fragment (such as a peptide) of an allergen molecule. The peptide typically comprises at least one epitope from an allergen The peptide may be from a naturally occurring protein which is recognised by a T cell that recognises a natural T cell epitope sequence froman allergen. The peptide may comprise a sequence which is recognised by a T cell which recognises an epitope from an allergen. Such sequences may have at least 70%, 80%, 90% homology to the original epitope sequence. In one embodiment the peptide comprises at least one MHC Class 11-binding T cell epitope from an allergen.
The peptide typically consists of less than 30 contiguous amino acids from an allergen molecule, or a variant thereof. The peptides may comprise, consist of, or consist essentially of the sequences shown in any of SEQ ID NOS: ito 65. Variants or analogues of the above peptides may also be used.
Variants and analogues ofpeptides Variant or analogue peptides are mentioned herein. Such variants or analogues typically comprise a sequence that binds to the same MHC class II molecule and/or is recognised by a T cell which recognises the corresponding epitope in the polypeptide of SEQ ID NOS: Ito 65.
Variants of SEQ ID NO's 1 to 65 may be fragments derived by truncation, e.g. by removal of one or more amino acids from the N and/or C-terminal ends of a polypeptide. Fragments may also be generated by one or more internal deletions, provided that the core 9 amino acids that makes up the T cell epitope is not substantially disrupted.
For example, a variant of SEQ ID NO: I may comprise a fragment of SEQ ID NO: 1, i.e. a shorter sequence. This may include a deletion of one, two, three or four amino acids from the N-terminal end of SEQ ID NO: 1 or from the C-terminal end of SEQ ID NO: 1. Such deletions may be made from both ends of SEQ ID NO: 1. A variant of SEQ ID NO: 1 may include additional amino acids (for example from the sequence of the parent protein from which the peptide derives) extending beyond the end(s) of SEQ ID NO: 1. A variant may include a combination of the deletions and additions discussed above. For example, amino acids may be deleted from one end * -46-of SEQ ID NO: 1, but additional amino acids from the full length parent protein sequence may be added at the other end of SEQ ID NO: I. The same discussion of variants above also applies to SEQ ID NOS: 2 to 65.
A variant peptide may include one or more amino acid substitutions from the amino acid sequence of any of SEQ ID NOS: 1 to 65 or a fragment thereof. A variant peptide may comprise sequence having at least 65% sequence identity to at least 9 or more contiguous amino acids in any of SEQ ID NOS: 1 to 65. More preferably a suitable variant may comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% amino acid identity to at least 9 contiguous amino acids of any of SEQ ID NO: ito 65. This level of amino acid identity may be seen at any section of the peptide, although it is preferably the core region. The level of amino acid identity is over at least 9 contiguous amino acids but it may be at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids, depending on the size of the peptides of comparison. Accordingly, any of the above-specified levels of identity may be across the entire length of sequence.
In connection with amino acid sequences, "sequence identity" refers to sequences which have the stated value when assessed using ClustalW (Thompson et al., 1994, supra) with the following parameters: Pairwise alignment parameters -Method: accurate, Matrix: PAM, Gap open penalty: 10.00, Gap extension penalty: 0.10; Multiple alignment parameters -Matrix: PAM, Gap open penalty: 10.00, % identity for delay: 30, Penalize end gaps: on, Gap separation distance: 0, Negative matrix: no, Gap extension penalty: 0.20, Residue-specific gap penalties: on, Hydrophilic gap penalties: on, Hydrophilic residues: GPSNDQEKR. Sequence identity at a particular residue is intended to include identical residues which have simply been denvatized.
A variant peptide may comprise 1, 2, 3, 4, 5 or more, or up to 10 amino acid substitutions from any of SEQ ID NOS: 1 to 65. Substitution variants preferably involve the replacement of one or more amino acids with the same number of amino acids and making conservative amino acid substitutions. For example, an amino acid may be substituted with an alternative amino acid having similar properties, for example, another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another
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hydrophobic amino acid, another polar amino acid, another aromatic amino acid or another aliphatic amino acid. Some properties of the 20 main amino acids which can be used to select suitable substituents are as follows: \la aliphatic, hydrophobic, neutral VIet hydrophobic, neutral ys polar, hydrophobic, neutral sn)olar, hydrophilic, neutral \sp polar, hydrophilic, charged (-) ro iydrophobic, neutral 31u polar, hydrophilic, charged (-) Gln)olar, hydrophilic, neutral he aromatic, hydrophobic, neutral rg)Olar, hydrophilic, charged (+) 3ly aliphatic, neutral Ser,olar, hydrophilic, neutral us aromatic, polar, hydrophilic, [hr olar, hydrophilic, neutral charged (+) [le aliphatic, hydrophobic, neutral Val aliphatic, hydrophobic, neutral bys polar, hydrophilic, charged(+) [rp aromatic, hydrophobic, neutral aliphatic, hydrophobic, neutral [yr aromatic, polar, hydrophobic Further variants include those in which instead of the naturally occurring amino acid the amino acid which appears in the sequence is a structural analog thereof. Amino acids used in the sequences may also be modified, e.g. labelled, providing the function of the peptide is not significantly adversely affected.
Where the peptide has a sequence that varies from the sequence of any of SEQ ID NOS: 1 to 65 or a fragment thereof, the substitutions may occur across the full length of the sequence, within the sequence of any of SEQ ID NOS: 1 to 65 or outside the sequence of any of SEQ ID NOS: ito 65. For example, the variations described herein, such as additions, deletions, substitutions and modifications, may occur within the sequence of any of SEQ ID NOS: ito 65. A variant peptide may comprise or consist essentially of the amino acid sequence of any of SEQ ID NOS: I to 65 in which one, two, three, four or more amino acid substitutions have been made. A variant peptide may comprise a fragment of the parent protein that is larger than any of SEQ ID NOS: 1 to 65. In this embodiment, the variations described * -48-herein, such as substitutions and modifications, may occur within andlor outside the sequence of any of SEQ ID NOS: Ito 65.
The variant peptides of the invention are 9 to 30 amino acids in length inclusive. Preferably, they may be from 9 to 20 or more preferably 13 to 17 amino acids in length. The peptides may be the same length as the peptide sequences in any one of SEQ ID NOS: I to 65.
The term "peptide" includes not only molecules in which amino acid residues are joined by peptide (-CO-NH-) linkages but also molecules in which the peptide bond is reversed. Such retro-inverso peptidomimetics may be made using methods known in the art, for example such as those described in Meziere et a! (1997) J. Immunol.1 59, 3230-323 7. This approach involves making pseudopeptides containing changes involving the backbone, and not the orientation of side chains. Meziere et a! (1997) show that, at least for MHC class II and T helper cell responses, these pseudopeptides are useful. Retro-inverse peptides, which contain NH-CO bonds instead of CO-NH peptide bonds, are much more resistant to proteolysis.
Similarly, the peptide bond may be dispensed with altogether provided that an appropriate linker moiety which retains the spacing between the carbon atoms of the amino acid residues is used; it is particularly preferred if the linker moiety has substantially the same charge distribution and substantially the same planarity as a peptide bond. It will also be appreciated that the peptide may conveniently be blocked at its N-or C-terminus so as to help reduce susceptibility to exoproteolytic digestion. For example, the N-terminal amino group of the peptides may be protected by reacting with a carboxylic acid and the C-terminal carboxyl group of the peptide may be protected by reacting with an amine. Other examples of modifications include glycosylation and phosphorylation. Another potential modification is that hydrogens on the side chain amines of R or K may be replaced with methylene groups (-NH2 - � -NH(Me) or -N(Me)2).
Analogues of peptides according to the invention may also include peptide variants that increase or decrease the peptide's half- life in vivo. Examples of analogues capable of increasing the half-life of peptides used according to the invention include peptoid analogues of the peptides, D-arnino acid derivatives of the peptides, and peptide-peptoid hybrids. A further embodiment of the variant
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polypeptides used according to the invention comprises D-aniino acid forms of the polypeptide. The preparation of polypeptides using D-amino acids rather than L-amino acids greatly decreases any unwanted breakdown of such an agent by normal metabolic processes, decreasing the amounts of agent which needs to be administered, along with the frequency of its administration.
The peptides provided by the present invention may be derived from splice variants of the parent proteins encoded by mRNA generated by alternative splicing of the primary transcripts encoding the parent protein chains. The peptides may also be derived from amino acid mutants, glycosylation variants and other covalent derivatives of the parent proteins which retain at least an MHC-binding property of the parent proteins. Exemplary derivatives include molecules wherein the peptides of the invention are covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid. Further included are naturally occurring variants of the parent proteins. Such a variant may be encoded by an allelic variant or represent an alternative splicing variant.
Variants as described above may be prepared during synthesis of the peptide or by post-production modification, or when the peptide is in recombinant form using the known techniques of site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids.
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Examples
Example I
Peptides from House Dust Mite allergens Peptides for use in the method of the invention are identified as set out below: MHC Class II binding search The aim of this study is to identify a distinct panel of peptides with strong affinities for the seven most common human MIHC Class II HLADRB1* allotypes (covering in total around 63% of the allotypes found in the average Caucasian population). In order to identify binding peptides in the House Dust Mite (I-IDM) allergens, Der p 1, Der p 2 and Der p 7, in vitro binding assays have been performed on a subset of peptides from these allergenic proteins. Peptides for testing in the binding assays were initially identified by an in silico approach known as "peptide threading" (earned out by Biovation, Ltd.,Aberdeen, Scotland, UK). This is a bioinformatic analysis of consecutive peptides from a sequence for the potential to be accommodated within the binding groove of MHC class II HLA-DR molecules. This subset of peptides was pre-screened for solubility in an aqueous, acidic milieu and a final panel of 44 peptides selected for testing in an in vitro MHC Class II binding assay.
Methods The assay employed is a competitive MHC class II binding assay, wherein each peptide is analysed for its ability to displace a known control binder from each of the human MHC class II allotypes investigated. The allotypes and control peptides used in this study are shown in Table 2: Allotype Control Peptide Seiuence DRBI *0101 Influenza haemagglutinin 307-319 PKYVKQNTLKLAT DRB1*0301 Myco. tuberculosis/leprae hsp 65 2-16 AKTIAYDEEARRGLE DRB 1*0401 Influenza haemagglutinin 307-319 PKYVKQNTLKLAT DRB 1*0701 Influenza haemagglutinin 307-319 PKYVKQNTLKLAT DRBI*1 101 Influenza haemagglutinin 307-3 19 PKYVKQNTLKLAT S -51-DRB * 1301 HLA-DQB 1*0603 21-36 TERVRLVTRHIYNREE DRBI* 1501 Human myelin basic protein 85-99 ENPVVHFFKNIVTPR DQB1*0602 Human Insulin B 1-15 FVNQHLCGSHLVEAL Table 2. Control peptides used in the in vitro binding assays Each of the 44 HDM peptides (which are shown in Tables 3A and 3B) were analysed in the competition assay and screened for relative binding compared to the control peptide. Due to the nature of the competitive assay the data for each peptide is represented as a ratio of its own 1C50 to that of the control peptide. Thus, a peptide that has an 1C50 value that is parity to the control peptide has an identical binding affinity, while peptides with a ratio less than one have a higher affinity and those with a ratio greater than one have a lower affinity.
Results Solubility in aqueous solution is an essential criterion for a peptide to be an effective therapeutic agent. Therefore, as a consequence of the solubility screen we will have eliminated very hydrophobic peptides with a high frequency of large hydrophobic amino acid residues in multiple binding registers. This is a characteristic of promiscuous HLADRB1* binders. The data from the binding assays is shown in Table 3B. The relative binding of each peptide is shown for each of the allotypes in the study. The data shows that 24 of the 44 peptides tested bound to one or more of the MHC Class II allotypes. A range of cross-reactivity is seen with 5 peptides binding only one allotype, 8 peptides binding two, 9 peptides binding three and two peptides binding four different M}IC Class II allotypes (red). It would also be expected that such peptides would have the ability to bind similar allotypes that have not been tested through the homology of M}IC structures. This can be seen in the cross-reactivity of peptides for DRIBI*0101, *0401, *0701 and *1101 in several cases here. Also shown is the solubility status of the peptide at the highest concentrations in the aqueous solution of the binding assay. The value illustrates the lowest concentration at which an insoluble white precipitate is seen. There appears to be no significant nonspecific effect of the formation of precipitate in the assays.
Several peptides that precipitate at high concentrations also bind to MHC class II; O -52-however, several also show no ability to compete with the control peptides. It is to be expected that peptides liable to form precipitates may exhibit high affinity and promiscuous binding due to the presence of many hydrophobic residues.
The % purity of the peptides is indicated in Table 3A. This is of significance as purities were seen to vary from 60-90%. This would have a considerable effect on the ability of a peptide to compete if it is relatively impure. For example, HDM23A and HDM32 show low affinity binding; however, they are of reduced purity (66.7% and 68.7% respectively) compared to other HDM peptides. Therefore, if purity is taken into consideration, they may in fact have an equivalent affinity to a peptide of a higher purity.
It can be seen that some MHC Class II allotypes bind to more peptides than others; this is probably to be expected as there is variability between the pocket positions in the different MHC class II binding grooves. There are however, also a number of well-characterised differences between the affinities of the various control peptides. Clearly a high affinity control peptide will be more difficult to displace by the competing HDM peptide resulting in the identification of fewer binding peptides.
This can be illustrated by the data presented here. For example, the Influenza Haemagglutinin 307-319 control peptide, has varying affinity according to the allotype, where DRB1*OlOl >*0401>*0701>* 1101. This is reflected in the number of binders to each of the allotypes, where DRB1*0101 has the lowest number of binders (5) and DRB I * 1101 has the highest( 14). Furthermore, the binding assay for DRB1* 1501 is very stringent due to the high affinity of Myelin Basic Protein 85-99 for this allotype. In the high stringency screen the Fel d I peptide EQVAQYKALPVVLENA, that was tested in an earlier study, gave a ratio of 0.97 indicating that high affinity binders could be identified at this stringency.
In addition, to identify lower affinity binders, the assay was also carried out under less stringent conditions. All the Der p binding peptides were seen to have a high ratio when tested against this allotype, showing they were low affinity binders compared to the control peptide. The DQA1*O1O2IDQBI*0602 binding assay uses a peptide from the B-chain of human insulin which is of lower affinity compared to those used in the DR assays. This dictates that the DQ assay is very sensitive and tends to produce very low ratio values for the strongest binders to this MHC Class II * -53-allotype. This sensitivity also accounts for the relatively higher number of DQ binding peptides within the panel screened. Finally, on closer analysis, the peptides identified as ligands for the DRB1*0l01,*0401, *0701 superfamily, are found to incorporate a motif that is characteristic of promiscuous binders to this family of allotypes where: P1 Y, F, W, L, 1, V, or M (Large aromatic or hydrophobic residue), P6 S, T, C, A, P, V, I, M (small, non-charged residue) Out of the 16 peptides (e.g. HDM 21B RGKPFQLEAVFEANQNT) identified as binders to all or a combination of these 3 allotypes, 14 (87.5%) contain this motif, which suggests that these are promiscuous binders with a range of affinities for the 1- 4-7 allotypes.
Conclusions
A range of peptides have been shown to have the capacity to bind the MHC Class II allotypes tested and can be tested for their ability to stimulate in vitro proliferation of CD4+ T cells and to stimulate T cell cytokine secretion.
Table 3A
Peptide Sequence Residues % Solubility test Precipitation __________ _____________ in parent purity _____________ in assay HDMOI IDLRQMRTVT 112-124 79.2 YES None
_________ PIR
HDMO2 RTVTPIRMQG 118-130 79.6 YES None
_________ GCG
HDMO3C RNQSLDLAEQ 149-167 60.1 YES None
_________ ELVDCASQH _________ _______ ___________ ___________
HDMO5 EYIQHNGVVQ 179-191 77.5 YES None
_________ ESY _________
HDMO6 RYVAREQSCR 193-205 79.7 YES None
_________ RPN
HDMO7 PNVNKIREAL 220-232 88.6 YES None
_________ AQT _________ _______ ___________ __________
HDMO8 NKIREALAQT 223-235 87.6 YES None
________ HSA
HDMO9A REALAQTHSA 226-239 69.6 YES 1000.tM __________ IAVI __________ ________ _____________ (2.9mg/mI) HDMII IGIKDLDAFR 240-252 77. 6 YES None
_________ HYD
fIDMI2 KDLDAFRHYD 243-255 72.9 YES None
_________ GRT
HDM13 RTIIQRDNGY 254-267 70.7 NO None
_________ QPNY _________ _______ ____________ ___________
HDM16A RNSWDTNWGD 287-300 70.00 YES None
_________ NGYG _________ _______ ____________ ___________
HDMI7 NSVNVPSELD 105-120 74.5 YES None
_________ LRSLRT _________ _______ ____________ ___________
HDMI9 DQVDVKDCAN 18-32 81.4 YES None
HEIKK _________ _______ ___________ __________
HDM2O CIIHRGKPFQ 44-56 77.4 YES None
LEA ________ ______ ___________ __________
HDM2I KPFQLEAVFE 50-64 88.7 YES 200iM _________ ANQNT _________ _______ ___________ (0.3 mg/mi) HDM21A KPFQLEAVFE 50-65 90.10 YES 50001. tM _________ ANQNTK _________ _______ ____________ (9.3 mg/mi) HDM21B RGKPFQLEAV 48-64 82.60 YES 1000.tM _________ FEANQNT _________ _______ ____________ (1.98mg/mi) HDM22A EAVFEANQNT 55-68 90.30 YES None
_________ KTAK _________ _______ ____________ ___________
HDM23A DGLEVDVPGI 76-88 66.7 YES None
__________ DPNACH __________ ________ _____________ ____________
HDM26A DGVLACAIAT 131-145 1000pM ________ HAKIR ________ _______ ___________ (1.5mg/mi) HDM27 AKIEIKASLD 67-79 65.9 YES 1000pM ________ GLE ________ _______ ___________ (1.4mg/mi) HDM28 KAVDEAVAAI 31-43 86.8 YES 1000pM ________ EKS ________ _______ ___________ (1.3mg/mi) HDM29 ETFDPMKVPD 44-56 84.7 YES None
_________ HSD _________ _______ ____________ ___________
HDM29A ETFDPMKVPD 44-57 91.7 YES None
H S DK __________ ________ _____________ ____________
HDM29B KSETFDPMKV 42-56 92.5 YES 1000pM _________ PDHSD _________ _______ ___________ (1.7mg/mi) HDM3O DKFERHIGII 56-68 81.4 YES 5000pM _________ DLK _________ _______ ____________ (7.9mg/mi) HDM31 IGIIDLKGEL 62-75 1000pM ________ DMRN ________ _______ ___________ (1.8mg/mi) HDM31A HIGIIDLKGE 61-75 66.40 YES 1000j.iM ________ LDMRN ________ _______ ___________ (1.7mg/mi) HDM32 IDLKGELDMR 65-77 68.7 YES 5000i.tM _________ NIQ _________ _______ ___________ (7.7mg/mi) HDM32A IDLKGELDMR 65-79 85.20 YES 5000pM ________ NIQVR ________ _______ ___________ (9.0mg/mi) HDM33 LDMRNIQVRG 71-83 70.3 YES None
_________ LKQ _________ _______ ___________ __________
HDM34 RNIQVRGLKQ 74-88 74.7 YES None
MKRVG _________ _______ ___________ __________
O
HDM35 RGLKQMKRVG 79-91 84.00 YES None
________ DAN ________ ______ ___________ __________
HDM36 KRVGDANVKS 85-97 82.9 YES None
__________ E DG _________ _______ ____________ ___________
HDM37 ANVKSEDGVV 90-102 76.5 YES None
__________ KAH __________ ________ _____________ ____________
HDM39 DDVVSMEYDL 109-121 84.9 NO* None
________ AYK ________ ______ ___________ __________
HDM39A HDDVVSMEYD 108-121 80.9 YES 1000.iM ________ LAYKL ________ ______ __________ (1.8mg/mi) HDM4OA VSMEYDLAYK 112-124 66.9 YES 1000.tM ________ LGDLH ________ ______ ___________ (1.8mg/mi) HDM48 TAIFQDTVRA 187-200 79.1 YES 1000tM _________ EMTK ________ _______ ___________ (1.6mg/mi) HDM49 DTVRAEMTRV 192-204 69.5 YES None
________ LAP ________ ______ ___________ __________
HDM5O KVLAPAFKKELE 200-212 90.8 YES None
R
HDM5I VDFKGELAMRNI 65-77 79.8 YES 1000pxM E (1.5mg/mi) HDM5IA VDFKGELANRNI 65-79 82.1 YES None
EAR
Table 3B
Peptide DRBI*01O1 DRB1*0301 DRB1*0401 DRBI*0701 DRB1*I101 DRBI*1301 DRB1*1501 DQA1O1O2 _________ ___________ ___________ ___________ ___________ ___________ ___________ ___________ DQB1 *0602 HDMO1 __________ 19.23 __________ 16 __________ __________ __________ ___________ HDMO2 ___________ ___________ ___________ ___________ ___________ ___________ 80 0.03 HDMO3C ________ ________ ________ ________ ________ ________ ________ 0.16 HDMO5 ________ ________ ________ ________ ________ ________ HDMO6 ___________ ___________ ___________ ___________ 30.36 ___________ ___________ 0.86 HDMO7 ________ ________ ________ ________ ________ ________ HDMO8 ________ ________ ________ ________ ________ ________ HDMO9A __________ __________ __________ 0.49 21.15 __________ 200 __________ HDM11 _______ _______ _______ _______ _______ _______ _______ _______ I-IDM12 ________ ________ ________ ________ ________ ________ HDM13 ________ _______ _______ ________ ________ ________ _______ ________ HDMI6A _______ _______ _______ _______ _______ _______ HDM17 _______ _______ _______ _______ _______ _______ _______ ________ HDM19 ________ _______ _______ ________ ________ ________ _______ ________ HDM2O _________ _________ _________ 1.1 28 __________ 242.11 2. 37 HDM21 92 _________ 11.15 _________ 11.73 _________ _________ __________ HDM21A 200 __________ 52.17 __________ 10.27 __________ __________ ___________ HDM21B 13.5 __________ 0.78 __________ 4.1 __________ __________ ___________ HDM22A ___________ ___________ 328.6 ___________ 80 ___________ ___________ ____________ HDM23A ___________ 347 ___________ ___________ ___________ ___________ ___________ 0.76 HDM26A 42.3 __________ __________ 16.28 __________ __________ __________ 0.61 HDM27 ________ ________ ________ ________ ________ ________ ________ ________ HDM28 ________ ________ ________ ________ ________ ________ ________ ________ HDM29 ________ ________ ________ ________ ________ ________ ________ ________ HDM29A ________ ________ ________ ________ ________ ________ ________ ________ HDM29B ________ ________ ________ ________ ________ ________ ________ ________ HDM3O ________ ________ ________ 6.2 ________ ________ ________ ________ HDM3I _______ _______ ________ ________ ________ ________ ________ ________ HDM31A _______ _______ ________ ________ ________ ________ ________ ________ HDM32A ________ ________ ________ ________ ________ ________ ________ _________ HDM33 __________ __________ __________ 46.51 41.5 263.16 __________ ___________ 1-1DM34 __________ __________ __________ __________ 3.38 3.7 769.23 ___________ HDM35 __________ __________ __________ __________ 1.26 __________ __________ ___________ HDM36 ________ ________ ________ ________ ________ ________ ________ ________ HDM37 ________ ________ ________ ________ ________ ________ ________ ________ HDM39 ________ ________ ________ ________ ________ ________ ________ ________ HDM39A __________ __________ 76.19 0.71 __________ __________ __________ 0.1 HDM4OA ___________ ___________ ___________ 2.29 6 ___________ ___________ ____________ HDM48 __________ 211.26 __________ 15.71 13.57 __________ __________ __________ HDM49 __________ __________ __________ __________ __________ __________ 671.43 1.7 HDM5O _______ _______ ________ ________ ________ ________ ________ ________ 1-IDM5I ___________ ___________ ___________ 20.93 30.91 ___________ ___________ ___________ Homology search The sequences of each of the 24 peptides identified above as MHC Class 11-binding were used to probe the sequence of the alternative protein in the dust mite allergen group from which the parent sequence derived. For example, peptide HDMOI (SEQ in Table 3A is from Der p 1, therefore the sequence of HDMO1 was used to probe for a homologous sequence in Der f 1. The same practice was applied for all 24 peptides identified above. The peptides identified in Example 1 and Example 2 are shown in
Tables 4 to 6
Table 4 -peptides from Group I HDM allegems
_______________
Peptide in Parent Sequence Residues SEQ ID Table 3A/B molecule ___________________________ in parent NO: HDMO1 Derpi IDLRQMRTVTPIR 112-124 1 ___________ Derf 1 LDLRSLRTVTPIR 113-1 25 25 HDMO2 Derpi RTVTPIRMQGGCG 118-130 2 ___________ Derf 1 RTVTPIRMQGGCG 119-131 26 HDMO3C Der p 1 RNQSLDLAEQELVDCASQH 149-167 3 ___________ Derf I RNTSLDLSEQELVDCASQH 150-168 27 HDMO6 Der p I RYVAREQSCRRPN 193-205 4 ____________ Der f I PYVAREQRCRRPN 194-206 28 HDMO9A Der p 1 REALAQTHSAIAVI 226-239 5 ___________ Derfl REALTQTHTAIAVI 227-240 29 Table 5 -peptides from Group 2 HDM allergens Peptide in Parent Sequence Residues SEQ ID Table 3A/B molecule ___________________________ in parent NO: HDM19 Der p2 DQVDVKDCA1HEIKK 18-32 6 __________ Derf2 DQVDVKDCANNEIKK 18-32 30 HDM2O Derp2 CIIHRGKPFQLEA 44-56 7 __________ Derf2 CIIHRGKPFTLEA 44-56 31 HDM21 Derp2 KPFQLEAVFEANQNT 50-64 8 __________ Der (2 KPFTLEALFDANQNT 50-64 32 _______________ _____________ -59-____________ ____________ HDM21A Derp2 KPFQLEAVFEANQNTK 50-65 9 _________ Derf2 KPFTLEALFDANQNTK 50-65 33 HDM21B Derp2 RGKPFQLEAVFEANQNT 48-64 10 __________ Der f 2 RGKPFTLEALFDANQNT 48-64 34 HDM22A Der p2 EAVFEANQNTKTAI< 55-68 11 ___________ Der f 2 EALFDANQNTKTAK 55-68 35 HDM23A Derp2 DGL. EVDVPGIDPNACH 76-88 12 ___________ Derf2 DGLEIDVPGIDTNACH 76-88 36 HDM26A Derp2 DGVLACAIATHAKIR 131-145 13 ___________ Derf2 NGVLACAIATHGKIR 131-145 37 Table 6 -peptides from Group 3 HDM allergens Peptide in Parent Sequence Residues SEQ ID Table 3A/B molecule ___________________________ in parent NO: HDM3O Der p 1 DKFERHIGIIDLK 56-68 14 ____________ Der f 7 DKPERHVGIVDFK 56-68 38 HDM32 Der p 7 IDLKGELDMRNIQ 65-77 15 ___________ Der f 7 VDFKGELAMRNIE 65-77 39 HDM33 Derp7 LDMRNIQVRGLKQ 71-83 16 ___________ Derf7 LAMRNTEARGLKQ 71-83 40 HDM34 Der p 7 RNIQVRGLKQMKRVG 74-88 17 ___________ Derf7 RNIEARGLKQMKRQG 74-88 41 HDM35 Der p 7 RGLKQt1KRVGDAN 79-91 18 ___________ Derf7 RGLKQMKRQGDAN 79-91 42 HDM39A Derp7 FIDDVVSMEYDLAYKL 108-122 19 ___________ Derf7 HDDIVSMEYDLAYKL 108-122 43 HDM4OA Derp7 VSMEYDLAYKLGDLH 112-126 20 ___________ Derf7 VSMEYDLAYKLGDLH 112-126 44 HDM48 Der p 7 TAIFQDTVRAEMTK 187-200 21 ___________ Derf7 TAIFQDTVRKEMTK 187-200 45 HDM49 Der p 7 DTVRAEMTKVLAP 192-204 22 ____________ Der f 7 DTVRKEMTKVLAP 1 92-204 46 HDM51 Der f 7 VDFKGELAHRNIE 65-77 23 ___________ Derp 7 IDLKGELDMRNIQ 65-77 15 HDM51A Derf 7 VDFKGELAMRNIEAR 65-79 24 ___________ Derp 7 IDLKGELDMRNIQVR 65-79 47
Example 2
Peptides from Cat allergens
1. Introduction
The following peptides from the Cat allergen Fel d 1 were selected on the basis of MI-IC binding (assay as described above) as being particularly suitable for use in the method of the invention: CPA VKRD VDLFLT (SEQ ID NO: 48); EQVAQYKALPVVLENA (SEQ ID NO: 49); KALPVVLENARILKNCV (SEQ ID NO: 50); RILKNCVDAKMTEEDKE (SEQ ID NO: 51); KENALSLLDKIYTSPL (SEQ ID NO: 52); TAMKKIQDCYVENGLI (SEQ ID NO: 53); SRVLDGLVMTTISSSK (SEQ ID NO: 54); The following experiments show that these peptides can be used individually or in combination to detect T cell responses to the Fel d I allergen.
2. Materials and Methods 2.1 Isolation of Peripheral Blood Mononuclear Cells Peripheral blood mononuclear cells (PBMC) were isolated from the heparinised blood sample obtained from the subject. PBMC's were isolated by Ficoll-Hypaque density gradient separation. Once isolated, the cells were used in the cytokine release assay.
2.2 Cytokine release assay Cytokine secretion profiles from PBMC's was analysed in response to the peptide stimulation. Supernatants from the cytokine release assay were tested for the presence of 3 cytokines, IFN--y, IL-lO and IL-13, using ELISA assays.
The cytokine release assay required 40x 106 PBMC's per subject. In more detail, 25Otl of a 200p.g/ml solution of the appropriate antigen or peptide concentration was distributed into the appropriate wells of 48 well plates. Plates were the incubated in a humidified 5% CO2 incubator at 37°C for a maximum of 4 hours. 250pJ of a 5x106 cell/mI PBMC suspension was then added to each well and the plates returned to the incubator for 5 days.
Following stimulation, samples of culture supernatant were harvested into 3 aliquots and frozen until the ELISA assays could be performed. One aliquot was tested for the presence of one cytokine (therefore all 3 aliquots were required to test for the 3 cytokines). The cytokine levels in the samples were determined by interpolation from standard curves also generated in the assay.
3. Results.
3.1 Cytokine Assay Overview Figure 3 summarises the percentage of individuals who mounted a detectable response to each of the peptides/antigens by production of the three cytokines measured.
The black bars represent production of IFN-y, the grey bars IL-13 and the white bars IL- 10. The positive control antigen PPD elicited a cytokine production in almost all individuals (IFN-y: 91%, IL-13: 97% and IL-lO: 96%). Whole cat allergen and the mixture of 7 peptides elicited a cytokine response in approximately 80% or more of subjects. Individual peptides elicited responses of differing frequency. In general cytokine production appeared to be a more sensitive method of detecting responses with larger percentages of individuals giving positive cytokine responses than proliferative responses. In most cases, IL-10 secretion was detected in the largest number of subjects and IFN-'y detected least frequently.
4. Figures 4.2 Cytokine Release Assay Figures 4 to 6 show, for each peptide/antigen, the percentage of individuals who made a response of any detectable magnitude (i.e. production of detectable IFN-'y, IL-13 or IL-b). The strength of those responses is then split into four levels of cytokine production. For example, 35% of the study population may have made an [FN-T response. Of that 35% of individuals, half (50%) made a very weak response, 20% a weak response, 15% a moderate response and 15% a strong response (giving a total of 100% of the responders). The boundaries of each cytokine level were arbitrarily assigned based on the detection range of the ELISA assay. The boundaries are different between IFN-y/IL- 10 and IL-13 since for WN-? and IL-lO the detection range was approximately 1-lOOpg/ml whereas the range for the IL-13 assay was approximately 0.5-5Opg/ml.
4.2.1 Interferon-rproduction Figure 4 shows the percentage of individuals producing IFN-y and the strength of the response following cell culture with peptide/antigen. IFN-y responses were detected between 26-44% of subjects in response to individual peptides. These responses were predominantly very low to low to moderate. Complex antigens induced more frequent responses (peptide mixture 80%, cat dander 79%, PPD 9 1%). These responses were low to moderate to high. PPD responses were particularly high (89 of PPD responses were above lOOpg/ml).
4.2.2 IL-13 Production Figure 5 demonstrates the percentage of individuals producing IL-I 3 and strength of the response following cell culture with peptide/antigen. IL-13 responses were detected in between 33-68% of subjects in response to individual peptides. These responses were predominantly very low to low, although a significant number of moderate responses were detected. This may reflect the Th2 nature of allergic
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sensitisation in these subjects. Complex antigens induced more frequent responses (peptide mixture 85%, cat dander 93%, PPD 97%). These responses were low to moderate to high.
4.2.3 IL-JO Production Figure 6 demonstrates the percentage of individuals producing IL-10 and strength of the response following cell culture with peptide/antigen. IL-JO responses were detected in between 46-75% of subjects in response to individual peptides. These responses were predominantly very low to low. Complex antigens induced more frequent responses (peptide mixture 93%, cat dander 96%, PPD 96%). These responses were low to moderate. Very few "high" IL-10 responses were observed.
5. Discussion 5.2 Cytokine Assays Cytokine measurement proved to be a sensitive method of measuring responses to the peptides. Production of each of the three cytokines varied with IL-lO generally being produced by a greater proportion of subjects than IL-13 and IFN-y. The lowest frequency of response was detected with IFN-y. The atopic allergic status of these subjects is likely to mean that the memory T cell response to Fel d I and its epitopes will be dominated by Th2 responses which may account for the less frequent Thi (IFN-y) response. The high frequency of IL-b responses was a surprise. IL-lO is considered to be a Th2 cytokine in the murine system but this is not well established in the human system. IL-b is generally regarded as a regulatory/immunosuppressive cytokine.
Previous reports have suggested that some peptide sequences may have intrinsic IL-b inducing properties. Such peptides were not observed in this study. The detection of such responses in other systems may simply reflect the nature of T cell priming to whole allergen which is recalled by culture of memory T cells with peptide. Thus, production of IL-b may be a recall response rather than the result of intrinsic IL-lO-inducing characteristics of the peptide.
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6. Conclusion
6.2 Cytokine assays The cytokine assay data shows that the peptides identified are suitable for use in the method of the invention. Other peptides derived from cat allergens which are suitable for use in the method of the invention are as follows: SEQ ID NO:55 MLA1 H2N EICPAVKRDVDLFLTGT COOH -Derived from Fel dl chain 1: SEQ ID NO:56 MLA2 l-12N LFLTGTPDEYVEQVAQY COOK Derived from Fel dl chain 1 SEQ ID NO:57 MLA6 H2N KMTEEDKENALSLLDK COOK Derived from Fel dl chain -SEQ ID NO:58 MLA7 H2N KENALSVLDKIYTSPL COOh Derived from Fel dl chain SEQ ID NO:59 MLA8 H2N VKMAETCPIFYDVFFA COOH Derived from Fel dl chain 2 SEQ ID NO:60 MLA9 j CPIFYDVFFP.VANGNEL COOH Derived from Fel dl chain 2 SEQ ID NO:61 MLA1O H2N GNELLLKLSLTKVNAT COOH Derived from Fel dl chain 2 SEQ ID NO:62 MLA1 1 H2N LTKVNATEPERTAMKK COOH Derived from Fel dl chain 2 SEQ ID NO:63 MLAI3 CYVENGLISRVLDGLV COOH Derived from Fel dl chain 2 SEQ ID NO:64 MLA15 H2N ISSSKDCMGEAVQNTV COOH Derived from Fel dl chain 2 SEQ ID NO:65 MLA16 H2N AVQNTVEDLKLNTLGR COOK Derived from Eel dl chain 2

Claims (15)

1. An in vitro method of determining whether an individual has or is at risk of a condition wherein the condition is classified as a non-allergic or intrinsic disorder, the method comprising testing whether the individual has T cells which respond to an allergen, thereby determining whether the individual has or is at risk of the condition, and additionally identifying the allergen as a trigger for the condition.
2. A method according to claim I wherein: -an IgE response is not detectable in the individual to one or more proteins associated with the condition, and/or -the individual has a negative skin-prick or negative skin-patch result to a proteins associated with the condition, and/or -the individual lacks symptoms or disease markers associated with an immune response.
3. A method according to claim I or 2 wherein a T-cell immune response to said protein is measured by contacting a peptide derived from the protein, or a variant thereof, with T cells in a sample taken from the subject, under conditions which allow the peptide and the T cells to interact; and determining whether or not any of the T cells are stimulated and thereby determining whether or not a T-cell immune response is present or absent, wherein optionally the peptide consists of 9 to 30 amino acids, and comprises at least one MHC class 11-binding T cell epitope.
4. A method according to any one of the preceding claims, wherein the individual is asthmatic, or is suspected of being asthmatic, or is considered to be at risk of developing asthma.
5. A method according to any one of the preceding claims, wherein the condition is intrinsic (non-allergic) asthma or intrinsic (non-allergic) rhinitis.
6. A method according to any one of claims 3 to 5 wherein the peptide or variant thereof is derived from an allergen selected from: a plant allergen (particularly a grass allergen), animal dander allergens, a mold or fungal allergen, a dust allergen, an environmental allergen or a food allergen.
7. A method according to claim 6 wherein the peptide or variant thereof derives from: cat dander protein Fel dl; House dust mite proteins Derpi, Derp2 and Derp7; Ragweed protein Amb a 1; Rye grass proteins Lol p1 and Lol p5; Timothy grass proteins PhI p1 and PhI p5; Alternaria alternate proteins Alt a I, Alt a 2 and Enolase (Alt a 6); Birch proteins Bet vi and P14; German Cockroach proteins Bla g 1, Bla g 2, Bla g 3, Bla g 4, Bla g 5 and Bla g 6; Mugwort protein Art v 1; Russian thistle proteins Salk I and Salk2.
8. A method according to claim 7 wherein the peptide consists of the sequence of anyoneofSEQIDNOS: ito 65.
9. A method according to any one of the preceding claims which is carried out as defined in claim 3 and the T cells are present in a population of PBMCs isolated from a blood or serum sample taken from the individual.
10. A method according to any one of the preceding claims wherein determining whether I cells of the individual respond to the protein is carried out by measuring the production of a cytokine by the T cells.
11. A method according to claim 10 wherein the production of a cytokine is detected by an ELISPOT assay.
12. A method according to claim 10 or 11 wherein the cytokine is IL-4, IL-9, IL-13 or IL-5, or any combination thereof.
13. A method of treating or preventing the subset of condition defined in any one of the preceding claims comprising determining whether an individual has or is at risk of
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the subset of the condition by the method of any one of the preceding claims, and if the individual has or is at risk of the subset of the condition tolerising the individual to said protein or administering to the individual an immunosuppressant
14. A method according to claim 13 wherein the condition is asthma and the immunosupressant is suitable for the prevention or treatment of a late asthmatic reaction (LAR), wherein the immunosuppressant is optionally an anti-CD4 antibody.
15. A method according to claim 13 wherein the condition is asthma and the immunosupressant is suitable for the prevention or treatment of an eosinophilic reaction, wherein the immunosuppressant is optionally an anti-eosinophil drug, preferably an anti-eotaxin antibody.
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US20150110820A1 (en) * 2012-05-11 2015-04-23 Circassia Limited Hydrochloride salt of peptide and its use in combination with other peptides for immunotherapy
US9657061B2 (en) * 2012-05-11 2017-05-23 Circassia Limited Hydrochloride salt of peptide and its use in combination with other peptides for immunotherapy
US11096994B2 (en) 2012-10-30 2021-08-24 Aravax Pty Ltd Immunotherapeutic molecules and uses thereof
US11266737B2 (en) 2013-09-25 2022-03-08 Aravax Pty Ltd Immunotherapeutic composition and uses thereof

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