WO2015077442A2 - Grass pollen immunogens and methods and uses for immune response modulation - Google Patents

Grass pollen immunogens and methods and uses for immune response modulation Download PDF

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WO2015077442A2
WO2015077442A2 PCT/US2014/066591 US2014066591W WO2015077442A2 WO 2015077442 A2 WO2015077442 A2 WO 2015077442A2 US 2014066591 W US2014066591 W US 2014066591W WO 2015077442 A2 WO2015077442 A2 WO 2015077442A2
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polypeptide
amino acid
seq
nos
sequence
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PCT/US2014/066591
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WO2015077442A9 (en
WO2015077442A3 (en
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Bjoern Peters
Alessandro Sette
Jason GREENBAUM
Ilka HOOF
Lars Harder CHRISTENSEN
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La Jolla Institute For Allergy And Immunology
Alk-Abelló A/S
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Publication of WO2015077442A2 publication Critical patent/WO2015077442A2/en
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Publication of WO2015077442A3 publication Critical patent/WO2015077442A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/35—Allergens
    • A61K39/36—Allergens from pollen

Definitions

  • the invention relates to pan grass pollen immunogens such as polypeptides, proteins and peptides, and methods and uses of such immunogens for modulating or relieving an immune response in a subject, such as treating a subject for an allergic immune response or inducing or promoting immunological tolerance to the immunogen or a non-grass pollen allergen in a subject.
  • immunogens such as polypeptides, proteins and peptides
  • methods and uses of such immunogens for modulating or relieving an immune response in a subject, such as treating a subject for an allergic immune response or inducing or promoting immunological tolerance to the immunogen or a non-grass pollen allergen in a subject.
  • Allergen-specific immunotherapy is a hyposensitizing immunotherapy introduced in clinical medicine almost a century ago for the treatment of an allergic immune response using the allergens that the subject is sensitized to.
  • An allergic immune response may be mediated by activated allergen-specific Th2 cells, which produce cytokines such as IL-4, IL- 5, and IL-13.
  • Th2 cells which produce cytokines such as IL-4, IL- 5, and IL-13.
  • Thl cells In healthy individuals, the allergen-specific T-cell response is mediated predominantly by Thl cells.
  • SIT may reduce the ratio of Th2:Thl cells and may alter the cytokine profile, reducing the production of IL-4, IL-5, and IL-13 and increasing the production of IFN-g in response to major allergens or allergen extracts.
  • SIT has several limitations, including safety concerns about giving patients allergenic substances. Because most SIT regimens involve the administration of whole, unfractionated, allergen extracts, adverse IgE-mediated events are a considerable risk. Significant efforts have been devoted to developing approaches to modulate allergen- specific T-cell responses without inducing IgE-meditated, immediate-type reactions. These approaches include developing hypoallergens that do not contain IgE-binding epitopes, allergens that are coupled to adjuvants and carriers of bacterial or viral origin or peptides that contain dominant T-cell epitopes and do not react with IgE in allergic individuals.
  • allergen-specific T-cells play an important role in allergic inflammation and that induction of antigen specific T regulatory cells (Tregs) or elimination of allergen-specific T helper type 2 cells (Th2) might be a prerequisite for the induction of specific tolerance. Yet, cross-reactivity among multiple pollen families at the T-cell level is less known.
  • NTGA novel Timothy Grass antigens
  • an immunogen derived from an allergenic pollen source is able to reduce an allergic immune response caused by an unrelated allergen via bystander suppression.
  • immunogens related to recently detected immunogens of Timothy grass pollen share high sequence conservation/homology across several different grass pollen families and are broadly reactive. Such immunogens have potential therapeutical utilization against immune responses triggered by pollen of a broad array of grass pollen families. Summary
  • pan-grass pollenimmunogens derived from previously detected NTGA's.
  • a pan-grass pollen immunogen consists of or contain as part of its sequence an amino acid sequence that is conserved across polypeptides detected in multiple grass pollen (e.g. (Kentucky blue grass (Poa pratensis, Poa p), Sweet vernal grass (Anthoxanthum odoratum, Ant o), Rye grass (Lollium Perenne, Lol p), Bermuda grass
  • the immunogens may contain conserved subsequences, e.g. T cell epitope-containing subsequences of previously detected NTGA's, which T cell epitope-containing subsequence is conserved across polypeptides detected in a grass pollen of the genera Phleum and at least in one other grass pollen species.
  • conserved subsequences e.g. T cell epitope-containing subsequences of previously detected NTGA's, which T cell epitope-containing subsequence is conserved across polypeptides detected in a grass pollen of the genera Phleum and at least in one other grass pollen species.
  • PG sequences or PG peptides have less than 3 mismatches to 15 contiguous amino acids of polypeptides detected in a Phi p grass pollen species and a non- Phi p grass pollen species described herein, for example Bermuda Grass pollen.
  • Table 1 shows examples on such conserved subsequences (PG peptides) derived from previously detected NTGA's.
  • the immunogens may be larger amino sequences containing one or more PG peptide of Table 1, for example wild type sequences of NTGA's.
  • Table 2 shows examples on wild type polypeptidesfound in Timothy grass pollen (Phi p), which contain at least one PG peptide.
  • Table 3 discloses wild type sequences of other grass pollen that as part of its sequence also contain one or more PG peptides or one with less 3 mismatches thereto. .
  • the immunogens may contain at least one T cell epitope as determined by the T cell response observed against immunogens of Tables 1, 2 or 3 in cultured PBMC's of grass pollen allergic donors. Furthermore, it was found that a T cell response of grass allergic donors to an immunogen of the invention may be cross reactive to non-grass pollen species, thereby indicating that grass pollen immunogens and its conserved homolog in non-grass pollen families share T cell epitopes.
  • the immunogens may contain at least one PG peptide disclosed in Table 8, e.g . a PG peptide with SEG ID NO: 246, 258 and 315. That is not to exclude that an immunogen may contain another peptide disclosed in Table 8.
  • the invention relates in a first aspect to a method for relieving an allergic immune response against a grass pollen allergen, wherein the grass pollen allergen is not a grass pollen allergen of the genus Phleum, in a subject in need thereof, comprising administering an effective amount of an immunogenic molecule, wherein said molecule comprises or consists of a) a polypeptide, which includes at least one amino acid sequence with 0, 1 or 2
  • SEQ ID NOs: 1-519 set out in Table 1 refers to PG peptides, which 15mer amino acid sequence is conserved across a Timothy grass pollen (Phi p) and at least a grass pollen Cyn d and Lol p.
  • SEQ ID NOs: 520-565 set out in Table 2 refers to full length sequences of NTGA's identified by combined transcriptomic and Mass Spectrometry analysis and which sequence contains regions conserved across at least a grass pollen and at least a grass pollen Cyn d and Lol p.
  • the polypeptides relates to NTGA 89 and the polypeptide of option a) includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 485-513; the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 563.
  • Other embodiments (A to AK) may be constructed the same way using the list below:
  • Embodiment Q 26 183-190 536 Embodiments: NTGA No: Polypeptide option a) Polypeptide option
  • a polypeptide of option a) includes one or more PG peptides from different NTGA's, so as to construct polypeptides with desirable properties.
  • one polypeptide may contain as part of its sequence an amino acid sequence of one or more PG peptides containing an immunodominant T cell epitope.
  • the invention also relates to a molecule for use in relieving an allergic immune response against a pollen allergen other than a grass pollen allergen in a subject, wherein said molecule comprises or consists of a) a polypeptide, which includes at least one amino acid sequence with 0, 1 or 2
  • the invention also relates to the use of a molecule for preparation of a medicament for relieving an allergic immune response against a pollen allergen other than a grass pollen allergen in a subject, wherein said molecule comprises or consists of a) a polypeptide, which includes at least one amino acid sequence with 0, 1 or 2
  • the invention relates in a further aspect to an immunogenic molecule, e.g . a molecule comprising of or consisting of a) a polypeptide, which includes at least one amino acid sequence with 0, 1 or 2
  • an immunogenic molecule may contain a conserved sequence of NTGA 89
  • a molecule comprises or consists of a polypeptide of option a) that includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 485- 513; or a polypeptide of option b) that comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 563
  • Other embodiments may be constructed the same way using the list above.
  • a cell expresses an immunogen.
  • a cell is a eukaryotic or prokaryotic cell and may be a mammalian, insect, fungal or bacterium cell.
  • An immunogen of the present invention is suitable as a reagent for example, for immunotherapy against various pollen allergies including a pollen allergy, which is not grass pollen allergy in a subject.
  • nucleic acid molecules encoding a polypeptide of option a) and b), or a molecule comprising polypeptide of option a) and b).
  • compositions for example pharmaceutical compositions comprising an immunogenic molecule of the invention.
  • a pharmaceutical composition is suitable for specific immunotherapy (e.g., treatment, desensitization, tolerance induction, bystander suppression).
  • a pharmaceutical composition is a vaccine, i.e. suitable formulated for the purpose of vaccination.
  • FIG. 2 Sensitization pattern of an immunogen of the invention (NTGA 86/51): It is shown that the in vitro T-cell response towards NTGA 86/51 is much weaker compared to the response to allergen Phi p 5.
  • Figures 3A-3C Tolerance induction investigated in mice. Figures show that prophylactic sublingual immunotherapy treatment (SLIT) with NTGA 86/51 in mice is capable of inducing tolerance towards the immunogen itself (3A) as well as towards Phi p extract (3B), as shown by the ability of NTGA 86/51 to reduce the proliferation of cells of splenocytes from treated mice compared to buffer (sham) treated mice.
  • SLIT prophylactic sublingual immunotherapy treatment
  • NTGA 6 is capable of inducing tolerance towards itself (3C) as observed by its ability to reduce proliferation of cells of splenocytes.
  • Figures 4A and B Bystander tolerance induction investigated in mice. As shown in Figure 4A, prophylactic SLIT treatment with NTGA 86/51 is capable of inducing direct tolerance (towards NTGA 86/51 itself), as demonstrated by reduced proliferation of splenocytes of NTGA 86/51-treated mice compared to buffer treated mice.
  • Figure 4B shows that SLIT treatment with OVA is also able to downregulate the NTGA 86/51 specific in vitro response, demonstrating bystander tolerance induction by OVA.
  • SLIT treatment with NTGA 86/51 is also able to induce bystander tolerance, as
  • conserved sequence is in the present context meant to include that a given sequence contains at least 15 contiguous amino acids within the sequence that has less than 3 mismatches compared to another sequence of 15 amino acids. Longer stretches of conserved sequences may contain several numbers of stretches of at least 15 contiguous amino acids having less than 3 mismatches compared to another sequence of 15 amino acids. In the present context, e.g. for the purpose of detecting a conserved sequence, the term “mismatch” is meant to include any substitution of an amino acid residue within the 15mer peptide.
  • the term "sensitized to” is generally meant to encompass that the subject has been exposed to an immunogen, e.g. an allergen or an antigen, in a manner that the individual's adaptive immune system displays memory to the immunogen, for example that the immunogen has induced detectable IgE antibodies against the immunogen and thus qualifies as an IgE-reactive antigen (allergen) and/or that T-cells stimulated in vitro are able to proliferate under the presence of the immunogen or fragments of the immunogen (e.g. linear peptides).
  • allergen an IgE-reactive antigen
  • T-cells stimulated in vitro are able to proliferate under the presence of the immunogen or fragments of the immunogen (e.g. linear peptides).
  • allergic immune response is meant to encompass a hypersensitivity immune response, e.g. type 1 immune response, such as typically an immune response that is associated with the production of IgE antibodies (i.e.
  • An allergic immune response may be associated with an allergic disease, for example atopic dermatitis, urticaria, contact dermatitis, allergic conjunctivitis, allergic rhinitis, allergic asthma, anaphylaxis, food allergy and hay fever.
  • grass pollen is meant to designate pollen of the plant family Poaceae, for example pollen of the plant genus Anthoxanthum, Cynodon, Dactylis, Festuca, Holcus, Hordeum, Lolium, Oryza, Paspalum, Phalaris, Phleum, Poa, Secale, Sorghum, Triticum and Zea.
  • an "immunogen” refers to a substance, including but not limited to a protein, polypeptide or peptide that modifies, e.g. elicits, induces, stimulates, promotes enhances or decreases, reduces, inhibits, suppresses, relieves an immune response when administered to a subject.
  • an immunogen may induce tolerance to itself in a subject.
  • An immune response elicited by an immunogen may include, but is not limited to, a B cell or a T cell response.
  • An immune response can include a cellular response with a particular pattern of lymphokine/cytokine production (e.g., Thl, Th2), a humoral response (e.g., antibody production, like IgE, IgG or IgA), or a combination thereof, to a particular immunogen.
  • a cellular response with a particular pattern of lymphokine/cytokine production e.g., Thl, Th2
  • a humoral response e.g., antibody production, like IgE, IgG or IgA
  • immunogens are antigens and allergens.
  • an antigen refers to a particular substance to which an immunoglobulin (Ig) isotype may be produced in response to the substance.
  • an immunoglobulin (Ig) refers to an antigen that induces an IgG antibody response.
  • an "IgE antigen” refers to an antigen that induces an IgE antibody response (and thus qualifies as an allergen) ;
  • an "IgA antigen” refers to a substance that induces an IgA antibody response, and so forth.
  • such an immunoglobulin (Ig) isotype produced in response to an antigen may also elicit production of other isotypes.
  • an IgG antigen may induce an IgG antibody response in combination with one more of an IgE, IgA, IgM or IgD antibody response. Accordingly, in certain embodiments, an IgG antigen may induce an IgG antibody response without inducing an IgE, IgA, IgM or IgD antibody response.
  • allergen refers to a particular type of a substance that can elicit production of IgE antibodies, such as in predisposed subjects. For example, if a subject previously exposed to an allergen (i.e. is sensitized or is hypersensitive) comes into contact with the allergen again, allergic asthma may develop due to a Th2 response characterized by an increased production of type 2 cytokines (e.g., IL-4, IL-5, IL-9, and/or IL-13) secreted by CD4+ T lymphocytes.
  • type 2 cytokines e.g., IL-4, IL-5, IL-9, and/or IL-13
  • subject is meant to designate a mammal having an adaptive immune system, such as a human, a domestic animal such as a dog, a cat, a horse or cattle.
  • the term "immunotherapy” is meant to encompass treatment of a disease by inducing, enhancing, or suppressing an immune response.
  • the therapeutically active agent is an immunogen, particularly an antigen, more particularly an allergen.
  • An immunogen may be a protein or a fragment thereof (e.g. immunogenic peptide).
  • Immunotherapy in connection with allergy usually encompasses repeated administration of a sufficient dose of the immunogen/antigen/allergen/ usually in microgram quantities, over a prolonged period of time, usually for more than 3 months, 6 months, 1 year, such as 2 or 3 years, during which period the immunogen may be administered daily or less frequent, such as several times a week, weekly, bi-weekly, or monthly, every second month or quarterly.
  • Immunotherapy can be effected by specific immunotherapy or may be effected by bystander tolerance induction.
  • specific immunotherapy in connection with allergy is meant to designate that immunotherapy is conducted with the administration of an immunogen to which the subject is sensitized to, particularly an immunogen to which the patient has raised specific IgE antibodies to, e.g. major allergens.
  • immunological tolerance refers to a) a decreased or reduced level of a specific immunological response (thought to be mediated at least in part by antigen-specific effector T lymphocytes, B lymphocytes, antibody, a combination); b) a delay in the onset or progression of a specific immunological response; or c) a reduced risk of the onset or progression of a specific immunological response to an antigen or allergen.
  • Specific immunological tolerance occurs when tolerance is preferentially invoked against certain antigens (allergens) in comparison with other antigens (allergens). Tolerance is an active antigen dependent process and differs from non-specific immunosuppression and immunodeficiency.
  • bystander tolerance induction in connection with allergy is meant to encompass that immunotherapy is conducted with the administration of an immunogen that elicits, induces, stimulates, promotes enhances or decreases, reduces, inhibits, suppresses, relieves an immune response against another unrelated immunogen, for example an allergen, e.g. major allergens of pollen.
  • an immunogen may induce tolerance to itself, and may be able to reactivate T regulatory cells specific to the immunogen to down-regulate an immune response caused by another unrelated immunogen, e.g. an allergen.
  • treatment refers to any type of treatment that conveys a benefit to a subject afflicted with allergy, including improvement in the condition of the subject (e.g., in one or more symptoms), delay in the onset of symptoms, slowing the progression of symptoms, or induce disease modification etc.
  • Typical symptoms of an allergic reaction is nasal symptoms in the form of itchy nose, sneezing, runny nose, blocked nose; conjunctival symptoms in the form of itchy eyes, red eyes, watery eyes; and respiratory symptoms in the form of decreased lung function.
  • the treatment may also give the benefit that the patient needs less concomitant treatment with corticosteroids or HI antihistamines to suppress the clinical symptoms.
  • treatment is not necessarily meant to imply cure or complete abolition of symptoms, but refers to any type of treatment that imparts a benefit to a patient.
  • Treatment may be initiated before the subject becomes sensitized to a protein. This may be realized by initiating immunotherapy before the subject has raised detectable serum IgE antibodies capable of binding specifically to the sensitizing protein or before any other biochemical marker indicative of an allergic immune response can be detected in biological samples isolated from the individual.
  • treatment may be initiated before the subject has evolved clinical symptoms of the allergic disease, such as symptoms of allergic rhinitis, allergic asthma or atopic dermatitis.
  • a therapeutically sufficient amount is meant to designate an amount effective to reduce, suppress, relieve or eliminate an allergic immune response, e.g. an amount sufficient to achieve the desirable reduction in clinical relevant symptoms or manifestations of the allergic immune response.
  • a therapeutically sufficient amount may be the accumulated dose of a polypeptide, a set of polypeptides administered during a course of immunotherapy in order to achieve the intended effect or it may be the maximal dose tolerated within a given period.
  • the total dose or accumulated dose may be divided into single doses administered daily, twice a week or more, weekly, every second or fourth week or monthly depending on the route of administration and the pharmaceutical formulation used. The total dose or accumulated dose may vary.
  • a single dose is in the microgram range, such as in the range of 5 to 500 microgram dependent on the nature of the polypeptide.
  • the term "patient responding to therapy,” such as “immunotherapy” is meant to designate that the patient has improvement in the symptoms of the allergic immune response caused by a pollen allergen. Symptoms may be the clinically symptoms of allergic rhinitis, allergic asthma allergic conjunctivitis, atopic dermatitis, food allergy and/or hay fever. Typically, the symptoms are the same as experienced with a flu/cold, sneezing, itching, congestion, coughing, feeling of fatigue, sleepiness and body aches.
  • nasal symptoms in the form of itchy nose For example nasal symptoms in the form of itchy nose, sneezing, runny nose, blocked nose; conjunctival symptoms in the form of itchy eyes, red eyes, watery eyes; and respiratory symptoms in the form of decreased lung function.
  • a responder may also be evaluated by monitoring the patient's reduced need for concomitant treatment with corticosteroids or H I antihistamines to suppress the clinical symptoms. Symptoms may be subjectively scored or in accordance with official guidelines used in clinical trials of SIT.
  • an immunogen refers to a substance that enhances the immune response to an immunogen. Depending on the nature of the adjuvant, it can promote either a cell-mediated immune response, humoral immune response or a mixture of the two.
  • an “epitope” refers to a region or part of an immunogen that elicits an immune response when administered to a subject.
  • an epitope is a T cell epitope, i.e., an epitope that elicits, stimulates, induces, promotes, increases or enhances a T cell activity, function or response.
  • An immunogen can be analyzed to determine whether it include at least one T cell epitope using any number of assays (e.g .
  • a T-cell epitope refers to an epitope that are MHC Class II binders (i .e. HLA-II binders), for example HLA-II binders shown in Table 9.
  • HLA-II binders MHC Class II binders
  • a subsequence may contain a T cell epitope, such as a Th2 cell epitope (Table 1) .
  • a subsequence or a polypeptide described herein may have HLA Class II binding properties.
  • HLA Class II binding can be predicted using NetMHCIIpan-3.0 tool (Karosiene, Edita, Michael Rasmussen, Thomas Bö, Ole Lund, Soren Buus, and Morten Nielsen. "NetMHCIIpan-3.0, a Common Pan-specific MHC Class II Prediction Method Including All Three Human MHC Class II Isotypes, HLA-DR, H LA-DP and HLA-DQ.” Immunogenetics) available at the internet site ⁇ URL: http://www.cbs.dtu.dk/services/NetMHCIIpan-3.0> .
  • immune response includes T cell (cellular) mediated and/or B cell (humoral) mediated immune responses, or both cellular and humoral responses.
  • exemplary immune responses include T cell responses, e.g ., lymphokine production, cytokine production and cellular cytotoxicity.
  • T-cell responses include Thl and/or Th2 responses.
  • immune response includes responses that are indirectly affected by T cell activation, e.g ., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., eosinophils, macrophages.
  • Immune cells involved in the immune response include lymphocytes, such as T cells (CD4+, CD8+, Thl and Th2 cells, memory T cells) and B cells; antigen presenting cells (e.g ., professional antigen presenting cells such as dendritic cells, macrophages, B lymphocytes, Langerhans cells, and non-professional antigen presenting cells such as keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes); natural killer (NK) cells; myeloid cells, such as macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • lymphocytes such as T cells (CD4+, CD8+, Thl and Th2 cells, memory T cells) and B cells
  • antigen presenting cells e.g ., professional antigen presenting cells such as dendritic cells, macrophages, B lymphocytes, Langerhans cells, and non-professional antigen presenting cells such
  • immunogens As disclosed herein, some immunogens (NTGA's) recently detected in Timothy grass pollen share substantial identity and similarity with immunogens detected in at least Cyn d and Poa p pollen.
  • NTGA's immunogens detected in at least Cyn d and Poa p pollen.
  • immunogens can be used to broadly treat a subject with or at risk of developing an allergy, allergic reaction, allergic immune response to a pollen allergen of a variety of pollen plant families, or broadly induce or promote tolerance of a subject to a pollen allergen of a variety of grass pollen families.
  • the immunogen is a molecule comprising or consisting of a) a polypeptide, which includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 1-519 set out in Table 1 (PG peptides).
  • the immunogen may contain at least one T cell epitope optionally a Th-2 cell epitope.
  • the polypeptide of option a includes at least one amino acid sequence with 0, 1 or 2 mismatches to a sequence selected from any one of SEQ ID NOs: 44, 8, 9, 10, 14, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 45, 47, 50, 51, 63, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 102, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 135, 136, 137, 154, 155, 162, 163, 166, 167, 168, 169, 1
  • polypeptide of option a) includes at least one amino acid sequence with 0, 1 or 2 mismatches to a sequence selected from any one of SEQ ID NOs 19, 20, 24, 25, 26, 28, 32, 37, 69, 71, 72, 73, 74, 78, 79, 80, 81, 82, 84, 86, 87, 88, 89, 90, 91, 93, 95, 99, 107, 166, 169, 173, 174, 176, 180, 193, 303, 313, 319, 321, 492, 505, 506, 510, 511, 513, 514, 518 and 519.
  • the number of amino acid mismatches is 0 or 1
  • the immunogen may be a molecule comprising or consisting of a) a polypeptide, which includes at least one amino acid sequence with 0 or 1 mismatches compared to a sequence selected from any one of SEQ ID NOs: 4, 8, 9, 10, 14, 19, 20, 21, 22, 24, 25, 26, 29, 30, 32, 33, 34, 35, 37, 38, 41, 42, 45, 65, 66, 67, 69, 71, 72, 73, 74, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 98, 99, 102, 106, 107, 109, 110, 116, 117, 135, 136154, 155, 162, 163, 166, 167, 168, 169, 170, 171, 172, 174, 175,
  • the polypeptide of option a) is derived from NTGA's 2, 6, 7, 49/54, 89, 53 and 91 that contains epitopes with high T cell reactivity
  • a polypeptide of option a) comprises a T cell epitope and includes at least one amino acid sequence with 0, 1 or 2 mismatches to a sequence selected from any one of SEQ ID NOs: 19, 20, 24, 25, 26, 32, 37, 69, 71, 72, 73, 74, 78, 79, 80, 81, 82, 84, 86, 87, 88, 89, 90, 91, 93, 95, 99, 319, 321, 506 and 510.
  • the immunogen is a molecule comprising or consisting of a polypeptide of option b) that are conserved across five grass pollen species and comprises several PG peptides in its sequence compared to other NTGA's investigated.
  • a polypeptide of option b) may comprise an amino acid sequence with at least 65% similarity or identity to a sequence selected from any of SEQ ID NOs: 520, 521, 523, 525, 526, 531, 533, 535, 538, 539, 541, 542, 557, 558, 559, 561, 563 and 564.
  • Immunogens eligible for relieving an allergic immune response to an allergen unrelated to the immunogen is thought, at least in part, to be mediated via bystander tolerance induction, which mechanism requires, at least in part, co-existence of the immune response triggering allergen and the unrelated immunogen at the target organ.
  • a polypeptide of option a) and option b) may be derived from a full length protein that co-releases/co-elutes with a major allergen of grass pollen.
  • a the full length sequence of a polypeptide of option a) and b) should be co-released from Phi p pollen as well as another interesting grass pollen, e.g. pollen of the plant genera Cynodon, and optionally also from grass pollen selected from any of the plant genera Anthoxanthum, Dactylis, Festuca, Lollium, Paspalum, Phalaris, Poa and Sorghum.
  • co-release or “co-elute” refers to an immunogen that starts release from a hydrated pollen within a period overlapping with a major allergen to which the allergic immune response is sought relieved. As major allergens start release from pollen within few minutes after hydration of pollen and continues to be released within the next 30 or 60 minutes, the term “co-release” or “co-elute” may refers to that an
  • immunogen of the invention starts being released from pollen within 30 minutes after hydration of the pollen.
  • the polypeptide of option b) comprises an amino acid sequence with at least 65% similarity or identity to a sequence selected from any of SEQ ID NOs:
  • polypeptide of option a includes at least one amino acid sequence with 0, 1 or 2
  • polypeptide defined herein may comprise one or more PG peptide
  • a polypeptide of option a) comprises two or more PG peptides, e.g. 2-25 PG peptides defined herein, e.g. 3-25, 4-25, 5-25, 6-25, 7-25 PG peptides, such as 2- 20, 3-20, 4-20, 5-20, 6-20 PG peptides or a corresponding sequence with 1 or 2
  • a polypeptide of option a) may include one or more immunodominant PG peptides, like those recognized by at least 3 subjects in a population of 20 subjects, e.g.
  • a polypeptide of option a) may include one or more sequences selected from any one of SEQ ID NOs: 19, 20, 24, 25, 26, 32, 37, 69, 71, 72, 73, 74, 78, 79, 80, 81, 82, 84, 86, 87, 88, 89, 90, 91, 93, 95, 99, 319, 321, 506 and 510, or a sequence with 0, 1 or 2 mismatches compared to the SEQ ID NOs: 19, 20, 24, 25, 26, 32, 37, 69, 71, 72, 73, 74, 78, 79, 80, 81, 82, 84, 86, 87, 88, 89, 90, 91, 93, 95, 99, 319, 321, 506 and 510.
  • a polypeptide of option a) may include one or more, such as from 2 to 25 PG peptides of different NTGAs able to be released in both Phi p and Cyn d pollens. Therefore, in still some embodiments, a polypeptide of option a) may include one or more sequences selected from any one of SEQ ID NOs: 1-18, 38-40, 41-55, 56-62, 63-89, 148-156, 169- 182, 183-190, 211-232, 270-277, 338-348, 292-296, 354-356, 409-419, 467-481, 485-513 and 519, or a sequence with 0, 1 or 2 mismatches compared to the SEQ ID NOs: 1-18, 38- 40, 41-55, 56-62, 63-89, 148-156, 169-182, 183-190, 211-232, 270-277, 338-348, 292- 296, 354-356, 409-419, 467-4
  • a polypeptide of option a) or option b) is derived from a full length protein that co-releases/co-elutes with a major allergen of grass pollen of the genera Phleum, Anthoxanthum, Cynodon, Lollium and/or Poa.
  • full length sequences that contain the same PG peptides may be used instead of the Phi p sequence set out in Table 2.
  • full length sequence of a polypeptide of option a) or b) may be a polypeptide present in, based upon or derived from a pollen of a plant family of Poaceae, optionally the genera of any of Phleum, Anthoxanthum, Cynodon, Lollium and Poa.
  • polypeptides are set out in Table 3.
  • a polypeptide of option b) may comprise an amino acid sequence having at least 65% identity to any of SEQ ID Nos: 566-688.
  • the polypeptide relates to NTGA 89, e.g. a polypeptide of option a) that includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 485-513; the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 563 or a homolog thereof in another grass pollen species, e.g. SEQ ID NOs: 683-687.
  • the polypeptide relates to NTGA 6, e.g.
  • polypeptide of option a) that includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 63-89;
  • polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 525 or a homolog thereof in another grass pollen species, e.g. SEQ ID NOs: 577-585.
  • the polypeptide relates to NTGA 24, e.g. a polypeptide of option a) that includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 169-182; the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 535 or a homolog thereof in another grass pollen species, e.g . SEQ ID NOs: 602.
  • the polypeptide relates to NTGA 24, e.g . a polypeptide of option a) that includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 198-210; the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 538 or a homolog thereof in another grass pollen species, e.g . SEQ ID NOs: 604.
  • the polypeptide relates to NTGA 91, e.g .
  • polypeptide of option a) that includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 519; the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 564 or a homolog thereof in another grass pollen species, e.g . SEQ ID NOs: 688.
  • the polypeptide relates to NTGA 39/51, e.g. a polypeptide of option a) that includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 270-277, 338- 348; the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 542 or a homolog thereof in another grass pollen species, e.g . SEQ ID NOs: 614 and 615.
  • the polypeptide relates to NTGA 86/51, e.g. a polypeptide of option a) that includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 467-481 ;
  • the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 561 or a homolog thereof in another grass pollen species, e.g . SEQ ID NOs: 679
  • the polypeptide relates to NTGA 49/54, e.g. a polypeptide of option a) that includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 301-305, 319- 322; the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 548 or or a homolog thereof in another grass pollen species, e.g . SEQ ID NOs: 620-630.
  • the immunogen is a molecule comprising or consisting of a polypeptide, which includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: SEQ ID NO's 321, 303, 414, 689, 690, 28, 691-713 set out in Table 8.
  • the immunogen may contain at least one T cell epitope optionally a Th-2 cell epitope.
  • the polypeptide includes at least one amino acid sequence with 0, 1 or 2 mismatches to a sequence selected from any one of SEQ ID NO's 321, 303, 414, 689, 690, 28, 691-713.
  • an immunogen of the present invention is an IgE reactive molecule, e.g. able to bind to IgE antibodies specific for the immunogen.
  • IgE reactivity towards an immunogen of the invention may only be conferred by a low fraction of an allergic population.
  • an immunogen of the invention do not fall under the usual definitions of a major allergen.
  • the immunogen is able to react with, bind to or induce IgG antibodies in a subject, at least in detectable levels.
  • the immunogen does not react with, bind to or induce IgG antibodies, at least in detectable levels. As demonstrated herein, an immunogen of the invention seems to be less immunogenic than a major allergen ( Figure 2).
  • a subject eligible for being treated with an immunogen of the invention may also be sensitized to a grass pollen allergen, for example a grass pollen allergen of a plant genus selected from any of Anthoxanthum, Cynodon, Phleum and Poa.
  • a grass pollen allergen of a plant genus selected from any of Anthoxanthum, Cynodon, Phleum and Poa.
  • immunogens of the present invention may be found in various grass pollen families and share high identity and similarity with a wild type immunogen in a grass pollen family other than of the genus Phleum.
  • a polypeptide of option b) comprises an amino acid sequence having at least 70% similarity or identity to a sequence selected from any one of SEQ ID NOs: 398-443, for example at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity or identity.
  • Table 3 Examples on wild type immunogens with high identity and similarity to the wild type NTGA's are shown in Table 3.
  • wild type proteins found in other grass pollen species and which contain as part of its sequence a sequence having less than 3 mismatches to a PG peptide disclosed herein.
  • wild type sequences comparable to NTGA 6 are found in at least Ant o, Cyn d, Lol p and Poa p and comprises SEQ ID NOs: 577-585.
  • a polypeptide of option b) may comprise an amino acid sequence having at least 70% similarity or identity to a sequence selected from any one of SEQ ID NOs: 566- 688, for example at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity or identity.
  • a polypeptide of option a) may have different lengths according to the desirable use, for example of about 15-800 or more amino acid residues in length, for example 15-750, 15- 700, 15-650, 15-600, 15-500 or more amino acid residues, for example 15-20, 15-25, 15- 30, 20-25, 25-30, 30-35, 35-40, 45-50, 50-60, 60-70, 70-80, 90-100, 100-125, 125-150, 150-175, 175-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-800 or more amino acid residues.
  • a polypeptide of option a) and a polypeptide of option d) has a length in the range of 15 to 30 amino acid residues, for example 15 to 25 amino acid residues.
  • a polypeptide of option a) is a longer polypeptide which comprises a secondary or tertiary structure, e.g. folded.
  • a polypeptide of option a) has a length in the range of 30 to 500 amino acid residues or more.
  • Polypeptides of option b) may have the same length as the wild type sequence of the NTGA of Table 2, or the homolog of Table 3, respectively or may be shorter or longer. It is considered that the length of the amino acid sequence of a polypeptide of option b) is no more than 800 amino acid residues, for example no more than 750, 700, 650, 600, 550, 500 or 450 amino acid residues. Also it may be considered that the length of a polypeptide of option b) has an amino acid sequence length that is 80% to 120% of the length of any one of SEQ ID NOs: 520-565 or of SEQ ID Nos: 566-688.
  • identity and “identical” and grammatical variations thereof, as used herein, mean that two or more referenced entities are the same (e.g., amino acid sequences). Thus, where two polypeptides are identical, they have the same amino acid sequence.
  • the identity can be over a defined area (region or domain) of the sequence, e.g. over the sequence length of a sequence disclosed in Tables 1, 2 and 3 or over a stretch thereof e.g. at least 15 contiguous amino acid residues.
  • identity can be over the length of the sequence overlapping the two polypeptides, when aligned with best fit with gaps permitted.
  • the polypeptide may be aligned with a sequence of Table 2 or 3 and the percent identity be calculated with reference to a sequence of Table 2 or 3.
  • Identity can be determined by comparing each position in aligned sequences.
  • a degree of identity between amino acid sequences is a function of the number of identical or matching amino acids at positions shared by the sequences, i.e. over a specified region.
  • Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, as are known in the art, including the Clustal Omega program, available at http://www.ebi.ac.uk/Tools/msa/clustalo/, the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl.
  • Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. Mol. Biol. 215:403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information (through the internet at http://www.ncbi.nlm.nih.gov/). Such algorithms that calculate percent sequence identity (homology) generally account for sequence gaps and mismatches over the comparison region or area.
  • a BLAST e.g., BLAST 2.0 search algorithm
  • BLAST 2.0 search algorithm
  • exemplary search parameters as follows: Mismatch -2; gap open 5; gap extension 2.
  • a BLASTP algorithm is typically used in combination with a scoring matrix, such as PAM100, PAM 250, BLOSUM 62 or BLOSUM 50.
  • FASTA e.g., FASTA2 and FASTA3
  • SSEARCH sequence comparison programs are also used to quantitate the extent of identity (Pearson et al., Proc. Natl. Acad. Sci.
  • a polypeptide sequence is a "homologue” of, or is “homologous” to, another sequence if the two sequences have substantial identity over a specified region and a functional activity of the sequences is preserved or conserved, at least in part (as used herein, the term
  • homologous polypeptides examples include polypeptides found in non- Timothy grass pollen and with high identity to the NTGA's disclosed in Table 2.
  • a homologous polypeptide may be found in grass pollen other than of the genera Phleum of the plant family Poaceae, e.g. the plant genera Anthoxanthum, Cynodon, Dactylis, Festuca, Holcus, Hordeum, Lollium, Oryza, Paspalum, Phalaris, Poa, Secale, Sorghum, Triticum and Zea.
  • Two polypeptide sequences are considered to be substantially identical if, when optimally aligned (with gaps permitted), they share at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, etc. identify over a specific region), for example, over all or a part of any amino acid sequence in Tables 1, 2, and 3, or if the sequences share defined functional motifs (e.g., epitopes).
  • the length of the sequence sharing the percent identity is at least 15, 16, 17, 18, 19, 20, etc. contiguous amino acids, e.g.
  • an "unrelated" or “non-homologous” sequence is considered to share less than 30% identity. More particularly, it may shares less than about 25 % identity, with a polypeptide of the invention over a specified region of homology.
  • An amino acid sequence set out in any of Tables 1, 2 and 3 may contain modifications resulting in greater or less activity or function, such as ability to elicit, stimulate, induce, promote, increase, enhance, activate, modulate, inhibit, decreases, suppress, or reduce an immune response (e.g. a T cell response) or elicit, stimulate, induce, promote, increase or enhance immunological tolerance (desensitize) to an immunogen of the invention or a pollen allergen.
  • an immune response e.g. a T cell response
  • an immune response e.g. a T cell response
  • elicit, stimulate, induce, promote, increase or enhance immunological tolerance desensitize
  • a modification includes deletions, including truncations and fragments; insertions and additions, substitutions, for example conservative substitutions, site-directed mutants and allelic variants.
  • Non-limiting examples of modifications include one or more amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-25, 25-30, 30-50, 50-100 or more residues), additions and insertions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-25, 25-30, 30-50, 50-100 or more residues) and deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-25, 25-30, 30-50, 50-100 or more) of a sequence set out in Tables 1, 2 and 3.
  • amino acid substitutions e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-25, 25-30, 30-50, 50-100 or more residues
  • additions and insertions e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-25, 25-30, 30-50, 50-100 or more
  • similarity and “similar” and grammatical variations thereof, as used herein, mean that two or more referenced amino acid sequences contains a limited number of conservative amino acid substitutions of the amino acid sequence.
  • a variety of criteria can be used to indicate whether amino acids at a particular position in a polypeptide are similar.
  • substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing.
  • a “conservative substitution” is the replacement of one amino acid by a biologically, chemically or structurally similar residue.
  • Biologically similar means that the substitution does not destroy a biological activity.
  • Structurally similar means that the amino acids have side chains with similar length, such as alanine, glycine and serine, or a similar size.
  • Chemical similarity means that the residues have the same charge, or are both hydrophilic or hydrophobic.
  • a conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain, which include amino acids with basic side chains (e.g., lysine, arginine, histidine); acidic side chains (e.g., aspartic acid, glutamic acid); uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, histidine); nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan).
  • Particular examples include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, serine for threonine, and the like.
  • Proline which is considered more difficult to classify, shares properties with amino acids that have aliphatic side chains (e.g., Leu, Val, He, and Ala).
  • substitution of glutamine for glutamic acid or asparagine for aspartic acid may be considered a similar substitution in that glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively.
  • Conservative changes can also include the substitution of a chemically derivatized moiety for a non-derivatized residue, for example, by reaction of a functional side group of an amino acid.
  • Variants and derivatives of polypeptides include forms having a limited number of one or more substituted residues.
  • a polypeptide of option a) and b) may be longer than the reference sequence set out in Tables 1, 2 and 3.
  • an addition can be one or more additional amino acid residues.
  • a polypeptide of option a) may contain amino acid residues in addition to the 15 amino acid residues of the PG peptide, and optionally, the additional amino acid residues may be identical to those present in the wild type NTGA from which the PG peptide derives from.
  • the polypeptide of option a) comprises one or more amino acid residues in addition to the 15 contiguous amino acids (PG peptide) set out in Table 1, wherein the additional amino acid residue(s) is/are selected from an amino acid residue or an amino acid sequence within the wild type protein of which the PG peptide is a part of (e.g. wild type sequences of Tables 2 or 3.
  • the wild type amino acid residue or wild type amino acid sequence to be added may be adjacent to, subtended, comprised within, overlapping with or is a part of the PG peptide sequence, when present in its natural biological context within the wild type protein.
  • An illustrative example is a PG peptide of NTGA 6 as set out in Table 1 that may be extended with amino acid residues from NTGA 6 set out in Table 2, or a homolog thereof set out in Table 3, such as amino acid residues adjacent to the PG sequence when aligned with NTGA 6 or the homolog thereof.
  • the additional amino acid residues may be added to the N- and/or C- terminal end of a sequence set out in Tables 1, 2 and 3, such as additional amino acids selected from amino acids flanking the N- and/or C- terminal ends when sequence is aligned with the source protein it is present in, based upon or derived from.
  • the additional amino acids may be the amino acids flanking the N- and/or C- terminal ends of the sequence when aligned to NTGA 6.
  • a polypeptide of option a) and b is derivatized. Specific non-limiting examples of derivatization are covalent or non-covalent attachment of another molecule.
  • a derivative is a fusion (chimeric) sequence, an amino acid sequence having one or more molecules not normally present in the wild type sequence covalently attached to the sequence.
  • chimeric and grammatical variations thereof, when used in reference to a sequence, means that the sequence contains one or more stretches that are derived from, obtained or isolated from, or based upon other physical or chemical entities.
  • a chimera of two or more different polypeptides may have one part a polypeptide, and a second part of the chimera may be from a different sequence, or unrelated protein sequence.
  • Another particular example of a derivatized polypeptide is one in which a second
  • heterologous sequence i.e., heterologous functional domain is attached (covalent or non- covalent binding) that confers a distinct or complementary function.
  • Heterologous functional domains are not restricted to amino acid residues.
  • a heterologous functional domain can consist of any of a variety of different types of small or large functional moieties.
  • Such moieties include nucleic acid, peptide, carbohydrate, lipid or small organic compounds, such as a drug (e.g., an antiviral), a metal (gold, silver), and radioisotope.
  • a tag such as T7 or polyhistidine can be attached in order to facilitate purification or detection of a protein, peptide, etc.
  • a 6-HIS tag may be added to the C- or N-terminal end of a polypeptide of option a), b), c) or d), e.g. the 6-HIS sequence GHHHHHHGSGMLDI, which optionally may remain in the immunogen when administered to a subject.
  • a polypeptide linked to a Tag containing histidines may easily be purified by use of a HIS tag affinity column.
  • polypeptides linked to a heterologous domain wherein the heterologous functional domain confers a distinct function on the polypeptide.
  • the polypeptide is derivatized for example to improve solubility, stability, bioavailability or biological activity.
  • tagged polypeptides and fusion proteins and modifications, including peptides having one or more non-amino acyl groups (q.v., sugar, lipid, etc.) covalently linked to the polypeptide and post-translational modifications.
  • Linkers such as amino acid or peptidomimetic sequences may be inserted between the sequence and the addition (e.g ., heterologous functional domain) so that the two entities maintain, at least in part, a distinct function or activity.
  • Linkers may have one or more properties that include a flexible conformation, an inability to form an ordered secondary structure or a hydrophobic or charged character, which could promote or interact with either domain.
  • Amino acids typically found in flexible protein regions include Gly, Asn and Ser. Other near neutral amino acids, such as Thr and Ala, may also be used in the linker sequence.
  • the length of the linker sequence may vary without significantly affecting a function or activity of the fusion protein (see, e.g ., U.S. Patent No.
  • Linkers further include chemical moieties and conjugating agents, such as sulfo-succinimidyl derivatives (sulfo-SMCC, sulfo-SMPB), disuccinimidyl suberate (DSS), disuccinimidyl glutarate (DSG) and disuccinimidyl tartrate (DST) .
  • sulfo-SMCC sulfo-succinimidyl derivatives
  • SSS disuccinimidyl suberate
  • DSG disuccinimidyl glutarate
  • DST disuccinimidyl tartrate
  • the invention provides polypeptides that are detectably labeled .
  • detectable labels include fluorophores, chromophores, radioactive isotopes (e.g ., S35, P32, 1125), electron-dense reagents, enzymes, ligands and receptors.
  • Enzymes are typically detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert a substrate such as 3,3-',5,5-'-tetramethylbenzidine (TMB) to a blue pigment, which can be quantified.
  • TMB 3,3-',5,5-'-tetramethylbenzidine
  • Modified polypeptides also include one or more D-amino acids substituted for L-amino acids (and mixtures thereof), structural and functional analogues, for example, peptidomimetics having synthetic or non-natural amino acids or amino acid analogues and derivatized forms. Modifications include cyclic structures such as an end-to-end amide bond between the amino and carboxy-terminus of the molecule or intra- or inter-molecular disulfide bond.
  • polypeptide of the invention may be modified to avoid oxidation, improve solubility in aqueous solution, avoid aggregation, overcome synthesis problems etc.
  • polypeptide amino acid sequence may include the following modifications:
  • a polypeptide may comprise one, two or more lysine or arginine amino acid residue(s) added to the N- or C-terminus of the peptide to be modified, which may improve the aqueous solubility.
  • a polypeptide of the invention may comprise one or more cysteine residues that are substituted with amino acid residues less prone to oxidation, e.g. serine or arginine.
  • Polypeptides may be provided in the form of a salt, for example as a pharmaceutically acceptable and/or a physiologically acceptable salt.
  • the salt may be an acid addition salt with an inorganic acid, an acid addition salt with an organic acid, a salt with a basic inorganic acid, a salt with a basic organic acid, a salt with an acidic or basic amino acid or a mixture thereof.
  • a salt such as a
  • pharmaceutically acceptable salt is an acetate salt.
  • the invention provides polypeptides and molecules in isolated and/or purified form.
  • isolated when used as a modifier of a composition, means that the compositions are made by the hand of man or are separated, completely or at least in part, from their naturally occurring in vivo environment. Generally, isolated compositions are substantially free of one or more materials with which they normally associate with in nature, for example, one or more protein, nucleic acid, lipid, carbohydrate, cell membrane.
  • isolated does not exclude alternative physical forms of the composition, such as fusions/chimeras, multimers/oligomers, modifications (e.g., phosphorylation, glycosylation, lipidation) or derivatized forms, or forms expressed in host cells produced by the hand of man.
  • an “isolated” composition e.g. polypeptides or molecules as defined herein
  • an isolated polypeptide that also is substantially pure or purified does not include polypeptides or polynucleotides present among millions of other sequences, such as polypeptide of an peptide library or nucleic acids in a genomic or cDNA library, for example.
  • compositions can be combined with one or more other molecules.
  • substantially pure or purified does not exclude combinations of compositions, such as combinations of polypeptides other antigens, agents, drugs or therapies.
  • Polypeptides can be prepared recombinantly, chemically synthesized, isolated from a biological material or source, and optionally modified, or any combination thereof.
  • a biological material or source would include an organism that produced or possessed any polypeptide or molecule set forth herein.
  • a biological material or source may further refer to a preparation in which the morphological integrity or physical state has been altered, modified or disrupted, for example, by dissection, dissociation, solubilization, fractionation, homogenization, biochemical or chemical extraction, pulverization, lyophilization, sonication or any other means of manipulating or processing a biological source or material.
  • Polypeptides of the invention can be prepared, for example, by substituting, deleting or adding one or more amino acid residues in the amino acid sequence and screening for biological activity, for example eliciting an immune response.
  • a skilled person will understand how to make such derivatives or variants, using standard molecular biology techniques and methods, described for example in Sambrook et al. (2001) Molecular Cloning: a Laboratory Manual, 3rd ed., Cold Spring Harbour Laboratory Press).
  • Polypeptides and molecules that are provided herein can be employed in various methods and uses. Such methods and uses include, for example, administration in vitro and in vivo of one or more polypeptides or molecules thereof.
  • the methods and uses provided include methods and uses of modulating an immune response (e.g. an allergic immune response), including, among others, methods and uses of relieving an immune response (e.g. allergic immune response), protecting and treating subjects against a disorder, disease (e.g. allergic disease); and methods and uses of providing immunotherapy, such as specific
  • methods and uses include administration or delivery of an immunogen provided herein to modulate an immune response in a subject, including, for example, modulating an immune response to a pollen allergen or the immunogen.
  • modulate means an alteration or effect on the term modified.
  • modulating involves decreasing, reducing, inhibiting, suppressing, relieving an immune response in a subject to an allergen or an immunogen provided herein.
  • modulating involves eliciting, stimulating, inducing, promoting, increasing or enhancing an immune response in a subject to an antigen or allergen.
  • the term “modulate” is used to modify the term "immune response against an allergen in a subject” this means that the immune response in the subject to the allergen or immunogen is altered or affected (e.g., decreased, reduced, inhibited, suppressed, limited, controlled, prevented, elicited, promoted, stimulated, increased, induced, enhanced, etc.
  • methods and uses of modulating an immune response against an allergen or immunogen as described herein may be used to provide a subject with protection against an allergic immune response or immune reaction to the allergen or immunogen, or symptoms or complications caused by or associated with the allergen or immunogen. Accordingly, in other embodiments, methods and uses include administering an immunogen of the invention to protect or treat a subject against an allergic immune response, or one or more symptoms caused by or associated with an allergen. In still other embodiments, methods and uses include administering or delivering an immunogen of the invention to elicit, stimulate, induce, promote, increase or enhance immunological tolerance of a subject to an allergen or immunogen disclosed herein.
  • a method or use includes administering to the subject an amount of an immunogen of the invention sufficient to provide the subject with protection against the allergic immune response, or symptoms caused by or associated with the allergen or immunogen.
  • Methods and uses of the invention include providing a subject with protection against an allergen or an immunogen, or symptoms caused by or associated with the subjects exposure to the allergen or immunogen, for example, vaccinating the subject to protect against an allergic immune response to the allergen or immunogen, for example with an immunogen provided herein.
  • methods and uses include protecting the subject against an allergic immune response by inducing tolerance of the subject (desensitizing) to the allergen, and optionally to the immunogen.
  • the terms "protection,” “protect” and grammatical variations thereof, when used in reference to an allergic immune response or symptoms caused by or associated with the exposure to allergen, means preventing an allergic immune response or symptoms caused by or associated with the exposure to the allergen, or reducing or decreasing susceptibility to an allergic immune response or one or more symptoms caused by or associated with the exposure to the allergen.
  • An allergic immune response includes but is not limited to an allergic reaction
  • allergic immune response may involve one or more of cell infiltration, production of antibodies, production of cytokines, lymphokines, chemokines, interferons and interleukins, cell growth and maturation factors (e.g., differentiation factors), cell proliferation, cell differentiation, cell accumulation or migration (chemotaxis) and cell, tissue or organ damage or remodeling.
  • an allergic immune response may include allergic rhinitis; atopic dermatitis; allergic conjunctivitis and asthma. Allergic responses can occur systemically, or locally in any region, organ, tissue, or cell.
  • an allergic immune response occurs in the skin, the upper respiratory tract, the lower respiratory tract, pancreas, thymus, kidney, liver, spleen, muscle, nervous system, skeletal joints, eye, mucosal tissue, gut or bowel.
  • Methods and uses herein include relieving, including treating, a subject for an allergic immune response, or one or more symptoms caused by or associated with an allergen. Such methods and uses include administering to a subject an amount of an immunogen sufficient to relieve, such as treat, the subject for the allergic immune response, or one or more symptoms caused by or associated with the allergen. Methods and uses of the invention include treating or administering a subject previously exposed to an allergen or immunogen. Thus, in certain embodiments, methods and uses are for treating or protecting a subject from an allergic immune response, or one or more symptoms caused by or associated with secondary or subsequent exposure to an allergen or an immunogen.
  • Immunogens described herein may elicit, stimulate, induce, promote, increase or enhance immunological tolerance to an allergen and/or to the immunogen. Methods and uses of the invention therefore further include inducing immunological tolerance of a subject to an allergen or the immunogen itself.
  • immunogens described herein can be effective in relieving, such as treating an allergic immune response, including but not limited to an allergic immune response following a secondary or subsequent exposure of a subject to an allergen.
  • a method or use includes administering to the subject an amount of an immunogen sufficient to induce tolerance in the subject to the allergen or immunogen itself.
  • the immunological tolerance elicited, stimulated, induced, promoted, increased or enhanced may involve modulation of T cell activity, including but not limited to CD4+ T cells, CD8+ T cells, Thl cells, Th2 cells and regulatory T cells.
  • immunological tolerance elicited, stimulated, induced, promoted, increased or enhanced from administration of the immunogen may involve modulation of the production or activity of pro-inflammatory or anti-inflammatory cytokines produced by T cells.
  • a method or use of inducing immunological tolerance in a subject to an allergen includes a reduction in occurrence, frequency, severity, progression, or duration of physiological conditions, disorders, illnesses, diseases, symptoms or complications caused by or associated an allergic response to the allergen in the subject.
  • inducing immunological tolerance can protect a subject against or treat a subject for an allergic immune response, or one or more symptoms caused by or associated with an allergen or the immunogen.
  • Methods and uses of the invention include treating a subject via immunotherapy, including specific immunotherapy.
  • a method or use includes administering to the subject an amount of an immunogen described herein.
  • an immunogen administered to a subject during specific immunotherapy to treat the subject is the same immunogen to which the subject has been sensitized or is hypersensitive (e.g., allergic).
  • an immunogen is administered to a subject to treat the subject to a different immunogen, e.g. a pollen allergen to which the subject has been sensitized or is hypersensitive (e.g., allergic).
  • the immunotherapeutic mechanism may involve bystander suppression of an allergic immune response caused by a pollen allergen by administering an unrelated immunogen, e.g.
  • an immunogen disclosed herein an immunogen disclosed herein.
  • an immunogen of the invention my induce bystander tolerance induction to an unrelated immunogen, e.g., a pollen allergen disclosed herein.
  • immunogens include T cell epitopes, such as Th2 cell epitopes.
  • the subject to be treated has a specific T-cell response to the immunogen before administering the first dose.
  • methods and uses of the invention include administering an amount of a an immunogen (e.g., a T cell epitope-containing immunogen) to a subject sufficient to provide the subject with protection against an allergic immune response, or one or more symptoms caused by or associated with an allergen.
  • a method includes administering an amount of an immunogen (e.g., a T cell epitope-containing immunogen) to a subject sufficient to relieve, e.g. treat, vaccinate or immunize the subject against an allergic immune response, or one or more symptoms caused by or associated with an allergen.
  • the specific T-cell response may be monitored by determining by way of contacting a sample of PBMCs obtained from the subject with the immunogens and measuring the IL-5 secretion or IL-5 mRNA gene expression in response to the immunogen.
  • a method or use includes administering to a subject an amount of a polypeptide of the invention or derivative thereof including an immunogenic molecule thereof, such as a T cell epitope, sufficient to modulate Th2 cell activity in the subject.
  • a polypeptide of the invention or derivative thereof including an immunogenic molecule thereof, such as a T cell epitope sufficient to modulate Th2 cell activity in the subject.
  • two or more immunogens may be administered to a subject, e.g. may be administered as a combination composition, or administered separately, such as concurrently or in series or sequentially.
  • methods and uses described herein comprises administration separately or as a combination: at least 2-25 polypeptides defined herein, or separately or as a combination of 3-25, 4-25, 5-25, 6-25, 7-25 polypeptides defined herein, or separately or as a combination of 2-20, 3-20, 4-20, 5-20, 6-20 defined herein, or separately or as a combination of 2-12, 3-12, 4-12, 5-12, 6-12, 7-12
  • polypeptides defined herein or separately or as a combination of 2-10, 3-10, 4-10, 5-10, 6- 10, 7-10 polypeptides defined herein.
  • a there may be administered to a subject, e.g. as a combination composition, one or more immunodominant PG peptides, like those recognized by at least 3 subjects in a population of 20 subjects, e.g.
  • composition comprising one more polypeptides of option a), wherein each polypeptide of option a may independently include one or more sequences selected from any one of SEQ ID NOs: 19, 20, 24, 25, 26, 32, 37, 69, 71, 72, 73, 74, 78, 79, 80, 81, 82, 84, 86, 87, 88, 89, 90, 91, 93, 95, 99, 319, 321 and 506, or a sequence with 0, 1 or 2 mismatches compared to the SEQ ID NOs: 19, 20, 24, 25, 26, 32, 37, 69, 71, 72, 73, 74, 78, 79, 80, 81, 82, 84, 86, 87, 88, 89, 90, 91, 93, 95, 99, 319, 321 and 506.
  • Compositions may comprise one or more polypeptides of option a that derives from NTGA's or homologs thereof that are co-released with major allergens from grass pollen, e.g.
  • each polypeptide of option a) may include one or more sequences selected from any one of SEQ ID NOs: 1-18, 38-40, 41-55, 56-62, 63-89, 148-156, 169-182, 183-190, 211-232, 270-277, 338-348, 292-296, 354-356, 409-419, 467-481, 485-513 and 519, or a sequence with 0, 1 or 2 mismatches compared to the SEQ ID NOs: 1-18, 38-40, 41-55, 56- 62, 63-89, 148-156, 169-182, 183-190, 211-232, 270-277, 338-348, 292-296, 354-356, 409-419, 467-481, 485-513 and 519.
  • Methods and uses of the invention therefore include any therapeutic or beneficial effect.
  • an allergic immune response, or one or more symptoms caused by or associated with an allergen is reduced, decreased, inhibited, limited, delayed or prevented.
  • Methods and uses of the invention moreover include reducing, decreasing, inhibiting, delaying or preventing onset, progression, frequency, duration, severity, probability or susceptibility of one or more adverse symptoms, disorders, illnesses, diseases or complications caused by or associated with an antigen/allergen.
  • methods and uses include improving, accelerating, facilitating, enhancing, augmenting, or hastening recovery of a subject from an allergic immune response, or one or more physiological conditions, symptoms or complications caused by or associated with an antigen/allergen.
  • methods and uses include stabilizing an allergic immune response, or one or more physiological conditions, symptoms or complications caused by or associated with an antigen/allergen.
  • a therapeutic or beneficial effect is therefore any objective or subjective measurable or detectable improvement or benefit provided to a particular subject.
  • a therapeutic or beneficial effect can but need not be complete ablation of all or any allergic immune response, or one or more symptoms caused by or associated with an allergen.
  • a satisfactory clinical endpoint is achieved when there is an incremental improvement or a partial reduction in an allergic immune response, or one or more symptoms caused by or associated with an allergen, or an inhibition, decrease, reduction, suppression, prevention, limit or control of worsening or progression of an allergic immune response, or one or more symptoms caused by or associated with an allergen, over a short or long duration (hours, days, weeks, months, etc.).
  • a therapeutic or beneficial effect also includes reducing or eliminating the need, dosage frequency or amount of a second therapeutic protocol or active such as another drug or other agent (e.g., anti-inflammatory) used for treating a subject having or at risk of having an allergic immune response, or one or more symptoms caused by or associated with an allergen.
  • an amount of an adjunct therapy such as a reduction or decrease of a treatment for an allergic immune response, or one or more symptoms caused by or associated with an allergen, or a specific immunotherapy, vaccination or immunization protocol is considered a beneficial effect.
  • reducing or decreasing an amount of the immunogen used for specific immunotherapy, vaccination or immunization of a subject to provide protection to the subject is considered a beneficial effect.
  • Methods and uses described herein may relieve one or more symptoms of an allergic immune response or delays the onset of symptoms, slow the progression of symptoms, or induce disease modification.
  • the following symptoms may be decreased or eliminated; nasal symptoms in the form of itchy nose, sneezing, runny nose, blocked nose; conjunctival symptoms in the form of itchy eyes, red eyes, watery eyes; and respiratory symptoms in the form of decreased lung function.
  • nasal symptoms in the form of itchy nose, sneezing, runny nose, blocked nose conjunctival symptoms in the form of itchy eyes, red eyes, watery eyes
  • respiratory symptoms in the form of decreased lung function may be decreased or eliminated.
  • an amount or dose of the immunogen to be administered can be determined by one skilled in the art.
  • the immunogen may be administered to the patient through any route known in the art, including, but not limited to oral, inhalation, sublingual, epicutaneous, intranasal, and/or parenteral routes (intravenous, intramuscular, subcutaneously, intradermal, and intraperitoneal).
  • Methods and uses of the invention include administration of an immunogen to a subject prior to contact by or exposure to an allergen; administration prior to, substantially contemporaneously with or after a subject has been contacted by or exposed to an allergen; and administration prior to, substantially contemporaneously with or after an allergic immune response, or one or more symptoms caused by or associated with an allergen.
  • a "sufficient amount” or “effective amount” or an “amount sufficient” or an “amount effective” refers to an amount that provides, in single (e.g., primary) or multiple (e.g., booster) doses, a long term or a short term detectable or measurable improvement in a given subject or any objective or subjective benefit to a given subject of any degree or for any time period or duration (e.g., for minutes, hours, days, months, years, or cured) .
  • An amount sufficient or an amount effective need not be therapeutically or prophylactically effective in each and every subject treated, nor a majority of subjects treated in a given group or population.
  • An amount sufficient or an amount effective means sufficiency or effectiveness in a particular subject, not a group of subjects or the general population. As is typical for such methods, different subjects will exhibit varied responses to a method of the invention, such as immunization, vaccination, specific immunotherapy and therapeutic treatments.
  • subject includes but is not limited to a subject at risk of allergen contact or exposure as well as a subject that has been contacted by or exposed to an allergen.
  • a subject also includes those having or at risk of having or developing an immune response to an antigen or an allergen .
  • Such subjects include mammalian animals (mammals), such domestic animal (dogs and cats), a farm animal (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), experimental animal (mouse, rat, rabbit, guinea pig) and humans.
  • Target subjects and subjects in need of treatment also include those at risk of allergen exposure or contact or at risk of having exposure or contact to an allergen. Accordingly, subjects include those at increased or elevated (high) risk of an allergic reaction; has, or has previously had or is at risk of developing hypersensitivity to an allergen; and those that have or have previously had or is at risk of developing asthma.
  • an allergic immune response e.g. preventing or treating an allergic immune response against a grass pollen allergen, which is not a Phi p grass pollen allergen by administering an immunogen described herein.
  • Non-Phi p grass pollen allergens are but not limited to pollen allergens of the plant family Poaceae, e.g. the plant genera Anthoxanthum, Cynodon, Dactylis, Festuca, Holcus,
  • Immunogens disclosed herein are conserved across a Phi p grass pollen and at least non-Phi p grass pollen species, e.g. Anthoxanthum, Cynodon, Dactylis, Lollium, and Poa.
  • the allergic immune response is not against a grass pollen allergen of the plant genus Phleum, e.g. Phleum Pratense.
  • immunogens of the invention are conserved across a Phi p grass pollen and at least Cyn d pollen, methods and uses described herein, comprises relieving an allergic immune response against Phi p grass pollen as well as against Cyn d pollen or another non-Phi p pollen, e.g. Lol P pollen, Poa p Pollen or Ant O pollen.
  • grass pollen allergens are but not limited to; Ant o 1, Cyn d 1, Cyn d 7, Cyn d 12, Cyn d 15, Cyn d 22w, Cyn d 23, Cyn d 24, Dac g 1, Dac g 2, Dac g 3, Dac g 4, Dac g 5, Fes p 4, Hoi I 1, Hoi I 5, Hor v 1, Hor v 5, Lol p 1, Lol p 2, Lol p 3, Lol p 4, Lol p 5, Lol p 11, Ory s 1, Pas n 1, Pha a 1, Pha a 5, Phi p 1, Phi p 2, Phi p 4, Phi p 5, Phi p , Phi p 7, Phi p 11, Phi p 12, Phi p 13, Poa p 1, Poa p 5, Sec c 1, Sec c 5, Sec c 38 and/or Sor h 1, of which group 1 allergens (e.g.
  • Ant o 1, Cyn d 1, Dac g 1, Hoi 1, Lol p 1, Pha a 1, Phi p 1 and Poa p 1) or group 5 allergens (Dac g 5, Lol p 5, Pha a 5, Phi p 5, Poa p 5) are considered major allergens important for the allergic immune response triggered by a grass pollen in a subject.
  • group 5 allergens Dac g 5, Lol p 5, Pha a 5, Phi p 5, Poa p 5
  • Many of the well known pollen allergens are major allergens and thought to be the most important allergens in eliciting an allergic immune in a subject.
  • the non Phi p grass pollen allergen at least is Ant o 1, Cyn d 1, Dac g 1, Hoi 1, Lol p 1, Pha a 1, and Poa p 1) or group 5 allergens (Dac g 5, Lol p 5, Pha a 5, Poa p 5).
  • “Prophylaxis” and grammatical variations thereof mean a method or use in which contact, administration or in vivo delivery to a subject is prior to contact with or exposure to an allergen. In certain situations it may not be known that a subject has been contacted with or exposed to an allergen, but administration or in vivo delivery to a subject can be performed prior to manifestation of an allergic immune response, or one or more symptoms caused by or associated with an allergen.
  • a subject can be provided protection against an allergic immune response, or one or more symptoms caused by or associated with an allergen or provided specific immunotherapy with a polypeptide of a derivative thereof, including an immunogenic molecule described herein.
  • a method or use can eliminate, prevent, inhibit, suppress, limit, decrease or reduce the probability of or susceptibility towards an allergic immune response, or one or more physiological conditions, symptoms or complications caused by or associated with an antigen/allergen.
  • “Prophylaxis” can also refer to a method or use in which contact, administration or in vivo delivery to a subject is prior to a secondary or subsequent exposure to an antigen/ allergen.
  • a subject may have had a prior contact or exposure to an allergen.
  • an acute allergic reaction may but need not be resolved.
  • Such a subject typically may have developed anti-allergen antibodies due to the prior exposure.
  • Immunization or vaccination by administration or in vivo delivery to such a subject, can be performed prior to a secondary or subsequent allergen exposure.
  • Such a method or use can eliminate, prevent, inhibit, suppress, limit, decrease or reduce the probability of or susceptibility towards a secondary or subsequent allergic immune response, or one or more symptoms caused by or associated with an allergen.
  • such a method or use includes providing specific immunotherapy to the subject to eliminate, prevent, inhibit, suppress, limit, decrease or reduce the probability of or susceptibility towards a secondary or subsequent allergic immune response, or one or more physiological conditions, symptoms or complications caused by or associated with an antigen/allergen.
  • Treatment of an allergic reaction or response can be at any time during the reaction or response.
  • An immunogen can be administered as a single or multiple dose e.g., one or more times hourly, daily, weekly, monthly or annually or between about 1 to 10 weeks, or for as long as appropriate (e.g. 3 months, 6 months or more, for example, to achieve a reduction in the onset, progression, severity, frequency, duration of one or more symptoms or complications associated with or caused by an allergic immune response, or one or more physiological conditions, symptoms or complications caused by or associated with an antigen/allergen.
  • methods and uses of the invention can be practiced one or more times (e.g., 1- 10, 1-5 or 1-3 times) an hour, day, week, month, or year.
  • Doses can be based upon current existing protocols, empirically determined, using animal disease models or optionally in human clinical trials.
  • Initial study doses can be based upon animal studies, e.g. a mouse, and the amount of the immunogen to be administered for being effective can be determined.
  • Exemplary non-limiting amounts (doses) are in a range of about 0.1 mg/kg to about 100 mg/kg, and any numerical value or range or value within such ranges.
  • doses can be administered, for example, 0.01-500 mg/kg, and any numerical value or range or value within such ranges.
  • the dose can be adjusted according to the mass of a subject, and will generally be in a range from about 1-10 ug/kg, 10-25 ug/kg, 25-50 ug/kg, 50-100 ug/kg, 100-500 ug/kg, 500-1,000 ug/kg, 1-5 mg/kg, 5-10 mg/kg, 10-20 mg/kg, 20-50 mg/kg, 50-100 mg/kg, 100-250 mg/kg, 250-500 mg/kg, or more, two, three, four, or more times per hour, day, week, month or annually.
  • a typical range will be from about 0.3 mg/kg to about 50 mg/kg, 0-25 mg/kg, or 1.0-10 mg/kg, or any numerical value or range or value within such ranges.
  • Doses can vary and depend upon whether the treatment is prophylactic or therapeutic, whether a subject has been previously exposed to the antigen/allergen, the onset, progression, severity, frequency, duration, probability of or susceptibility of the symptom, condition, pathology or complication, or vaccination or specific immunotherapy to which treatment is directed, the clinical endpoint desired, previous or simultaneous treatments, the general health, age, gender, race or immunological competency of the subject and other factors that will be appreciated by the skilled artisan. The skilled artisan will appreciate the factors that may influence the dosage and timing required to provide an amount sufficient for providing a therapeutic or prophylactic benefit. Immunogens of the invention can be provided in compositions, and in turn such
  • compositions can be used in accordance with the invention methods and uses. Such compositions, methods and uses include pharmaceutical compositions and formulations.
  • a pharmaceutical composition includes one or more immunogens.
  • such compositions and formulations may be a vaccine, including but not limited to a vaccine to protect against an allergic immune response, or one or more symptoms caused by or associated with an allergen.
  • a pharmaceutical comprises an immunogen of the invention and a pharmaceutically acceptable ingredient or carrier.
  • the term "pharmaceutically acceptable” and “physiologically acceptable” mean a biologically acceptable formulation, gaseous, liquid or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery or contact.
  • Such formulations include solvents (aqueous or non aqueous), solutions (aqueous or non aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery.
  • Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents.
  • Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules and crystals.
  • Supplementary active compounds e.g., preservatives, antibacterial, antiviral and antifungal agents
  • compositions may be lyophilized so as to enhance stability and ease of transportation.
  • the composition may be sterile.
  • Pharmaceutical compositions can be formulated to be compatible with a particular route of administration.
  • pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.
  • Exemplary routes of administration for contact or in vivo delivery for which a composition can optionally be formulated include inhalation, intranasal, oral, buccal, sublingual, subcutaneous, intradermal, epicutaneous, rectal, transdermal, or intralymphatic.
  • the pharmaceutical composition is aqueous and, in other embodiments, the composition is non-aqueous solutions, suspensions or emulsions of the peptide/protein, which compositions are typically sterile and can be isotonic with the biological fluid or organ of the intended recipient.
  • Non-limiting illustrative examples include water, saline, dextrose, fructose, ethanol, vegetable or synthetic oils.
  • a composition can take the form of for example a solid dosage form, e.g. tablets or capsules, optionally formulated as fast- integrating tablets/capsules or slow-release tablets/capsules.
  • the tablet is a freeze-dried, optionally fast-disintegrating tablet suitable for being administered under the tongue.
  • a solid dosage form optionally is sterile, optionally anhydrous.
  • the pharmaceutical composition may also be formulated into a "unit dosage form".
  • a unit dosage form refers to physically discrete units suited as dosages for the subject to be treated; each unit containing a predetermined quantity of a peptide/protein optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle or filling agent) which, when administered in one or more doses, is calculated to produce a desired effect.
  • Unit dosage forms also include, for example, ampules and vials, which may include a composition in a freeze-dried or lyophilized state; a sterile liquid carrier, for example, can be added prior to administration or delivery in vivo.
  • Unit dosage forms additionally include, for example, ampules and vials with liquid compositions disposed therein. Individual unit dosage forms can be included in multi-dose kits or containers. Pharmaceutical formulations can be packaged in single or multiple unit dosage form for ease of administration and uniformity of dosage.
  • immunogens can be mixed with adjuvants.
  • Monophosphoryl lipid A and derivatives thereof e.g., 3 De-O-acylated monophosphoryl lipid A, aka 3D-MPL or d3-MPL, to indicate that position 3 of the reducing end glucosamine is de- O-acylated, 3D-MPL consisting of the tri and tetra acyl congeners), and enterobacterial lipopolysaccharides (LPS); plant derived saponins and derivatives thereof, for example Quil A (isolated from the Quilaja Saponaria Molina tree, see, e.g., "Saponin adjuvants", Archiv. fur dierare Virusforschung, Vol. 44, Springer Verlag, Berlin, p243-254; U.S. Patent No. 5,057,540), and fragments of Quil A which retain adjuvant activity without associated toxicity, for example QS7 and QS21 (also known as QA7 and QA21), as described in
  • W096/33739 for example; surfactants such as, soya lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone; oligonucleotides such as CpG (WO 96/02555, and WO 98/16247), polyriboA and polyriboU; block copolymers; and
  • immunostimulatory cytokines such as GM-CSF and IL-1, and Muramyl tripeptide (MTP).
  • MTP Muramyl tripeptide
  • Cosolvents may be added to the composition.
  • cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters.
  • cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol,
  • polypropylene glycol glycol ether
  • glycol ether glycol ether
  • glycerol glycol ether
  • polyoxyethylene alcohols polyoxyethylene fatty acid esters.
  • Supplementary compounds e.g., preservatives, antioxidants, antimicrobial agents including biocides and biostats such as antibacterial, antiviral and antifungal agents
  • Pharmaceutical compositions may therefore include preservatives, anti-oxidants and antimicrobial agents.
  • Preservatives can be used to inhibit microbial growth or increase stability of ingredients thereby prolonging the shelf life of the pharmaceutical formulation.
  • Suitable preservatives include, for example, EDTA, EGTA, benzalkonium chloride or benzoic acid or benzoates, such as sodium benzoate.
  • Antioxidants include, for example, ascorbic acid, vitamin A, vitamin E, tocopherols, and similar vitamins or provitamins.
  • An antimicrobial agent or compound directly or indirectly inhibits, reduces, delays, halts, eliminates, arrests, suppresses or prevents contamination by or growth, infectivity, replication, proliferation, reproduction, of a pathogenic or non- pathogenic microbial organism.
  • Classes of antimicrobials include antibacterial, antiviral, antifungal and
  • Antimicrobials include agents and compounds that kill or destroy (-cidal) or inhibit (-static) contamination by or growth, infectivity, replication, proliferation,
  • compositions, methods and uses of the invention are known in the art (see, e.g. Remington: The Science and Practice of Pharmacy (David B. Troy, Paul Beringer Lippincott Williams & Wilkins) 2006).
  • compositions can be formulated to be compatible with a particular route of administration.
  • pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes (For example excipients recorded in a
  • Exemplary routes of administration for contact or in vivo delivery include inhalation, respiration, intranasal, intubation, intrapulmonary instillation, oral, buccal, intrapulmonary, intradermal, topical, dermal, parenteral, sublingual, subcutaneous, intravascular, intrathecal, intraarticular, intracavity, transdermal, iontophoretic, intraocular, opthalmic, optical, intravenous (i.v.), intramuscular, intraglandular, intraorgan, or intralymphatic.
  • inhalation respiration, intranasal, intubation, intrapulmonary instillation, oral, buccal, intrapulmonary, intradermal, topical, dermal, parenteral, sublingual, subcutaneous, intravascular, intrathecal, intraarticular, intracavity, transdermal, iontophoretic, intraocular, opthalmic, optical, intravenous (i.v.), intramuscular, intraglandular, intraorgan, or intralymphatic.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous solutions, suspensions or emulsions of the active compound, which preparations are typically sterile and can be isotonic with the blood of the intended recipient.
  • Non-limiting illustrative examples include water, saline, dextrose, fructose, ethanol, animal, vegetable or synthetic oils.
  • Methods and uses of the invention may be practiced by any mode of administration or delivery, or by any route, systemic, regional and local administration or delivery.
  • Exemplary administration and delivery routes include intravenous (i.v.), intraperitoneal (i.p.), intrarterial, intramuscular, parenteral, subcutaneous, intra-pleural, topical, dermal, intradermal, transdermal, transmucosal, intra-cranial, intra-spinal, rectal, oral (alimentary), mucosal, inhalation, respiration, intranasal, intubation, intrapulmonary, intrapulmonary instillation, buccal, sublingual, intravascular, intrathecal, intracavity, iontophoretic, intraocular, ophthalmic, optical, intraglandular, intraorgan, or intralymphatic.
  • a composition can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (for example, pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (for example, lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (for example, magnesium stearate, talc or silica); disintegrants (for example, potato starch or sodium starch glycolate); or wetting agents (for example, sodium lauryl sulphate).
  • binding agents for example, pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers for example, lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants for example, magnesium stearate, talc or silica
  • disintegrants for example, potato starch or sodium starch glycolate
  • wetting agents for example, sodium lauryl sulphate
  • suspending agents for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents for example, lecithin or acacia
  • non-aqueous vehicles for example, almond oil, oily esters, ethyl alcohol or fractionated vegetable oils
  • preservatives for example, methyl or propyl-p-hydroxybenzoates or sorbic acid
  • compositions peptides, proteins, antigens, allergens
  • substituents described herein are disclosed by the application to the same extent as if each composition or group of compositions was set forth individually. Thus, selection of particular peptides, proteins, antigens, allergens, etc. is clearly within the scope of the invention.
  • any concentration range, percentage range, ratio range or other integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • a range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the use of a range expressly includes all possible subranges, all individual numerical values within that range, and all numerical values or numerical ranges including integers within such ranges and fractions of the values or the integers within ranges unless the context clearly indicates otherwise.
  • reference to a range of 90-100% includes 91-99%, 92-98%, 93-95%, 91-98%, 91-97%, 91-96%, 91-95%, 91-94%, 91-93%, and so forth.
  • Reference to a range of 90-100% includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%,
  • Reference to a range of 5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, 100-150, and 150-175, includes ranges such as 5-20, 5-30, 5-40, 5-50, 5-75, 5- 100, 5-150, 5-171, and 10-30, 10-40, 10-50, 10-75, 10-100, 10-150, 10-175, and 20-40, 20-50, 20-75, 20-100, 20-150, 20-175, and so forth.
  • reference to a series of ranges of 2-72 hours, 2-48 hours, 4-24 hours, 4-18 hours and 6-12 hours includes ranges of 2-6 hours, 2, 12 hours, 2-18 hours, 2-24 hours, etc., and 4-27 hours, 4-48 hours, 4-6 hours, etc.
  • Table 1 indicates for each of the PG peptides (SEQ ID NOs 1-519) in which other grass pollen species (across Cyn d and Lol p or across Cyn d, Lol p, Ant o and Poa p) a matching peptide with either less than 3, less than 2 or zero mismatches are found.
  • Table 2 shows wild type sequences of NTGA's detected by combined transcriptomic analysis and Mass spectrometry analysis of grass pollen extracts.
  • ASLKPEFVDIIKSATVKSSS SEQ NTGA Phi p wild type sequence (SEQ ID NO 520-565)
  • Table 3 shows full length sequences of homologs to NTGA's of Table 2 found in other grass pollen species.

Abstract

The invention relates to pan grass pollen immunogens such as polypeptides, proteins and peptides, and methods and uses of such immunogens for modulating or relieving an immune response in a subject. For example, the pan grass pollen immunogens can be used for treating a subject for an allergic immune response or inducing or promoting immunological tolerance to the immunogen or a non-grass pollen allergen in a subject.

Description

GRASS POLLEN IMMUNOGENS AND METHODS AND USES FOR IMMUNE RESPONSE
MODULATION
Government support
This invention was made with government support under contract NIH- NIAIDHHSN272200700048C awarded by the National Institutes of Health. The government has certain rights in the invention.
Field of the invention
The invention relates to pan grass pollen immunogens such as polypeptides, proteins and peptides, and methods and uses of such immunogens for modulating or relieving an immune response in a subject, such as treating a subject for an allergic immune response or inducing or promoting immunological tolerance to the immunogen or a non-grass pollen allergen in a subject.
Introduction
Patients with pollen allergies are typically poly-sensitized as evidenced by positive RAST- and/or skin prick tests to multiple grass pollen allergens.
Allergen-specific immunotherapy (SIT) is a hyposensitizing immunotherapy introduced in clinical medicine almost a century ago for the treatment of an allergic immune response using the allergens that the subject is sensitized to. An allergic immune response may be mediated by activated allergen-specific Th2 cells, which produce cytokines such as IL-4, IL- 5, and IL-13. In healthy individuals, the allergen-specific T-cell response is mediated predominantly by Thl cells. SIT may reduce the ratio of Th2:Thl cells and may alter the cytokine profile, reducing the production of IL-4, IL-5, and IL-13 and increasing the production of IFN-g in response to major allergens or allergen extracts.
Despite its efficacy, SIT has several limitations, including safety concerns about giving patients allergenic substances. Because most SIT regimens involve the administration of whole, unfractionated, allergen extracts, adverse IgE-mediated events are a considerable risk. Significant efforts have been devoted to developing approaches to modulate allergen- specific T-cell responses without inducing IgE-meditated, immediate-type reactions. These approaches include developing hypoallergens that do not contain IgE-binding epitopes, allergens that are coupled to adjuvants and carriers of bacterial or viral origin or peptides that contain dominant T-cell epitopes and do not react with IgE in allergic individuals. Several investigators have suggested that immunotherapy with a single grass species (such as Timothy grass), is sufficient to also treat allergies to other grass pollens due to observed cross reactivity at the IgE level. However, other studies indicate that there is limited cross- reactivity of Timothy grass major allergens to other Pooideae grasses at the T cell epitope level.
It is firmly established that allergen-specific T-cells play an important role in allergic inflammation and that induction of antigen specific T regulatory cells (Tregs) or elimination of allergen-specific T helper type 2 cells (Th2) might be a prerequisite for the induction of specific tolerance. Yet, cross-reactivity among multiple pollen families at the T-cell level is less known.
It was recently shown that a large fraction of Timothy Grass-specific T cells target epitopes contained in novel Timothy Grass antigens (NTGA). NTGA's are unrelated to the known allergens of Timothy grass which mainly are identified based on their high IgE reactivity. International patent application, WO2013/119863 Al, relates to novel antigens (NTGA's) derived from Timothy grass pollen.
It has also recently been shown and described in International patent application
WO2012/049310 that an immunogen derived from an allergenic pollen source is able to reduce an allergic immune response caused by an unrelated allergen via bystander suppression. As disclosed herein, immunogens related to recently detected immunogens of Timothy grass pollen (NTGA's) share high sequence conservation/homology across several different grass pollen families and are broadly reactive. Such immunogens have potential therapeutical utilization against immune responses triggered by pollen of a broad array of grass pollen families. Summary
Disclosed herein are immunogens, also named pan-grass pollenimmunogens, derived from previously detected NTGA's. A pan-grass pollen immunogen consists of or contain as part of its sequence an amino acid sequence that is conserved across polypeptides detected in multiple grass pollen (e.g. (Kentucky blue grass (Poa pratensis, Poa p), Sweet vernal grass (Anthoxanthum odoratum, Ant o), Rye grass (Lollium Perenne, Lol p), Bermuda grass
(Cynodon dactylon, Cyn d)).In some embodiments, the immunogens may contain conserved subsequences, e.g. T cell epitope-containing subsequences of previously detected NTGA's, which T cell epitope-containing subsequence is conserved across polypeptides detected in a grass pollen of the genera Phleum and at least in one other grass pollen species. These are herein named PG sequences or PG peptides and have less than 3 mismatches to 15 contiguous amino acids of polypeptides detected in a Phi p grass pollen species and a non- Phi p grass pollen species described herein, for example Bermuda Grass pollen. Table 1 shows examples on such conserved subsequences (PG peptides) derived from previously detected NTGA's. In other embodiments, the immunogens may be larger amino sequences containing one or more PG peptide of Table 1, for example wild type sequences of NTGA's. Table 2 shows examples on wild type polypeptidesfound in Timothy grass pollen (Phi p), which contain at least one PG peptide. Table 3 discloses wild type sequences of other grass pollen that as part of its sequence also contain one or more PG peptides or one with less 3 mismatches thereto. .
In certain embodiments, the immunogens may contain at least one T cell epitope as determined by the T cell response observed against immunogens of Tables 1, 2 or 3 in cultured PBMC's of grass pollen allergic donors. Furthermore, it was found that a T cell response of grass allergic donors to an immunogen of the invention may be cross reactive to non-grass pollen species, thereby indicating that grass pollen immunogens and its conserved homolog in non-grass pollen families share T cell epitopes. It was in general demonstrated (tendency) that T cells previously stimulated with a PG peptide produced a T cell response in response to different non-grass pollen extracts when the mismatch of the PG+ peptide compared to a subsequence of a polypeptide in the non-grass pollen extract was less than 3 mismatches (Table 8, Figure 1) . Therefore, in certain embodiments, the immunogens may contain at least one PG peptide disclosed in Table 8, e.g . a PG peptide with SEG ID NO: 246, 258 and 315. That is not to exclude that an immunogen may contain another peptide disclosed in Table 8. Therefore, the invention relates in a first aspect to a method for relieving an allergic immune response against a grass pollen allergen, wherein the grass pollen allergen is not a grass pollen allergen of the genus Phleum, in a subject in need thereof, comprising administering an effective amount of an immunogenic molecule, wherein said molecule comprises or consists of a) a polypeptide, which includes at least one amino acid sequence with 0, 1 or 2
mismatches compared to a sequence selected from any one of SEQ ID NOs: 1-519; or b) a polypeptide comprising an amino acid sequence having at least 65% sequence similarity or identity to a sequence selected from any one of SEQ ID NOs: 520-565. SEQ ID NOs: 1-519 set out in Table 1 refers to PG peptides, which 15mer amino acid sequence is conserved across a Timothy grass pollen (Phi p) and at least a grass pollen Cyn d and Lol p.
SEQ ID NOs: 520-565 set out in Table 2 refers to full length sequences of NTGA's identified by combined transcriptomic and Mass Spectrometry analysis and which sequence contains regions conserved across at least a grass pollen and at least a grass pollen Cyn d and Lol p.
Below is shown embodiments specifically related to each of the conserved NTGA's identified. For example in embodiment AM the polypeptides relates to NTGA 89 and the polypeptide of option a) includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 485-513; the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 563. Other embodiments (A to AK) may be constructed the same way using the list below:
Embodiments: NTGA No: Polypeptide option a) Polypeptide option
PG Sequence of Table 1 : b)
Full length sequence of Table 2:
Embodiment A 1 1-18 520
Embodiment B 2 19-37 521
Embodiment C 3 38-40 522
Embodiment D 4 41-55 523
Embodiment E 5/64 56-62 524
Embodiment F 6 63-89 525
Embodiment G 7 89-99 526
Embodiment H 8 100 527
Embodiment I 9 101-108 528
Embodiment J 10 109-111 529
Embodiment K 11 112-119 530
Embodiment L 13 120-134 531
Embodiment M 19 143-147 532
Embodiment N 20 148-156 533
Embodiment O 22 162-168 534
Embodiment P 24 169-182 535
Embodiment Q 26 183-190 536 Embodiments: NTGA No: Polypeptide option a) Polypeptide option
PG Sequence of Table 1 : b)
Full length sequence of Table 2:
Embodiment R 27 191-195 537
Embodiment S 29 198-210 538
Embodiment T 30 211-232 539
Embodiment u 32 236-240 540
Embodiment V 34 242-251 541
Embodiment X 39 _59 270-277,338-348 542
Embodiment Y 47 292-296 546
Embodiment z 49/54 301-305, 319-322 548
Embodiment AA 51 307-311 551
Embodiment AB 52 312, 313 552
Embodiment AC 53 314-318 553
Embodiment AD 56 325-328 554
Embodiment AE 62 354-356 555
Embodiment AF 65 360-364 556
Embodiment AG 73 391-407 557
Embodiment AH 76 409-419 558
Embodiment AI 77 420-433 559
Embodiment AJ 83 440-456
Embodiment AK 86/51 467-481 561
Embodiment AL 87 482-484 562
Embodiment AM 89 485-513 563
Embodiment AN 90 514-518
Embodiment AO 91 519 564
In other embodiments, a polypeptide of option a) includes one or more PG peptides from different NTGA's, so as to construct polypeptides with desirable properties. For example one polypeptide may contain as part of its sequence an amino acid sequence of one or more PG peptides containing an immunodominant T cell epitope. The invention also relates to a molecule for use in relieving an allergic immune response against a pollen allergen other than a grass pollen allergen in a subject, wherein said molecule comprises or consists of a) a polypeptide, which includes at least one amino acid sequence with 0, 1 or 2
mismatches compared to a sequence selected from any one of SEQ ID NOs: 1-519; or b) a polypeptide comprising an amino acid sequence having at least 65% sequence similarity or identity to a sequence selected from any one of SEQ ID NOs: 520-565.
The invention also relates to the use of a molecule for preparation of a medicament for relieving an allergic immune response against a pollen allergen other than a grass pollen allergen in a subject, wherein said molecule comprises or consists of a) a polypeptide, which includes at least one amino acid sequence with 0, 1 or 2
mismatches compared to a sequence selected from any one of SEQ ID NOs: 1-519; or b) a polypeptide comprising an amino acid sequence having at least 65% sequence similarity or identity to a sequence selected from any one of SEQ ID NOs: 520-565.
The invention relates in a further aspect to an immunogenic molecule, e.g . a molecule comprising of or consisting of a) a polypeptide, which includes at least one amino acid sequence with 0, 1 or 2
mismatches compared to a sequence selected from any one of SEQ ID NOs: 1-519; or b) a polypeptide comprising an amino acid sequence having at least 65% sequence similarity or identity to a sequence selected from any one of SEQ ID NOs: 520-565. For example, an immunogenic molecule may contain a conserved sequence of NTGA 89
(embodiment AM of the above table) . Thus, in one particular aspect, a molecule comprises or consists of a polypeptide of option a) that includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 485- 513; or a polypeptide of option b) that comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 563 Other embodiments (A to AK) may be constructed the same way using the list above.
Also provided are cells expressing an immunogen described herein. In various embodiments, a cell expresses an immunogen. In certain aspects, a cell is a eukaryotic or prokaryotic cell and may be a mammalian, insect, fungal or bacterium cell. An immunogen of the present invention is suitable as a reagent for example, for immunotherapy against various pollen allergies including a pollen allergy, which is not grass pollen allergy in a subject.
In other embodiments, there are provided nucleic acid molecules encoding a polypeptide of option a) and b), or a molecule comprising polypeptide of option a) and b).
In additional aspects, there are provided compositions, for example pharmaceutical compositions comprising an immunogenic molecule of the invention. In one embodiment, a pharmaceutical composition is suitable for specific immunotherapy (e.g., treatment, desensitization, tolerance induction, bystander suppression). In certain embodiments, a pharmaceutical composition is a vaccine, i.e. suitable formulated for the purpose of vaccination.
Brief Description of the Drawings
Figure 1: Conservation in transcriptome predicts peptide cross-reactivity. For each peptide, TG allergic donors were selected that reacted to the peptide after expanding PBMCs in vitro with TG extract. PBMCs were stimulated with individual peptides for 14 days and IL- 5 responses were measured by ELISPOT to i) the peptide itself, ii) TG extract, iii) non-TG extracts (e.g. Amb a, Que, Ole e, Bet v, Cyn d, Ant o, Poa P, Lol p), iv) pools of pre-defined peptide pools (P20 and P19) that did or did not contain the peptide as relevant and irrelevant controls. T cell cultures that did not induce a robust response (> =200 SFC) to the peptide itself were excluded. Reponses to extracts and peptide pools are expressed as the relative fraction of the response to the peptide itself, and capped at 100%.
Figure 2: Sensitization pattern of an immunogen of the invention (NTGA 86/51): It is shown that the in vitro T-cell response towards NTGA 86/51 is much weaker compared to the response to allergen Phi p 5. Figures 3A-3C: Tolerance induction investigated in mice. Figures show that prophylactic sublingual immunotherapy treatment (SLIT) with NTGA 86/51 in mice is capable of inducing tolerance towards the immunogen itself (3A) as well as towards Phi p extract (3B), as shown by the ability of NTGA 86/51 to reduce the proliferation of cells of splenocytes from treated mice compared to buffer (sham) treated mice. In addition, it was shown that NTGA 6 is capable of inducing tolerance towards itself (3C) as observed by its ability to reduce proliferation of cells of splenocytes. Figures 4A and B: Bystander tolerance induction investigated in mice. As shown in Figure 4A, prophylactic SLIT treatment with NTGA 86/51 is capable of inducing direct tolerance (towards NTGA 86/51 itself), as demonstrated by reduced proliferation of splenocytes of NTGA 86/51-treated mice compared to buffer treated mice. Furthermore, Figure 4B shows that SLIT treatment with OVA is also able to downregulate the NTGA 86/51 specific in vitro response, demonstrating bystander tolerance induction by OVA. Likewise, SLIT treatment with NTGA 86/51 is also able to induce bystander tolerance, as
demonstrated by the decreased OVA-specific in vitro proliferation of splenocytes from NTGA 86/51-SLIT treated mice compared to buffer treated mice.
Detailed description
The term "a" or "an" refers to an indefinite number and shall not only be interpreted as "one" but also may be interpreted to mean "some", "several" or one or more.
The term "conserved sequence" is in the present context meant to include that a given sequence contains at least 15 contiguous amino acids within the sequence that has less than 3 mismatches compared to another sequence of 15 amino acids. Longer stretches of conserved sequences may contain several numbers of stretches of at least 15 contiguous amino acids having less than 3 mismatches compared to another sequence of 15 amino acids. In the present context, e.g. for the purpose of detecting a conserved sequence, the term "mismatch" is meant to include any substitution of an amino acid residue within the 15mer peptide.
The term "sensitized to" is generally meant to encompass that the subject has been exposed to an immunogen, e.g. an allergen or an antigen, in a manner that the individual's adaptive immune system displays memory to the immunogen, for example that the immunogen has induced detectable IgE antibodies against the immunogen and thus qualifies as an IgE-reactive antigen (allergen) and/or that T-cells stimulated in vitro are able to proliferate under the presence of the immunogen or fragments of the immunogen (e.g. linear peptides). The term "allergic immune response" is meant to encompass a hypersensitivity immune response, e.g. type 1 immune response, such as typically an immune response that is associated with the production of IgE antibodies (i.e. IgE-mediated immune response) and/or production of cytokines usually produced by Th2 cells. An allergic immune response may be associated with an allergic disease, for example atopic dermatitis, urticaria, contact dermatitis, allergic conjunctivitis, allergic rhinitis, allergic asthma, anaphylaxis, food allergy and hay fever. The term "grass pollen" is meant to designate pollen of the plant family Poaceae, for example pollen of the plant genus Anthoxanthum, Cynodon, Dactylis, Festuca, Holcus, Hordeum, Lolium, Oryza, Paspalum, Phalaris, Phleum, Poa, Secale, Sorghum, Triticum and Zea.
As used herein, an "immunogen" refers to a substance, including but not limited to a protein, polypeptide or peptide that modifies, e.g. elicits, induces, stimulates, promotes enhances or decreases, reduces, inhibits, suppresses, relieves an immune response when administered to a subject. For example, an immunogen may induce tolerance to itself in a subject. An immune response elicited by an immunogen may include, but is not limited to, a B cell or a T cell response. An immune response can include a cellular response with a particular pattern of lymphokine/cytokine production (e.g., Thl, Th2), a humoral response (e.g., antibody production, like IgE, IgG or IgA), or a combination thereof, to a particular immunogen. Particular immunogens are antigens and allergens.
The term "an antigen" refers to a particular substance to which an immunoglobulin (Ig) isotype may be produced in response to the substance. For example, an "IgG antigen" refers to an antigen that induces an IgG antibody response. Likewise, an "IgE antigen" refers to an antigen that induces an IgE antibody response (and thus qualifies as an allergen) ; an "IgA antigen" refers to a substance that induces an IgA antibody response, and so forth. In certain embodiments, such an immunoglobulin (Ig) isotype produced in response to an antigen may also elicit production of other isotypes. For example, an IgG antigen may induce an IgG antibody response in combination with one more of an IgE, IgA, IgM or IgD antibody response. Accordingly, in certain embodiments, an IgG antigen may induce an IgG antibody response without inducing an IgE, IgA, IgM or IgD antibody response.
The term "allergen" refers to a particular type of a substance that can elicit production of IgE antibodies, such as in predisposed subjects. For example, if a subject previously exposed to an allergen (i.e. is sensitized or is hypersensitive) comes into contact with the allergen again, allergic asthma may develop due to a Th2 response characterized by an increased production of type 2 cytokines (e.g., IL-4, IL-5, IL-9, and/or IL-13) secreted by CD4+ T lymphocytes. The term "subject" is meant to designate a mammal having an adaptive immune system, such as a human, a domestic animal such as a dog, a cat, a horse or cattle.
The term "immunotherapy" is meant to encompass treatment of a disease by inducing, enhancing, or suppressing an immune response. Typically, the therapeutically active agent is an immunogen, particularly an antigen, more particularly an allergen. An immunogen may be a protein or a fragment thereof (e.g. immunogenic peptide). Immunotherapy in connection with allergy usually encompasses repeated administration of a sufficient dose of the immunogen/antigen/allergen/ usually in microgram quantities, over a prolonged period of time, usually for more than 3 months, 6 months, 1 year, such as 2 or 3 years, during which period the immunogen may be administered daily or less frequent, such as several times a week, weekly, bi-weekly, or monthly, every second month or quarterly.
Immunotherapy can be effected by specific immunotherapy or may be effected by bystander tolerance induction.
The term "specific immunotherapy" in connection with allergy is meant to designate that immunotherapy is conducted with the administration of an immunogen to which the subject is sensitized to, particularly an immunogen to which the patient has raised specific IgE antibodies to, e.g. major allergens.
As used herein, the term "immunological tolerance" refers to a) a decreased or reduced level of a specific immunological response (thought to be mediated at least in part by antigen-specific effector T lymphocytes, B lymphocytes, antibody, a combination); b) a delay in the onset or progression of a specific immunological response; or c) a reduced risk of the onset or progression of a specific immunological response to an antigen or allergen. "Specific" immunological tolerance occurs when tolerance is preferentially invoked against certain antigens (allergens) in comparison with other antigens (allergens). Tolerance is an active antigen dependent process and differs from non-specific immunosuppression and immunodeficiency.
The term "bystander tolerance induction" in connection with allergy is meant to encompass that immunotherapy is conducted with the administration of an immunogen that elicits, induces, stimulates, promotes enhances or decreases, reduces, inhibits, suppresses, relieves an immune response against another unrelated immunogen, for example an allergen, e.g. major allergens of pollen. For example, an immunogen may induce tolerance to itself, and may be able to reactivate T regulatory cells specific to the immunogen to down-regulate an immune response caused by another unrelated immunogen, e.g. an allergen. The term "treatment" refers to any type of treatment that conveys a benefit to a subject afflicted with allergy, including improvement in the condition of the subject (e.g., in one or more symptoms), delay in the onset of symptoms, slowing the progression of symptoms, or induce disease modification etc. Typical symptoms of an allergic reaction is nasal symptoms in the form of itchy nose, sneezing, runny nose, blocked nose; conjunctival symptoms in the form of itchy eyes, red eyes, watery eyes; and respiratory symptoms in the form of decreased lung function. The treatment may also give the benefit that the patient needs less concomitant treatment with corticosteroids or HI antihistamines to suppress the clinical symptoms. As used herein, "treatment" is not necessarily meant to imply cure or complete abolition of symptoms, but refers to any type of treatment that imparts a benefit to a patient. Treatment may be initiated before the subject becomes sensitized to a protein. This may be realized by initiating immunotherapy before the subject has raised detectable serum IgE antibodies capable of binding specifically to the sensitizing protein or before any other biochemical marker indicative of an allergic immune response can be detected in biological samples isolated from the individual. Furthermore, treatment may be initiated before the subject has evolved clinical symptoms of the allergic disease, such as symptoms of allergic rhinitis, allergic asthma or atopic dermatitis.
The phrase "therapeutically sufficient amount" or "sufficient amount" is meant to designate an amount effective to reduce, suppress, relieve or eliminate an allergic immune response, e.g. an amount sufficient to achieve the desirable reduction in clinical relevant symptoms or manifestations of the allergic immune response. For example, a therapeutically sufficient amount may be the accumulated dose of a polypeptide, a set of polypeptides administered during a course of immunotherapy in order to achieve the intended effect or it may be the maximal dose tolerated within a given period. The total dose or accumulated dose may be divided into single doses administered daily, twice a week or more, weekly, every second or fourth week or monthly depending on the route of administration and the pharmaceutical formulation used. The total dose or accumulated dose may vary. It is expected that a single dose is in the microgram range, such as in the range of 5 to 500 microgram dependent on the nature of the polypeptide. The term "patient responding to therapy," such as "immunotherapy" is meant to designate that the patient has improvement in the symptoms of the allergic immune response caused by a pollen allergen. Symptoms may be the clinically symptoms of allergic rhinitis, allergic asthma allergic conjunctivitis, atopic dermatitis, food allergy and/or hay fever. Typically, the symptoms are the same as experienced with a flu/cold, sneezing, itching, congestion, coughing, feeling of fatigue, sleepiness and body aches. For example nasal symptoms in the form of itchy nose, sneezing, runny nose, blocked nose; conjunctival symptoms in the form of itchy eyes, red eyes, watery eyes; and respiratory symptoms in the form of decreased lung function. A responder may also be evaluated by monitoring the patient's reduced need for concomitant treatment with corticosteroids or H I antihistamines to suppress the clinical symptoms. Symptoms may be subjectively scored or in accordance with official guidelines used in clinical trials of SIT.
The term "adjuvant" refers to a substance that enhances the immune response to an immunogen. Depending on the nature of the adjuvant, it can promote either a cell-mediated immune response, humoral immune response or a mixture of the two. As used herein an "epitope" refers to a region or part of an immunogen that elicits an immune response when administered to a subject. In particular embodiments, an epitope is a T cell epitope, i.e., an epitope that elicits, stimulates, induces, promotes, increases or enhances a T cell activity, function or response. An immunogen can be analyzed to determine whether it include at least one T cell epitope using any number of assays (e.g . T cell proliferation assays, lymphokine secretion assays, T cell non-responsiveness studies, etc.) . In the context of the present invention, a T-cell epitope refers to an epitope that are MHC Class II binders (i .e. HLA-II binders), for example HLA-II binders shown in Table 9. As shown herein a subsequence may contain a T cell epitope, such as a Th2 cell epitope (Table 1) . A subsequence or a polypeptide described herein may have HLA Class II binding properties. HLA Class II binding can be predicted using NetMHCIIpan-3.0 tool (Karosiene, Edita, Michael Rasmussen, Thomas Blicher, Ole Lund, Soren Buus, and Morten Nielsen. "NetMHCIIpan-3.0, a Common Pan-specific MHC Class II Prediction Method Including All Three Human MHC Class II Isotypes, HLA-DR, H LA-DP and HLA-DQ." Immunogenetics) available at the internet site <URL: http://www.cbs.dtu.dk/services/NetMHCIIpan-3.0> . As used herein, the term "immune response" includes T cell (cellular) mediated and/or B cell (humoral) mediated immune responses, or both cellular and humoral responses. Exemplary immune responses include T cell responses, e.g ., lymphokine production, cytokine production and cellular cytotoxicity. T-cell responses include Thl and/or Th2 responses. In addition, the term immune response includes responses that are indirectly affected by T cell activation, e.g ., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., eosinophils, macrophages. Immune cells involved in the immune response include lymphocytes, such as T cells (CD4+, CD8+, Thl and Th2 cells, memory T cells) and B cells; antigen presenting cells (e.g ., professional antigen presenting cells such as dendritic cells, macrophages, B lymphocytes, Langerhans cells, and non-professional antigen presenting cells such as keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes); natural killer (NK) cells; myeloid cells, such as macrophages, eosinophils, mast cells, basophils, and granulocytes.
As disclosed herein, some immunogens (NTGA's) recently detected in Timothy grass pollen share substantial identity and similarity with immunogens detected in at least Cyn d and Poa p pollen. Thus, such immunogens can be used to broadly treat a subject with or at risk of developing an allergy, allergic reaction, allergic immune response to a pollen allergen of a variety of pollen plant families, or broadly induce or promote tolerance of a subject to a pollen allergen of a variety of grass pollen families.
Thus, by the present invention it is now possible to relieve an immune response of a multisensitized subject caused by grass pollen allergens of different grass families by administering an immunogen described herein. Likewise, it is also now possible to treat subjects with different grass pollen allergies using the same immunogen(s).
In certain embodiments, the immunogen is a molecule comprising or consisting of a) a polypeptide, which includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 1-519 set out in Table 1 (PG peptides). The immunogen may contain at least one T cell epitope optionally a Th-2 cell epitope. Thus, in some embodiments, the polypeptide of option a includes at least one amino acid sequence with 0, 1 or 2 mismatches to a sequence selected from any one of SEQ ID NOs: 44, 8, 9, 10, 14, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 45, 47, 50, 51, 63, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 102, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 135, 136, 137, 154, 155, 162, 163, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 187, 191, 192, 193, 194, 195, 197, 199, 200, 203, 220, 236, 237, 239, 240, 244, 250, 251, 252, 253, 254, 273, 274, 278, 279, 285, 293, 294, 301, 303, 305, 308, 309, 310, 312, 313, 314, 317, 318, 319, 320, 321, 322, 327, 341, 343, 345, 346, 347, 348, 354, 356, 357, 359, 360, 361, 362, 379, 385, 398, 404, 409, 413, 414, 416, 417, 419, 426, 427, 432, 433, 435, 436, 437, 439, 440, 441, 443, 444, 445, 446, 447, 448, 451, 452, 453, 454, 455, 456, 457, 460, 461, 463, 481, 485, 487, 489, 490, 492, 502, 504, 505, 506, 507, 508, 509, 510, 511, 513, 514, 515, 517, 518 and 519.
In methods and uses described herein, one may consider using an immunogen recognized by a greater number of individuals, for example, the immunogen may be recognized by more than two subjects in a population of 20 subjects, e.g. wherein polypeptide of option a) includes at least one amino acid sequence with 0, 1 or 2 mismatches to a sequence selected from any one of SEQ ID NOs 19, 20, 24, 25, 26, 28, 32, 37, 69, 71, 72, 73, 74, 78, 79, 80, 81, 82, 84, 86, 87, 88, 89, 90, 91, 93, 95, 99, 107, 166, 169, 173, 174, 176, 180, 193, 303, 313, 319, 321, 492, 505, 506, 510, 511, 513, 514, 518 and 519.
In some embodiments, the number of amino acid mismatches is 0 or 1, for example the immunogen may be a molecule comprising or consisting of a) a polypeptide, which includes at least one amino acid sequence with 0 or 1 mismatches compared to a sequence selected from any one of SEQ ID NOs: 4, 8, 9, 10, 14, 19, 20, 21, 22, 24, 25, 26, 29, 30, 32, 33, 34, 35, 37, 38, 41, 42, 45, 65, 66, 67, 69, 71, 72, 73, 74, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 98, 99, 102, 106, 107, 109, 110, 116, 117, 135, 136154, 155, 162, 163, 166, 167, 168, 169, 170, 171, 172, 174, 175, 177, 178, 179,
180, 182, 187, 192, 193, 194, 199, 220, 236, 237, 239, 240, 244, 252, 254, 273, 274, 278, 279, 285, 305, 309, 310, 312, 319, 320, 321, 327, 345, 346, 347, 348, 354, 357, 359, 360, 361, 379, 385, 398, 409, 413, 416, 417, 419, 426, 427, 432, 433, 436, 440, 441, 443, 444, 445, 446, 447, 448, 453, 454, 455, 456, 457, 460, 461, 463, 481, 485, 487, 489, 502, 504, 506, 507, 508, 509, 510, 515 and 517.
In still some embodiments, the polypeptide of option a) is derived from NTGA's 2, 6, 7, 49/54, 89, 53 and 91 that contains epitopes with high T cell reactivity, for example a polypeptide of option a) comprises a T cell epitope and includes at least one amino acid sequence with 0, 1 or 2 mismatches to a sequence selected from any one of SEQ ID NOs: 19, 20, 24, 25, 26, 32, 37, 69, 71, 72, 73, 74, 78, 79, 80, 81, 82, 84, 86, 87, 88, 89, 90, 91, 93, 95, 99, 319, 321, 506 and 510.
In certain embodiments, the immunogen is a molecule comprising or consisting of a polypeptide of option b) that are conserved across five grass pollen species and comprises several PG peptides in its sequence compared to other NTGA's investigated. Thus, a polypeptide of option b) may comprise an amino acid sequence with at least 65% similarity or identity to a sequence selected from any of SEQ ID NOs: 520, 521, 523, 525, 526, 531, 533, 535, 538, 539, 541, 542, 557, 558, 559, 561, 563 and 564.
Immunogens eligible for relieving an allergic immune response to an allergen unrelated to the immunogen is thought, at least in part, to be mediated via bystander tolerance induction, which mechanism requires, at least in part, co-existence of the immune response triggering allergen and the unrelated immunogen at the target organ.
Therefore, a polypeptide of option a) and option b) may be derived from a full length protein that co-releases/co-elutes with a major allergen of grass pollen. In the present context, where multiple grass pollen allergies should be treated using one product, a the full length sequence of a polypeptide of option a) and b) should be co-released from Phi p pollen as well as another interesting grass pollen, e.g. pollen of the plant genera Cynodon, and optionally also from grass pollen selected from any of the plant genera Anthoxanthum, Dactylis, Festuca, Lollium, Paspalum, Phalaris, Poa and Sorghum.
In the present context, the term "co-release" or "co-elute" refers to an immunogen that starts release from a hydrated pollen within a period overlapping with a major allergen to which the allergic immune response is sought relieved. As major allergens start release from pollen within few minutes after hydration of pollen and continues to be released within the next 30 or 60 minutes, the term "co-release" or "co-elute" may refers to that an
immunogen of the invention starts being released from pollen within 30 minutes after hydration of the pollen.
Thus, in some embodiments, the polypeptide of option b) comprises an amino acid sequence with at least 65% similarity or identity to a sequence selected from any of SEQ ID
NOs: 520, 522, 523, 524, 525, 533, 535, 536, 539, 542, 546, 555, 558, 561, 563 and 564.
(NTGA's 1, 3, 4, 6, 5/64, 20, 24, 26, 30, 39/59, 47, 62, 76, 86/51, 89 and 91) and the polypeptide of option a) includes at least one amino acid sequence with 0, 1 or 2
mismatches to a sequence selected from any one of SEQ ID NOs: 1-18, 38-40, 41-55, 56- 62, 63-89, 148-156, 169-182, 183-190, 211-232, 270-277, 338-348, 292-296, 354-356,
409-419, 467-481, 485-513 and 519. (PG peptides derived from NTGA's 1, 3, 4, 6, 5/64,
20, 24, 26, 30, 39/59, 47, 62, 76, 86/51, 89 and 91.
As mentioned a polypeptide defined herein may comprise one or more PG peptide
sequences or a corresponding sequence with 1 or 2 mismatches compared to the PG peptide. In certain embodiments, a polypeptide of option a) comprises two or more PG peptides, e.g. 2-25 PG peptides defined herein, e.g. 3-25, 4-25, 5-25, 6-25, 7-25 PG peptides, such as 2- 20, 3-20, 4-20, 5-20, 6-20 PG peptides or a corresponding sequence with 1 or 2
mismatches compared to the PG peptide. For example, a polypeptide of option a) may include one or more immunodominant PG peptides, like those recognized by at least 3 subjects in a population of 20 subjects, e.g. a polypeptide of option a) may include one or more sequences selected from any one of SEQ ID NOs: 19, 20, 24, 25, 26, 32, 37, 69, 71, 72, 73, 74, 78, 79, 80, 81, 82, 84, 86, 87, 88, 89, 90, 91, 93, 95, 99, 319, 321, 506 and 510, or a sequence with 0, 1 or 2 mismatches compared to the SEQ ID NOs: 19, 20, 24, 25, 26, 32, 37, 69, 71, 72, 73, 74, 78, 79, 80, 81, 82, 84, 86, 87, 88, 89, 90, 91, 93, 95, 99, 319, 321, 506 and 510. Furthermore, a polypeptide of option a) may include one or more, such as from 2 to 25 PG peptides of different NTGAs able to be released in both Phi p and Cyn d pollens. Therefore, in still some embodiments, a polypeptide of option a) may include one or more sequences selected from any one of SEQ ID NOs: 1-18, 38-40, 41-55, 56-62, 63-89, 148-156, 169- 182, 183-190, 211-232, 270-277, 338-348, 292-296, 354-356, 409-419, 467-481, 485-513 and 519, or a sequence with 0, 1 or 2 mismatches compared to the SEQ ID NOs: 1-18, 38- 40, 41-55, 56-62, 63-89, 148-156, 169-182, 183-190, 211-232, 270-277, 338-348, 292- 296, 354-356, 409-419, 467-481, 485-513 and 519.
In still other embodiment, a polypeptide of option a) or option b) is derived from a full length protein that co-releases/co-elutes with a major allergen of grass pollen of the genera Phleum, Anthoxanthum, Cynodon, Lollium and/or Poa.
It should be understood that full length sequences that contain the same PG peptides (with less than 1-3 mismatches) may be used instead of the Phi p sequence set out in Table 2. For example, full length sequence of a polypeptide of option a) or b) may be a polypeptide present in, based upon or derived from a pollen of a plant family of Poaceae, optionally the genera of any of Phleum, Anthoxanthum, Cynodon, Lollium and Poa. Exemplary
polypeptides are set out in Table 3. Thus a polypeptide of option b) may comprise an amino acid sequence having at least 65% identity to any of SEQ ID Nos: 566-688.
In more specific embodiments of the invention, the polypeptide relates to NTGA 89, e.g. a polypeptide of option a) that includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 485-513; the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 563 or a homolog thereof in another grass pollen species, e.g. SEQ ID NOs: 683-687. In still more specific embodiments of the invention, the polypeptide relates to NTGA 6, e.g. a polypeptide of option a) that includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 63-89; the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 525 or a homolog thereof in another grass pollen species, e.g. SEQ ID NOs: 577-585.
In still more specific embodiments of the invention, the polypeptide relates to NTGA 24, e.g. a polypeptide of option a) that includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 169-182; the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 535 or a homolog thereof in another grass pollen species, e.g . SEQ ID NOs: 602.
In still more specific embodiments of the invention, the polypeptide relates to NTGA 24, e.g . a polypeptide of option a) that includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 198-210; the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 538 or a homolog thereof in another grass pollen species, e.g . SEQ ID NOs: 604. In still more specific embodiments of the invention, the polypeptide relates to NTGA 91, e.g . a polypeptide of option a) that includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 519; the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 564 or a homolog thereof in another grass pollen species, e.g . SEQ ID NOs: 688.
In still more specific embodiments of the invention, the polypeptide relates to NTGA 39/51, e.g. a polypeptide of option a) that includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 270-277, 338- 348; the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 542 or a homolog thereof in another grass pollen species, e.g . SEQ ID NOs: 614 and 615.
In still more specific embodiments of the invention, the polypeptide relates to NTGA 86/51, e.g. a polypeptide of option a) that includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 467-481 ; the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 561 or a homolog thereof in another grass pollen species, e.g . SEQ ID NOs: 679
In still more specific embodiments of the invention, the polypeptide relates to NTGA 49/54, e.g. a polypeptide of option a) that includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 301-305, 319- 322; the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 548 or or a homolog thereof in another grass pollen species, e.g . SEQ ID NOs: 620-630. In certain embodiments, the immunogen is a molecule comprising or consisting of a polypeptide, which includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: SEQ ID NO's 321, 303, 414, 689, 690, 28, 691-713 set out in Table 8. The immunogen may contain at least one T cell epitope optionally a Th-2 cell epitope. Thus, in some embodiments, the polypeptide includes at least one amino acid sequence with 0, 1 or 2 mismatches to a sequence selected from any one of SEQ ID NO's 321, 303, 414, 689, 690, 28, 691-713.
In some embodiments, an immunogen of the present invention is an IgE reactive molecule, e.g. able to bind to IgE antibodies specific for the immunogen. However, IgE reactivity towards an immunogen of the invention may only be conferred by a low fraction of an allergic population. Thus, an immunogen of the invention do not fall under the usual definitions of a major allergen. In some embodiments, the immunogen is able to react with, bind to or induce IgG antibodies in a subject, at least in detectable levels. In still other embodiments, the immunogen does not react with, bind to or induce IgG antibodies, at least in detectable levels. As demonstrated herein, an immunogen of the invention seems to be less immunogenic than a major allergen (Figure 2).
As mentioned, a subject eligible for being treated with an immunogen of the invention may also be sensitized to a grass pollen allergen, for example a grass pollen allergen of a plant genus selected from any of Anthoxanthum, Cynodon, Phleum and Poa. As disclosed herein, immunogens of the present invention may be found in various grass pollen families and share high identity and similarity with a wild type immunogen in a grass pollen family other than of the genus Phleum. For example, a polypeptide of option b) comprises an amino acid sequence having at least 70% similarity or identity to a sequence selected from any one of SEQ ID NOs: 398-443, for example at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity or identity. Examples on wild type immunogens with high identity and similarity to the wild type NTGA's are shown in Table 3. Here is disclosed wild type proteins found in other grass pollen species and which contain as part of its sequence a sequence having less than 3 mismatches to a PG peptide disclosed herein. For example, wild type sequences comparable to NTGA 6 are found in at least Ant o, Cyn d, Lol p and Poa p and comprises SEQ ID NOs: 577-585.
It follows that a polypeptide of option b) may comprise an amino acid sequence having at least 70% similarity or identity to a sequence selected from any one of SEQ ID NOs: 566- 688, for example at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity or identity. A polypeptide of option a) may have different lengths according to the desirable use, for example of about 15-800 or more amino acid residues in length, for example 15-750, 15- 700, 15-650, 15-600, 15-500 or more amino acid residues, for example 15-20, 15-25, 15- 30, 20-25, 25-30, 30-35, 35-40, 45-50, 50-60, 60-70, 70-80, 90-100, 100-125, 125-150, 150-175, 175-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-800 or more amino acid residues. One may consider utilizing short linear peptides, which when administered to a subject need not to be processed by an antigen presenting cells to interact with a relevant T cell receptor, but rather freely loaded onto a MHC class II molecule to interact with the relevant T cell receptor. Thus, in some embodiments, a polypeptide of option a) and a polypeptide of option d) has a length in the range of 15 to 30 amino acid residues, for example 15 to 25 amino acid residues. In other embodiments, a polypeptide of option a) is a longer polypeptide which comprises a secondary or tertiary structure, e.g. folded. Thus, in other embodiments, a polypeptide of option a) has a length in the range of 30 to 500 amino acid residues or more.
Polypeptides of option b) may have the same length as the wild type sequence of the NTGA of Table 2, or the homolog of Table 3, respectively or may be shorter or longer. It is considered that the length of the amino acid sequence of a polypeptide of option b) is no more than 800 amino acid residues, for example no more than 750, 700, 650, 600, 550, 500 or 450 amino acid residues. Also it may be considered that the length of a polypeptide of option b) has an amino acid sequence length that is 80% to 120% of the length of any one of SEQ ID NOs: 520-565 or of SEQ ID Nos: 566-688.
The term "identity" and "identical" and grammatical variations thereof, as used herein, mean that two or more referenced entities are the same (e.g., amino acid sequences). Thus, where two polypeptides are identical, they have the same amino acid sequence. The identity can be over a defined area (region or domain) of the sequence, e.g. over the sequence length of a sequence disclosed in Tables 1, 2 and 3 or over a stretch thereof e.g. at least 15 contiguous amino acid residues. Moreover, the identity can be over the length of the sequence overlapping the two polypeptides, when aligned with best fit with gaps permitted. For example, to determine whether a polypeptide has at least 65% similarity or identity to a sequence set out in Tables 2 or 3, the polypeptide may be aligned with a sequence of Table 2 or 3 and the percent identity be calculated with reference to a sequence of Table 2 or 3.
Identity can be determined by comparing each position in aligned sequences. A degree of identity between amino acid sequences is a function of the number of identical or matching amino acids at positions shared by the sequences, i.e. over a specified region. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, as are known in the art, including the Clustal Omega program, available at http://www.ebi.ac.uk/Tools/msa/clustalo/, the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerized implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI, U.S.A.). Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. Mol. Biol. 215:403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information (through the internet at http://www.ncbi.nlm.nih.gov/). Such algorithms that calculate percent sequence identity (homology) generally account for sequence gaps and mismatches over the comparison region or area. For example, a BLAST (e.g., BLAST 2.0) search algorithm (see, e.g., Altschul et al., J. Mol. Biol. 215:403 (1990), publicly available through NCBI) has exemplary search parameters as follows: Mismatch -2; gap open 5; gap extension 2. For polypeptide sequence comparisons, a BLASTP algorithm is typically used in combination with a scoring matrix, such as PAM100, PAM 250, BLOSUM 62 or BLOSUM 50. FASTA (e.g., FASTA2 and FASTA3) and SSEARCH sequence comparison programs are also used to quantitate the extent of identity (Pearson et al., Proc. Natl. Acad. Sci. USA 85: 2444 (1988); Pearson, Methods Mol Biol. 132: 185 (2000); and Smith et al., J. Mol. Biol. 147: 195 (1981)). Programs for quantitating protein structural similarity using Delaunay-based topological mapping have also been developed (Bostick et al., Biochem Biophys Res Commun. 304: 320 (2003)).
A polypeptide sequence is a "homologue" of, or is "homologous" to, another sequence if the two sequences have substantial identity over a specified region and a functional activity of the sequences is preserved or conserved, at least in part (as used herein, the term
'homologous' does not infer nor exclude evolutionary relatedness). Examples of "homologous polypeptides" of the invention include polypeptides found in non- Timothy grass pollen and with high identity to the NTGA's disclosed in Table 2. For example, a homologous polypeptide may be found in grass pollen other than of the genera Phleum of the plant family Poaceae, e.g. the plant genera Anthoxanthum, Cynodon, Dactylis, Festuca, Holcus, Hordeum, Lollium, Oryza, Paspalum, Phalaris, Poa, Secale, Sorghum, Triticum and Zea. Two polypeptide sequences are considered to be substantially identical if, when optimally aligned (with gaps permitted), they share at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, etc. identify over a specific region), for example, over all or a part of any amino acid sequence in Tables 1, 2, and 3, or if the sequences share defined functional motifs (e.g., epitopes). In particular aspects, the length of the sequence sharing the percent identity is at least 15, 16, 17, 18, 19, 20, etc. contiguous amino acids, e.g.
more than 25, 30, 35, 40, 45 or 50 or more contiguous amino acids, including the entire length of a reference sequence of Tables 1, 2 and 3.
An "unrelated" or "non-homologous" sequence is considered to share less than 30% identity. More particularly, it may shares less than about 25 % identity, with a polypeptide of the invention over a specified region of homology.
An amino acid sequence set out in any of Tables 1, 2 and 3 may contain modifications resulting in greater or less activity or function, such as ability to elicit, stimulate, induce, promote, increase, enhance, activate, modulate, inhibit, decreases, suppress, or reduce an immune response (e.g. a T cell response) or elicit, stimulate, induce, promote, increase or enhance immunological tolerance (desensitize) to an immunogen of the invention or a pollen allergen.
A modification includes deletions, including truncations and fragments; insertions and additions, substitutions, for example conservative substitutions, site-directed mutants and allelic variants.
Non-limiting examples of modifications include one or more amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-25, 25-30, 30-50, 50-100 or more residues), additions and insertions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-25, 25-30, 30-50, 50-100 or more residues) and deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-25, 25-30, 30-50, 50-100 or more) of a sequence set out in Tables 1, 2 and 3.
The term "similarity" and "similar" and grammatical variations thereof, as used herein, mean that two or more referenced amino acid sequences contains a limited number of conservative amino acid substitutions of the amino acid sequence. A variety of criteria can be used to indicate whether amino acids at a particular position in a polypeptide are similar. In making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing.
A "conservative substitution" is the replacement of one amino acid by a biologically, chemically or structurally similar residue. Biologically similar means that the substitution does not destroy a biological activity. Structurally similar means that the amino acids have side chains with similar length, such as alanine, glycine and serine, or a similar size.
Chemical similarity means that the residues have the same charge, or are both hydrophilic or hydrophobic. For example, a conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain, which include amino acids with basic side chains (e.g., lysine, arginine, histidine); acidic side chains (e.g., aspartic acid, glutamic acid); uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, histidine); nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). Particular examples include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, serine for threonine, and the like. Proline, which is considered more difficult to classify, shares properties with amino acids that have aliphatic side chains (e.g., Leu, Val, He, and Ala). In certain circumstances, substitution of glutamine for glutamic acid or asparagine for aspartic acid may be considered a similar substitution in that glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively. Conservative changes can also include the substitution of a chemically derivatized moiety for a non-derivatized residue, for example, by reaction of a functional side group of an amino acid. Variants and derivatives of polypeptides include forms having a limited number of one or more substituted residues.
As mentioned, a polypeptide of option a) and b) may be longer than the reference sequence set out in Tables 1, 2 and 3.
An addition can be one or more additional amino acid residues. For example, a polypeptide of option a) may contain amino acid residues in addition to the 15 amino acid residues of the PG peptide, and optionally, the additional amino acid residues may be identical to those present in the wild type NTGA from which the PG peptide derives from. Thus, in some embodiments, the polypeptide of option a) comprises one or more amino acid residues in addition to the 15 contiguous amino acids (PG peptide) set out in Table 1, wherein the additional amino acid residue(s) is/are selected from an amino acid residue or an amino acid sequence within the wild type protein of which the PG peptide is a part of (e.g. wild type sequences of Tables 2 or 3. For example, the wild type amino acid residue or wild type amino acid sequence to be added may be adjacent to, subtended, comprised within, overlapping with or is a part of the PG peptide sequence, when present in its natural biological context within the wild type protein. An illustrative example is a PG peptide of NTGA 6 as set out in Table 1 that may be extended with amino acid residues from NTGA 6 set out in Table 2, or a homolog thereof set out in Table 3, such as amino acid residues adjacent to the PG sequence when aligned with NTGA 6 or the homolog thereof.
The additional amino acid residues may be added to the N- and/or C- terminal end of a sequence set out in Tables 1, 2 and 3, such as additional amino acids selected from amino acids flanking the N- and/or C- terminal ends when sequence is aligned with the source protein it is present in, based upon or derived from. Thus, where a sequence derives from NTGA 6, the additional amino acids may be the amino acids flanking the N- and/or C- terminal ends of the sequence when aligned to NTGA 6. In one embodiment, a polypeptide of option a) and b is derivatized. Specific non-limiting examples of derivatization are covalent or non-covalent attachment of another molecule. Specific examples include glycosylation, acetylation, phosphorylation, amidation, formylation, ubiquitination, and derivatization by protecting/blocking groups and any of numerous chemical modifications. In particular embodiments, a derivative is a fusion (chimeric) sequence, an amino acid sequence having one or more molecules not normally present in the wild type sequence covalently attached to the sequence. The term "chimeric" and grammatical variations thereof, when used in reference to a sequence, means that the sequence contains one or more stretches that are derived from, obtained or isolated from, or based upon other physical or chemical entities. For example, a chimera of two or more different polypeptides may have one part a polypeptide, and a second part of the chimera may be from a different sequence, or unrelated protein sequence.
Another particular example of a derivatized polypeptide is one in which a second
heterologous sequence, i.e., heterologous functional domain is attached (covalent or non- covalent binding) that confers a distinct or complementary function. Heterologous functional domains are not restricted to amino acid residues. Thus, a heterologous functional domain can consist of any of a variety of different types of small or large functional moieties. Such moieties include nucleic acid, peptide, carbohydrate, lipid or small organic compounds, such as a drug (e.g., an antiviral), a metal (gold, silver), and radioisotope. For example, a tag such as T7 or polyhistidine can be attached in order to facilitate purification or detection of a protein, peptide, etc. For example, a 6-HIS tag may be added to the C- or N-terminal end of a polypeptide of option a), b), c) or d), e.g. the 6-HIS sequence GHHHHHHGSGMLDI, which optionally may remain in the immunogen when administered to a subject. Thus, a polypeptide linked to a Tag containing histidines may easily be purified by use of a HIS tag affinity column.
Accordingly, there are provided polypeptides linked to a heterologous domain, wherein the heterologous functional domain confers a distinct function on the polypeptide.
In some embodiments, the polypeptide is derivatized for example to improve solubility, stability, bioavailability or biological activity. For example, tagged polypeptides and fusion proteins; and modifications, including peptides having one or more non-amino acyl groups (q.v., sugar, lipid, etc.) covalently linked to the polypeptide and post-translational modifications.
Linkers, such as amino acid or peptidomimetic sequences may be inserted between the sequence and the addition (e.g ., heterologous functional domain) so that the two entities maintain, at least in part, a distinct function or activity. Linkers may have one or more properties that include a flexible conformation, an inability to form an ordered secondary structure or a hydrophobic or charged character, which could promote or interact with either domain. Amino acids typically found in flexible protein regions include Gly, Asn and Ser. Other near neutral amino acids, such as Thr and Ala, may also be used in the linker sequence. The length of the linker sequence may vary without significantly affecting a function or activity of the fusion protein (see, e.g ., U.S. Patent No. 6,087,329) . Linkers further include chemical moieties and conjugating agents, such as sulfo-succinimidyl derivatives (sulfo-SMCC, sulfo-SMPB), disuccinimidyl suberate (DSS), disuccinimidyl glutarate (DSG) and disuccinimidyl tartrate (DST) .
Further non-limiting examples of derivatives are detectable labels. Thus, in another embodiment, the invention provides polypeptides that are detectably labeled . Specific examples of detectable labels include fluorophores, chromophores, radioactive isotopes (e.g ., S35, P32, 1125), electron-dense reagents, enzymes, ligands and receptors. Enzymes are typically detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert a substrate such as 3,3-',5,5-'-tetramethylbenzidine (TMB) to a blue pigment, which can be quantified. Modified polypeptides also include one or more D-amino acids substituted for L-amino acids (and mixtures thereof), structural and functional analogues, for example, peptidomimetics having synthetic or non-natural amino acids or amino acid analogues and derivatized forms. Modifications include cyclic structures such as an end-to-end amide bond between the amino and carboxy-terminus of the molecule or intra- or inter-molecular disulfide bond.
A polypeptide of the invention may be modified to avoid oxidation, improve solubility in aqueous solution, avoid aggregation, overcome synthesis problems etc. For example the polypeptide amino acid sequence may include the following modifications:
• a glutamate residue present at the N- terminus of a peptide replaced with
pyroglutamate;
• addition of one or more lysine amino residue(s) at the N- or C- terminus of the peptide;
• addition of one or more arginine amino residue(s) at the N- or C- terminus of the peptide;
• one or more modifications selected from the following : (a) any cysteine residues in the wild type sequence of the peptide are replaced with serine or 2-aminobutyric acid; (b) hydrophobic residues in the up to three amino acids at the N or C terminus of the wild type sequence of the peptide are deleted; (c) any two consecutive amino acids comprising the sequence Asp-Gly in the up to four amino acids at the N or C terminus of the wild type sequence of the peptide are deleted; and/or (d) one or more positively charged residues are added at the N- and/or C-terminus.
In particular, a polypeptide may comprise one, two or more lysine or arginine amino acid residue(s) added to the N- or C-terminus of the peptide to be modified, which may improve the aqueous solubility.
In particular, a polypeptide of the invention may comprise one or more cysteine residues that are substituted with amino acid residues less prone to oxidation, e.g. serine or arginine.
Polypeptides may be provided in the form of a salt, for example as a pharmaceutically acceptable and/or a physiologically acceptable salt. For example, the salt may be an acid addition salt with an inorganic acid, an acid addition salt with an organic acid, a salt with a basic inorganic acid, a salt with a basic organic acid, a salt with an acidic or basic amino acid or a mixture thereof. In particular embodiments of the invention a salt, such as a
pharmaceutically acceptable salt, is an acetate salt. The invention provides polypeptides and molecules in isolated and/or purified form.
The term "isolated," when used as a modifier of a composition, means that the compositions are made by the hand of man or are separated, completely or at least in part, from their naturally occurring in vivo environment. Generally, isolated compositions are substantially free of one or more materials with which they normally associate with in nature, for example, one or more protein, nucleic acid, lipid, carbohydrate, cell membrane. The term "isolated" does not exclude alternative physical forms of the composition, such as fusions/chimeras, multimers/oligomers, modifications (e.g., phosphorylation, glycosylation, lipidation) or derivatized forms, or forms expressed in host cells produced by the hand of man.
An "isolated" composition (e.g. polypeptides or molecules as defined herein) can also be "substantially pure" or "purified" when free of most or all of the materials with which it typically associates with in nature. Thus, an isolated polypeptide that also is substantially pure or purified does not include polypeptides or polynucleotides present among millions of other sequences, such as polypeptide of an peptide library or nucleic acids in a genomic or cDNA library, for example.
A "substantially pure" or "purified" composition can be combined with one or more other molecules. Thus, "substantially pure" or "purified" does not exclude combinations of compositions, such as combinations of polypeptides other antigens, agents, drugs or therapies.
Polypeptides can be prepared recombinantly, chemically synthesized, isolated from a biological material or source, and optionally modified, or any combination thereof. A biological material or source would include an organism that produced or possessed any polypeptide or molecule set forth herein. A biological material or source may further refer to a preparation in which the morphological integrity or physical state has been altered, modified or disrupted, for example, by dissection, dissociation, solubilization, fractionation, homogenization, biochemical or chemical extraction, pulverization, lyophilization, sonication or any other means of manipulating or processing a biological source or material.
Polypeptides of the invention can be prepared, for example, by substituting, deleting or adding one or more amino acid residues in the amino acid sequence and screening for biological activity, for example eliciting an immune response. A skilled person will understand how to make such derivatives or variants, using standard molecular biology techniques and methods, described for example in Sambrook et al. (2001) Molecular Cloning: a Laboratory Manual, 3rd ed., Cold Spring Harbour Laboratory Press). Polypeptides and molecules that are provided herein can be employed in various methods and uses. Such methods and uses include, for example, administration in vitro and in vivo of one or more polypeptides or molecules thereof. The methods and uses provided include methods and uses of modulating an immune response (e.g. an allergic immune response), including, among others, methods and uses of relieving an immune response (e.g. allergic immune response), protecting and treating subjects against a disorder, disease (e.g. allergic disease); and methods and uses of providing immunotherapy, such as specific
immunotherapy against an allergic immune response, e.g. allergy.
In particular embodiments, methods and uses include administration or delivery of an immunogen provided herein to modulate an immune response in a subject, including, for example, modulating an immune response to a pollen allergen or the immunogen.
As used herein, the term "modulate," means an alteration or effect on the term modified. In certain embodiments, modulating involves decreasing, reducing, inhibiting, suppressing, relieving an immune response in a subject to an allergen or an immunogen provided herein. In other embodiments, modulating involves eliciting, stimulating, inducing, promoting, increasing or enhancing an immune response in a subject to an antigen or allergen. Thus, where the term "modulate" is used to modify the term "immune response against an allergen in a subject" this means that the immune response in the subject to the allergen or immunogen is altered or affected (e.g., decreased, reduced, inhibited, suppressed, limited, controlled, prevented, elicited, promoted, stimulated, increased, induced, enhanced, etc.
Methods and uses of modulating an immune response against an allergen or immunogen as described herein may be used to provide a subject with protection against an allergic immune response or immune reaction to the allergen or immunogen, or symptoms or complications caused by or associated with the allergen or immunogen. Accordingly, in other embodiments, methods and uses include administering an immunogen of the invention to protect or treat a subject against an allergic immune response, or one or more symptoms caused by or associated with an allergen. In still other embodiments, methods and uses include administering or delivering an immunogen of the invention to elicit, stimulate, induce, promote, increase or enhance immunological tolerance of a subject to an allergen or immunogen disclosed herein.
In various embodiments, there are provided methods and uses of providing a subject with protection against an allergic immune response, or one or more symptoms caused by or associated with an allergen or immunogen disclosed herein. In various aspects, a method or use includes administering to the subject an amount of an immunogen of the invention sufficient to provide the subject with protection against the allergic immune response, or symptoms caused by or associated with the allergen or immunogen.
Methods and uses of the invention include providing a subject with protection against an allergen or an immunogen, or symptoms caused by or associated with the subjects exposure to the allergen or immunogen, for example, vaccinating the subject to protect against an allergic immune response to the allergen or immunogen, for example with an immunogen provided herein. In certain embodiments, methods and uses include protecting the subject against an allergic immune response by inducing tolerance of the subject (desensitizing) to the allergen, and optionally to the immunogen. As used herein, the terms "protection," "protect" and grammatical variations thereof, when used in reference to an allergic immune response or symptoms caused by or associated with the exposure to allergen, means preventing an allergic immune response or symptoms caused by or associated with the exposure to the allergen, or reducing or decreasing susceptibility to an allergic immune response or one or more symptoms caused by or associated with the exposure to the allergen.
An allergic immune response includes but is not limited to an allergic reaction,
hypersensitivity, an inflammatory response or inflammation. In certain embodiments allergic immune response may involve one or more of cell infiltration, production of antibodies, production of cytokines, lymphokines, chemokines, interferons and interleukins, cell growth and maturation factors (e.g., differentiation factors), cell proliferation, cell differentiation, cell accumulation or migration (chemotaxis) and cell, tissue or organ damage or remodeling. In particular aspects, an allergic immune response may include allergic rhinitis; atopic dermatitis; allergic conjunctivitis and asthma. Allergic responses can occur systemically, or locally in any region, organ, tissue, or cell. In particular aspects, an allergic immune response occurs in the skin, the upper respiratory tract, the lower respiratory tract, pancreas, thymus, kidney, liver, spleen, muscle, nervous system, skeletal joints, eye, mucosal tissue, gut or bowel.
Methods and uses herein include relieving, including treating, a subject for an allergic immune response, or one or more symptoms caused by or associated with an allergen. Such methods and uses include administering to a subject an amount of an immunogen sufficient to relieve, such as treat, the subject for the allergic immune response, or one or more symptoms caused by or associated with the allergen. Methods and uses of the invention include treating or administering a subject previously exposed to an allergen or immunogen. Thus, in certain embodiments, methods and uses are for treating or protecting a subject from an allergic immune response, or one or more symptoms caused by or associated with secondary or subsequent exposure to an allergen or an immunogen.
Immunogens described herein may elicit, stimulate, induce, promote, increase or enhance immunological tolerance to an allergen and/or to the immunogen. Methods and uses of the invention therefore further include inducing immunological tolerance of a subject to an allergen or the immunogen itself. Thus, for example, immunogens described herein can be effective in relieving, such as treating an allergic immune response, including but not limited to an allergic immune response following a secondary or subsequent exposure of a subject to an allergen. In one embodiment, a method or use includes administering to the subject an amount of an immunogen sufficient to induce tolerance in the subject to the allergen or immunogen itself. In particular aspects, the immunological tolerance elicited, stimulated, induced, promoted, increased or enhanced may involve modulation of T cell activity, including but not limited to CD4+ T cells, CD8+ T cells, Thl cells, Th2 cells and regulatory T cells. For example, immunological tolerance elicited, stimulated, induced, promoted, increased or enhanced from administration of the immunogen, may involve modulation of the production or activity of pro-inflammatory or anti-inflammatory cytokines produced by T cells.
In additional embodiments, a method or use of inducing immunological tolerance in a subject to an allergen includes a reduction in occurrence, frequency, severity, progression, or duration of physiological conditions, disorders, illnesses, diseases, symptoms or complications caused by or associated an allergic response to the allergen in the subject. Thus, in certain embodiments, inducing immunological tolerance can protect a subject against or treat a subject for an allergic immune response, or one or more symptoms caused by or associated with an allergen or the immunogen.
Methods and uses of the invention include treating a subject via immunotherapy, including specific immunotherapy. In one embodiment, a method or use includes administering to the subject an amount of an immunogen described herein. In one aspect, an immunogen administered to a subject during specific immunotherapy to treat the subject is the same immunogen to which the subject has been sensitized or is hypersensitive (e.g., allergic). In another non-limiting aspect, an immunogen is administered to a subject to treat the subject to a different immunogen, e.g. a pollen allergen to which the subject has been sensitized or is hypersensitive (e.g., allergic). Thus, the immunotherapeutic mechanism may involve bystander suppression of an allergic immune response caused by a pollen allergen by administering an unrelated immunogen, e.g. an immunogen disclosed herein. Thus, an immunogen of the invention my induce bystander tolerance induction to an unrelated immunogen, e.g., a pollen allergen disclosed herein. As described herein, immunogens include T cell epitopes, such as Th2 cell epitopes. In methods and uses herein, the subject to be treated has a specific T-cell response to the immunogen before administering the first dose.
Accordingly, methods and uses of the invention include administering an amount of a an immunogen (e.g., a T cell epitope-containing immunogen) to a subject sufficient to provide the subject with protection against an allergic immune response, or one or more symptoms caused by or associated with an allergen. In another embodiment, a method includes administering an amount of an immunogen (e.g., a T cell epitope-containing immunogen) to a subject sufficient to relieve, e.g. treat, vaccinate or immunize the subject against an allergic immune response, or one or more symptoms caused by or associated with an allergen.
The specific T-cell response may be monitored by determining by way of contacting a sample of PBMCs obtained from the subject with the immunogens and measuring the IL-5 secretion or IL-5 mRNA gene expression in response to the immunogen.
In accordance with the invention, methods and uses of modulating anti-allergen activity of T cells, including but not limited to CD8+ T cells, CD4+ T cells, Thl cells or Th2 cells, in a subject are provided. In one embodiment, a method or use includes administering to a subject an amount of a polypeptide of the invention or derivative thereof including an immunogenic molecule thereof, such as a T cell epitope, sufficient to modulate Th2 cell activity in the subject. In certain embodiments, two or more immunogens may be administered to a subject, e.g. may be administered as a combination composition, or administered separately, such as concurrently or in series or sequentially. For example, methods and uses described herein comprises administration separately or as a combination: at least 2-25 polypeptides defined herein, or separately or as a combination of 3-25, 4-25, 5-25, 6-25, 7-25 polypeptides defined herein, or separately or as a combination of 2-20, 3-20, 4-20, 5-20, 6-20 defined herein, or separately or as a combination of 2-12, 3-12, 4-12, 5-12, 6-12, 7-12
polypeptides defined herein, or separately or as a combination of 2-10, 3-10, 4-10, 5-10, 6- 10, 7-10 polypeptides defined herein. For example, a there may be administered to a subject, e.g. as a combination composition, one or more immunodominant PG peptides, like those recognized by at least 3 subjects in a population of 20 subjects, e.g. composition comprising one more polypeptides of option a), wherein each polypeptide of option a may independently include one or more sequences selected from any one of SEQ ID NOs: 19, 20, 24, 25, 26, 32, 37, 69, 71, 72, 73, 74, 78, 79, 80, 81, 82, 84, 86, 87, 88, 89, 90, 91, 93, 95, 99, 319, 321 and 506, or a sequence with 0, 1 or 2 mismatches compared to the SEQ ID NOs: 19, 20, 24, 25, 26, 32, 37, 69, 71, 72, 73, 74, 78, 79, 80, 81, 82, 84, 86, 87, 88, 89, 90, 91, 93, 95, 99, 319, 321 and 506.
Compositions may comprise one or more polypeptides of option a that derives from NTGA's or homologs thereof that are co-released with major allergens from grass pollen, e.g.
wherein each polypeptide of option a) may include one or more sequences selected from any one of SEQ ID NOs: 1-18, 38-40, 41-55, 56-62, 63-89, 148-156, 169-182, 183-190, 211-232, 270-277, 338-348, 292-296, 354-356, 409-419, 467-481, 485-513 and 519, or a sequence with 0, 1 or 2 mismatches compared to the SEQ ID NOs: 1-18, 38-40, 41-55, 56- 62, 63-89, 148-156, 169-182, 183-190, 211-232, 270-277, 338-348, 292-296, 354-356, 409-419, 467-481, 485-513 and 519.
Methods and uses of the invention therefore include any therapeutic or beneficial effect. In various methods embodiments, an allergic immune response, or one or more symptoms caused by or associated with an allergen is reduced, decreased, inhibited, limited, delayed or prevented. Methods and uses of the invention moreover include reducing, decreasing, inhibiting, delaying or preventing onset, progression, frequency, duration, severity, probability or susceptibility of one or more adverse symptoms, disorders, illnesses, diseases or complications caused by or associated with an antigen/allergen. In further various particular embodiments, methods and uses include improving, accelerating, facilitating, enhancing, augmenting, or hastening recovery of a subject from an allergic immune response, or one or more physiological conditions, symptoms or complications caused by or associated with an antigen/allergen. In yet additional various embodiments, methods and uses include stabilizing an allergic immune response, or one or more physiological conditions, symptoms or complications caused by or associated with an antigen/allergen. A therapeutic or beneficial effect is therefore any objective or subjective measurable or detectable improvement or benefit provided to a particular subject. A therapeutic or beneficial effect can but need not be complete ablation of all or any allergic immune response, or one or more symptoms caused by or associated with an allergen. Thus, a satisfactory clinical endpoint is achieved when there is an incremental improvement or a partial reduction in an allergic immune response, or one or more symptoms caused by or associated with an allergen, or an inhibition, decrease, reduction, suppression, prevention, limit or control of worsening or progression of an allergic immune response, or one or more symptoms caused by or associated with an allergen, over a short or long duration (hours, days, weeks, months, etc.). A therapeutic or beneficial effect also includes reducing or eliminating the need, dosage frequency or amount of a second therapeutic protocol or active such as another drug or other agent (e.g., anti-inflammatory) used for treating a subject having or at risk of having an allergic immune response, or one or more symptoms caused by or associated with an allergen. For example, reducing an amount of an adjunct therapy, such as a reduction or decrease of a treatment for an allergic immune response, or one or more symptoms caused by or associated with an allergen, or a specific immunotherapy, vaccination or immunization protocol is considered a beneficial effect. In addition, reducing or decreasing an amount of the immunogen used for specific immunotherapy, vaccination or immunization of a subject to provide protection to the subject is considered a beneficial effect. Methods and uses described herein may relieve one or more symptoms of an allergic immune response or delays the onset of symptoms, slow the progression of symptoms, or induce disease modification. For example, the following symptoms may be decreased or eliminated; nasal symptoms in the form of itchy nose, sneezing, runny nose, blocked nose; conjunctival symptoms in the form of itchy eyes, red eyes, watery eyes; and respiratory symptoms in the form of decreased lung function. Furthermore, the beneficial effect of methods and uses described herein may be observed by the patient's need for less concomitant treatment with corticosteroids or HI antihistamines to suppress the symptoms.
When an immunogen is administered to induce tolerance, an amount or dose of the immunogen to be administered, and the period of time required to achieve a desired outcome or result (e.g., to desensitize or develop tolerance to the allergen or immunogen) can be determined by one skilled in the art. The immunogen may be administered to the patient through any route known in the art, including, but not limited to oral, inhalation, sublingual, epicutaneous, intranasal, and/or parenteral routes (intravenous, intramuscular, subcutaneously, intradermal, and intraperitoneal). Methods and uses of the invention include administration of an immunogen to a subject prior to contact by or exposure to an allergen; administration prior to, substantially contemporaneously with or after a subject has been contacted by or exposed to an allergen; and administration prior to, substantially contemporaneously with or after an allergic immune response, or one or more symptoms caused by or associated with an allergen. As used herein, a "sufficient amount" or "effective amount" or an "amount sufficient" or an "amount effective" refers to an amount that provides, in single (e.g., primary) or multiple (e.g., booster) doses, a long term or a short term detectable or measurable improvement in a given subject or any objective or subjective benefit to a given subject of any degree or for any time period or duration (e.g., for minutes, hours, days, months, years, or cured) .
An amount sufficient or an amount effective need not be therapeutically or prophylactically effective in each and every subject treated, nor a majority of subjects treated in a given group or population. An amount sufficient or an amount effective means sufficiency or effectiveness in a particular subject, not a group of subjects or the general population. As is typical for such methods, different subjects will exhibit varied responses to a method of the invention, such as immunization, vaccination, specific immunotherapy and therapeutic treatments.
The term "subject" includes but is not limited to a subject at risk of allergen contact or exposure as well as a subject that has been contacted by or exposed to an allergen. A subject also includes those having or at risk of having or developing an immune response to an antigen or an allergen . Such subjects include mammalian animals (mammals), such domestic animal (dogs and cats), a farm animal (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), experimental animal (mouse, rat, rabbit, guinea pig) and humans.
Target subjects and subjects in need of treatment also include those at risk of allergen exposure or contact or at risk of having exposure or contact to an allergen. Accordingly, subjects include those at increased or elevated (high) risk of an allergic reaction; has, or has previously had or is at risk of developing hypersensitivity to an allergen; and those that have or have previously had or is at risk of developing asthma.
As mentioned , methods and uses described herein, relates to relieving an allergic immune response, e.g. preventing or treating an allergic immune response against a grass pollen allergen, which is not a Phi p grass pollen allergen by administering an immunogen described herein.
Non-Phi p grass pollen allergens are but not limited to pollen allergens of the plant family Poaceae, e.g. the plant genera Anthoxanthum, Cynodon, Dactylis, Festuca, Holcus,
Hordeum, Lollium, Oryza, Paspalum, Phalaris, Poa, Secale, Sorghum, Triticum and Zea.
Immunogens disclosed herein are conserved across a Phi p grass pollen and at least non-Phi p grass pollen species, e.g. Anthoxanthum, Cynodon, Dactylis, Lollium, and Poa. In a particular embodiment, the allergic immune response is not against a grass pollen allergen of the plant genus Phleum, e.g. Phleum Pratense.
As immunogens of the invention are conserved across a Phi p grass pollen and at least Cyn d pollen, methods and uses described herein, comprises relieving an allergic immune response against Phi p grass pollen as well as against Cyn d pollen or another non-Phi p pollen, e.g. Lol P pollen, Poa p Pollen or Ant O pollen.
Examples on grass pollen allergens are but not limited to; Ant o 1, Cyn d 1, Cyn d 7, Cyn d 12, Cyn d 15, Cyn d 22w, Cyn d 23, Cyn d 24, Dac g 1, Dac g 2, Dac g 3, Dac g 4, Dac g 5, Fes p 4, Hoi I 1, Hoi I 5, Hor v 1, Hor v 5, Lol p 1, Lol p 2, Lol p 3, Lol p 4, Lol p 5, Lol p 11, Ory s 1, Pas n 1, Pha a 1, Pha a 5, Phi p 1, Phi p 2, Phi p 4, Phi p 5, Phi p , Phi p 7, Phi p 11, Phi p 12, Phi p 13, Poa p 1, Poa p 5, Sec c 1, Sec c 5, Sec c 38 and/or Sor h 1, of which group 1 allergens (e.g. Ant o 1, Cyn d 1, Dac g 1, Hoi 1, Lol p 1, Pha a 1, Phi p 1 and Poa p 1) or group 5 allergens (Dac g 5, Lol p 5, Pha a 5, Phi p 5, Poa p 5) are considered major allergens important for the allergic immune response triggered by a grass pollen in a subject. Many of the well known pollen allergens are major allergens and thought to be the most important allergens in eliciting an allergic immune in a subject. Thus, in some embodiments, the non Phi p grass pollen allergen at least is Ant o 1, Cyn d 1, Dac g 1, Hoi 1, Lol p 1, Pha a 1, and Poa p 1) or group 5 allergens (Dac g 5, Lol p 5, Pha a 5, Poa p 5).
"Prophylaxis" and grammatical variations thereof mean a method or use in which contact, administration or in vivo delivery to a subject is prior to contact with or exposure to an allergen. In certain situations it may not be known that a subject has been contacted with or exposed to an allergen, but administration or in vivo delivery to a subject can be performed prior to manifestation of an allergic immune response, or one or more symptoms caused by or associated with an allergen. For example, a subject can be provided protection against an allergic immune response, or one or more symptoms caused by or associated with an allergen or provided specific immunotherapy with a polypeptide of a derivative thereof, including an immunogenic molecule described herein. In such case, a method or use can eliminate, prevent, inhibit, suppress, limit, decrease or reduce the probability of or susceptibility towards an allergic immune response, or one or more physiological conditions, symptoms or complications caused by or associated with an antigen/allergen.
"Prophylaxis" can also refer to a method or use in which contact, administration or in vivo delivery to a subject is prior to a secondary or subsequent exposure to an antigen/ allergen. In such a situation, a subject may have had a prior contact or exposure to an allergen. In such subjects, an acute allergic reaction may but need not be resolved. Such a subject typically may have developed anti-allergen antibodies due to the prior exposure.
Immunization or vaccination, by administration or in vivo delivery to such a subject, can be performed prior to a secondary or subsequent allergen exposure. Such a method or use can eliminate, prevent, inhibit, suppress, limit, decrease or reduce the probability of or susceptibility towards a secondary or subsequent allergic immune response, or one or more symptoms caused by or associated with an allergen. In certain embodiments, such a method or use includes providing specific immunotherapy to the subject to eliminate, prevent, inhibit, suppress, limit, decrease or reduce the probability of or susceptibility towards a secondary or subsequent allergic immune response, or one or more physiological conditions, symptoms or complications caused by or associated with an antigen/allergen.
Treatment of an allergic reaction or response can be at any time during the reaction or response. An immunogen can be administered as a single or multiple dose e.g., one or more times hourly, daily, weekly, monthly or annually or between about 1 to 10 weeks, or for as long as appropriate (e.g. 3 months, 6 months or more, for example, to achieve a reduction in the onset, progression, severity, frequency, duration of one or more symptoms or complications associated with or caused by an allergic immune response, or one or more physiological conditions, symptoms or complications caused by or associated with an antigen/allergen.
Accordingly, methods and uses of the invention can be practiced one or more times (e.g., 1- 10, 1-5 or 1-3 times) an hour, day, week, month, or year. The skilled artisan will know when it is appropriate to delay or discontinue administration. Doses can be based upon current existing protocols, empirically determined, using animal disease models or optionally in human clinical trials. Initial study doses can be based upon animal studies, e.g. a mouse, and the amount of the immunogen to be administered for being effective can be determined. Exemplary non-limiting amounts (doses) are in a range of about 0.1 mg/kg to about 100 mg/kg, and any numerical value or range or value within such ranges. Greater or lesser amounts (doses) can be administered, for example, 0.01-500 mg/kg, and any numerical value or range or value within such ranges. The dose can be adjusted according to the mass of a subject, and will generally be in a range from about 1-10 ug/kg, 10-25 ug/kg, 25-50 ug/kg, 50-100 ug/kg, 100-500 ug/kg, 500-1,000 ug/kg, 1-5 mg/kg, 5-10 mg/kg, 10-20 mg/kg, 20-50 mg/kg, 50-100 mg/kg, 100-250 mg/kg, 250-500 mg/kg, or more, two, three, four, or more times per hour, day, week, month or annually. A typical range will be from about 0.3 mg/kg to about 50 mg/kg, 0-25 mg/kg, or 1.0-10 mg/kg, or any numerical value or range or value within such ranges. Doses can vary and depend upon whether the treatment is prophylactic or therapeutic, whether a subject has been previously exposed to the antigen/allergen, the onset, progression, severity, frequency, duration, probability of or susceptibility of the symptom, condition, pathology or complication, or vaccination or specific immunotherapy to which treatment is directed, the clinical endpoint desired, previous or simultaneous treatments, the general health, age, gender, race or immunological competency of the subject and other factors that will be appreciated by the skilled artisan. The skilled artisan will appreciate the factors that may influence the dosage and timing required to provide an amount sufficient for providing a therapeutic or prophylactic benefit. Immunogens of the invention can be provided in compositions, and in turn such
compositions can be used in accordance with the invention methods and uses. Such compositions, methods and uses include pharmaceutical compositions and formulations. In certain embodiments, a pharmaceutical composition includes one or more immunogens. In particular, aspects, such compositions and formulations may be a vaccine, including but not limited to a vaccine to protect against an allergic immune response, or one or more symptoms caused by or associated with an allergen.
A pharmaceutical comprises an immunogen of the invention and a pharmaceutically acceptable ingredient or carrier.
As used herein the term "pharmaceutically acceptable" and "physiologically acceptable" mean a biologically acceptable formulation, gaseous, liquid or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery or contact. Such formulations include solvents (aqueous or non aqueous), solutions (aqueous or non aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery.
Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules and crystals. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral and antifungal agents) can also be incorporated into the compositions.
A composition may be lyophilized so as to enhance stability and ease of transportation. For the purpose of being used as a vaccine, the composition may be sterile. Pharmaceutical compositions can be formulated to be compatible with a particular route of administration. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes. Exemplary routes of administration for contact or in vivo delivery for which a composition can optionally be formulated include inhalation, intranasal, oral, buccal, sublingual, subcutaneous, intradermal, epicutaneous, rectal, transdermal, or intralymphatic.
In some embodiments, the pharmaceutical composition is aqueous and, in other embodiments, the composition is non-aqueous solutions, suspensions or emulsions of the peptide/protein, which compositions are typically sterile and can be isotonic with the biological fluid or organ of the intended recipient. Non-limiting illustrative examples include water, saline, dextrose, fructose, ethanol, vegetable or synthetic oils.
For oral, buccal or sublingual administration, a composition can take the form of for example a solid dosage form, e.g. tablets or capsules, optionally formulated as fast- integrating tablets/capsules or slow-release tablets/capsules. In some embodiments, the tablet is a freeze-dried, optionally fast-disintegrating tablet suitable for being administered under the tongue. A solid dosage form optionally is sterile, optionally anhydrous.
The pharmaceutical composition may also be formulated into a "unit dosage form". As used herein a unit dosage form refers to physically discrete units suited as dosages for the subject to be treated; each unit containing a predetermined quantity of a peptide/protein optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle or filling agent) which, when administered in one or more doses, is calculated to produce a desired effect. Unit dosage forms also include, for example, ampules and vials, which may include a composition in a freeze-dried or lyophilized state; a sterile liquid carrier, for example, can be added prior to administration or delivery in vivo. Unit dosage forms additionally include, for example, ampules and vials with liquid compositions disposed therein. Individual unit dosage forms can be included in multi-dose kits or containers. Pharmaceutical formulations can be packaged in single or multiple unit dosage form for ease of administration and uniformity of dosage.
To increase an immune response, immunological tolerance or protection against an allergic immune response, or one or more symptoms caused by or associated with an allergen, immunogens can be mixed with adjuvants.
Adjuvants include, for example: oil (mineral or organic) emulsion adjuvants such as Freund's complete (CFA) and incomplete adjuvant (IFA) (WO 95/17210; WO 98/56414; WO 99/12565; WO 99/11241; and U.S. Patent No. 5,422,109); metal and metallic salts, such as aluminum and aluminum salts, such as aluminum phosphate or aluminum hydroxide, alum (hydrated potassium aluminum sulfate); bacterially derived compounds, such as
Monophosphoryl lipid A and derivatives thereof (e.g., 3 De-O-acylated monophosphoryl lipid A, aka 3D-MPL or d3-MPL, to indicate that position 3 of the reducing end glucosamine is de- O-acylated, 3D-MPL consisting of the tri and tetra acyl congeners), and enterobacterial lipopolysaccharides (LPS); plant derived saponins and derivatives thereof, for example Quil A (isolated from the Quilaja Saponaria Molina tree, see, e.g., "Saponin adjuvants", Archiv. fur die gesamte Virusforschung, Vol. 44, Springer Verlag, Berlin, p243-254; U.S. Patent No. 5,057,540), and fragments of Quil A which retain adjuvant activity without associated toxicity, for example QS7 and QS21 (also known as QA7 and QA21), as described in
W096/33739, for example; surfactants such as, soya lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone; oligonucleotides such as CpG (WO 96/02555, and WO 98/16247), polyriboA and polyriboU; block copolymers; and
immunostimulatory cytokines such as GM-CSF and IL-1, and Muramyl tripeptide (MTP). Additional examples of adjuvants are described, for example, in "Vaccine Design--the subunit and adjuvant approach" (Edited by Powell, M. F. and Newman, M. J.; 1995,
Pharmaceutical Biotechnology (Plenum Press, New York and London, ISBN 0-306-44867-X) entitled "Compendium of vaccine adjuvants and excipients" by Powell, M. F. and Newman M. Cosolvents may be added to the composition. Non-limiting examples of cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters. Non-limiting examples of cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol,
polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters.
Supplementary compounds (e.g., preservatives, antioxidants, antimicrobial agents including biocides and biostats such as antibacterial, antiviral and antifungal agents) can also be incorporated into the compositions. Pharmaceutical compositions may therefore include preservatives, anti-oxidants and antimicrobial agents.
Preservatives can be used to inhibit microbial growth or increase stability of ingredients thereby prolonging the shelf life of the pharmaceutical formulation. Suitable preservatives are known in the art and include, for example, EDTA, EGTA, benzalkonium chloride or benzoic acid or benzoates, such as sodium benzoate. Antioxidants include, for example, ascorbic acid, vitamin A, vitamin E, tocopherols, and similar vitamins or provitamins.
An antimicrobial agent or compound directly or indirectly inhibits, reduces, delays, halts, eliminates, arrests, suppresses or prevents contamination by or growth, infectivity, replication, proliferation, reproduction, of a pathogenic or non- pathogenic microbial organism. Classes of antimicrobials include antibacterial, antiviral, antifungal and
antiparasitics. Antimicrobials include agents and compounds that kill or destroy (-cidal) or inhibit (-static) contamination by or growth, infectivity, replication, proliferation,
reproduction of the microbial organism. Pharmaceutical formulations and delivery systems appropriate for the compositions, methods and uses of the invention are known in the art (see, e.g. Remington: The Science and Practice of Pharmacy (David B. Troy, Paul Beringer Lippincott Williams & Wilkins) 2006).
Pharmaceutical compositions can be formulated to be compatible with a particular route of administration. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes (For example excipients recorded in a
Pharmacopiea). Exemplary routes of administration for contact or in vivo delivery, which a composition can optionally be formulated, include inhalation, respiration, intranasal, intubation, intrapulmonary instillation, oral, buccal, intrapulmonary, intradermal, topical, dermal, parenteral, sublingual, subcutaneous, intravascular, intrathecal, intraarticular, intracavity, transdermal, iontophoretic, intraocular, opthalmic, optical, intravenous (i.v.), intramuscular, intraglandular, intraorgan, or intralymphatic.
Formulations suitable for parenteral administration include aqueous and non-aqueous solutions, suspensions or emulsions of the active compound, which preparations are typically sterile and can be isotonic with the blood of the intended recipient. Non-limiting illustrative examples include water, saline, dextrose, fructose, ethanol, animal, vegetable or synthetic oils.
Methods and uses of the invention may be practiced by any mode of administration or delivery, or by any route, systemic, regional and local administration or delivery. Exemplary administration and delivery routes include intravenous (i.v.), intraperitoneal (i.p.), intrarterial, intramuscular, parenteral, subcutaneous, intra-pleural, topical, dermal, intradermal, transdermal, transmucosal, intra-cranial, intra-spinal, rectal, oral (alimentary), mucosal, inhalation, respiration, intranasal, intubation, intrapulmonary, intrapulmonary instillation, buccal, sublingual, intravascular, intrathecal, intracavity, iontophoretic, intraocular, ophthalmic, optical, intraglandular, intraorgan, or intralymphatic.
For oral administration, a composition can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (for example, pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (for example, lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (for example, magnesium stearate, talc or silica); disintegrants (for example, potato starch or sodium starch glycolate); or wetting agents (for example, sodium lauryl sulphate). The tablets can be coated by methods known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by
conventional means with pharmaceutically acceptable additives such as suspending agents (for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (for example, lecithin or acacia); non-aqueous vehicles (for example, almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (for example, methyl or propyl-p-hydroxybenzoates or sorbic acid).
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.
All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the invention is not entitled to antedate such publication by virtue of prior invention.
As used in this specification and the appended claims, the use of an indefinite article or the singular forms "a," "an" and "the" include plural reference unless the context clearly dictates otherwise. In addition, it should be understood that the individual peptides, proteins, antigens, allergens (referred to collectively as compositions), or groups of compositions, modeled or derived from the various components or combinations of the compositions, and substituents described herein, are disclosed by the application to the same extent as if each composition or group of compositions was set forth individually. Thus, selection of particular peptides, proteins, antigens, allergens, etc. is clearly within the scope of the invention.
As used in this specification and the appended claims, the terms "comprise", "comprising", "comprises" and other forms of these terms are intended in the non-limiting inclusive sense, that is, to include particular recited elements or components without excluding any other element or component. Unless defined otherwise all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. As used herein, "about" means + or - 5%. The use of the wild type (e.g., "or") should be understood to mean one, both, or any combination thereof of the wild types, i.e., "or" can also refer to "and."
As used in this specification and the appended claims, any concentration range, percentage range, ratio range or other integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. For example, although numerical values are often presented in a range format throughout this document, a range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the use of a range expressly includes all possible subranges, all individual numerical values within that range, and all numerical values or numerical ranges including integers within such ranges and fractions of the values or the integers within ranges unless the context clearly indicates otherwise. This
construction applies regardless of the breadth of the range and in all contexts throughout this patent document. Thus, to illustrate, reference to a range of 90-100% includes 91-99%, 92-98%, 93-95%, 91-98%, 91-97%, 91-96%, 91-95%, 91-94%, 91-93%, and so forth. Reference to a range of 90-100%, includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%,
92.5%, etc., and so forth. Reference to a range of 5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, 100-150, and 150-175, includes ranges such as 5-20, 5-30, 5-40, 5-50, 5-75, 5- 100, 5-150, 5-171, and 10-30, 10-40, 10-50, 10-75, 10-100, 10-150, 10-175, and 20-40, 20-50, 20-75, 20-100, 20-150, 20-175, and so forth. Further, for example, reference to a series of ranges of 2-72 hours, 2-48 hours, 4-24 hours, 4-18 hours and 6-12 hours, includes ranges of 2-6 hours, 2, 12 hours, 2-18 hours, 2-24 hours, etc., and 4-27 hours, 4-48 hours, 4-6 hours, etc.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The invention is further exemplified by way of the following non-limited examples.
References
Oseroff C, Sidney J, Kotturi MF, Kolla R, Alam R, Broide DH, et al. Molecular determinants of T cell epitope recognition to the common Timothy grass allergen. Journal of immunology 2010; 185:943-55.
P. Wang, J. Sidney, C. Dow, B. Mothe, A. Sette, B. Peters. A systematic assessment of MHC class II peptide binding predictions and evaluation of a consensus approach. PLoS Comput Biol, 4 (2008), p. el000048
P. Wang, J. Sidney, Y. Kim, A. Sette, O. Lund, M. Nielsen, et al. Peptide binding predictions for HLA DR, DP and DQ molecules. BMC Bioinform, 11 (2010), p. 568
Karosiene, Edita, Michael Rasmussen, Thomas Blicher, Ole Lund, Soren Buus, and Morten Nielsen. "NetMHCIIpan-3.0, a Common Pan-specific MHC Class II Prediction Method
Including All Three Human MHC Class II Isotypes, HLA-DR, H LA-DP and HLA-DQ."
Immunogenetics
Tables
Table 1
Table 1 indicates for each of the PG peptides (SEQ ID NOs 1-519) in which other grass pollen species (across Cyn d and Lol p or across Cyn d, Lol p, Ant o and Poa p) a matching peptide with either less than 3, less than 2 or zero mismatches are found. The number of TG grass allergic donors (n = 20) with an in vitro T cell response to the TG peptide sequence is also shown.
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Table 2
Table 2 shows wild type sequences of NTGA's detected by combined transcriptomic analysis and Mass spectrometry analysis of grass pollen extracts.
SEQ NTGA Phi p wild type sequence (SEQ ID NO 520-565)
ID No
No
520 1 MAATIQSVKARQIFDSRGNPTVEVDVCCSDGTFARAAVPSGASTGVYEALELRDGGSDY
LGKGVLKAVDNVNSIIGPALIGKDPTEQTELDNFMVHQLDGTKNEWGWCKQKLGANAI
LAVSL.AVCKAGALVKKIPLYQHIANLAGNKQLVLPVPAFNVINGGSHAGNKLAMQEFMIL
PTGASSFKEAMKMGVEVYHNLKSVIKKKYGQDATNVGDEGGFAPNIQENKEGLELLKTA
IEKAGYTGKVVIGMDVAASEFYGEKDQTYDLNFKEENNDGSQKISGDSLKNVYKSFVSE
YPIVSIEDPFDQDDWVHYAKMTEEIGEQVQIVGDDLLVTNPTRVAKAIAEKSCNALLLKV
NQIGSVTESIEAVKMSKRAGWGVMTSHRSGETEDTFIADLAVGLSTGQIKTGAPCRSER
LAKYNQLLRIEEELGAAAVYAGLKFRAPVEPY
521 2 MASPALISDTDQWKALQAHVGAIHKTHLRDLMADADRCKALTAEFEGVFLDYSRQQAT
TETVDKLFKLAEAAKLKEKIAKMFNGDKINSTENRSVLHVALRAPRDAVINSDGVNVVPE
VWAVIDKIKQFSETFRSGSWVGATGKPLTNVVSVGIGGSFLGPLFVHTALQTDPEAAES
AKGRQLRFLANVDPVDVARSIKDLDPETTLVVVVSKTFTTAETMLNARTIKEWIVSSLGP
QAVSKHMIAVSTNLKLVKEFGIDPNNAFAFWDWVGGRYSVCSAVGVLPLSLQYGFPIVQ
RFLEGASSIDNHFRTASFEKNIPVLLGLLSVWNVSFLGYPARAILPYSQALEKLAPHIQQL
SMESNGKGVSIDGVPLPYEAGEIDFGEPGTNGQHSFYQLIHQGRVIPCDFIGVIKSQQPV
YLKGETSNHDELMSNFFAQPDALASRKTPAPLRSENVSENLIPHKTFKGNRPSLSFLLSSL
SAYEIGQLLAIYEHRIAVQGFIWGINSFDQWGVELGKSLASQVRKQLHASRMEGKPVEG
F N PSSAS LI_ARYLA VE PSTPYDTTVLP KV
522 3 MDDHKEHKEKEHTGGNPEVNEEEEEDEEAKRAVLLGPQVPLKEQLELDKDDESLRRWK
EQLLGQVDTEQLGETAEPEVKVVDLTILSPDRPDLVLPIPFVADEKGYAFALKDGSTYSFR FSFIVSNNIVSGLKYTNTVWKTGVRVENQKMMLGTFSPQPEPYIYVGEEETTPAGIFARG SYSAKLKFVDDDGKVYLEMSYYFEIRKDWPTGQ
523 4 YIKLMKTIFDFESIKKLLASPKFSFCFDGLHGVAGAYAKRMFVDELGASESSLLNCVPKED
FGGGHPDPNLTYAKELVERMGLGKSSSNVEPPEFGAAADGDADRNMVLGKRFFVTPSD
SVAIIAANAVQSIPYFASGLKGVARSMPTSAALDVVAKNLNLKFFEVPTGWKFFGNLMDA
GMCSVCGEESFGTGSDHIREKDGIWAVLAWLSIIAYKN KDNLGGDKLVSVEDIVLQHW
ATYGRHYYTRYDYENVDAEAAKELMANLVKMQSALSDVNKLIKEIQPDVAEVVSADEFE
YKDPVDGSVSKHQGIRYLFGDGSRLVFRLSGTGSVGATIRIYIEQYEKDSSKTGRESSDA
LSPLVDVALKLSKIKEYTGRSAPTVIT
524 5/64 MAAKCYPTVSDEYLAAVAKARRKLRGLIAEKNCAPLMLRIAWHSAGTFDVATKTGGPFG
TMRCPAEL.AHGANAGLDIAVRLLEPIKEQVPILSYADFYQLAGVVAVEITGGPEVPFHPGR
QDKTEPPPEGRLPDATLGSDHLRQVFTAQMGLSDQDIVALSGGHTLGRCHKERSGFEG
AWTANPLIFDNSYFTELLTGEKEGLLQLPTDKTLLTDPAFRPLVEKYAADEDAFFADYAEA
HLKLSELGFGE
525 6 MADEKLAKLREAVAGLPQISDNEKSGFISLVSRYLSGEEEHIEWPKIHTPTDEVVVPYDT
VDAPPEDLEATKALLDKLAVLKLNGGLGTTMGCTGPKSVIEVRNGFTFLDLIVLQIESLNK
KYGSNVPLLLMNSFNTHEDTLKIVEKYANSSIDIHTFNQSQYPRVVADEFLPWPSKGKTD
KDGWYPPGHGDIFPSLMNSGKLDLLLSQGKEYVFIANSDNLGAIVDMKILNHLIHKQNE
YCMEVTPKTLADVKGGTLISYEGRVQLLEIAQVPDAHVDEFKSIEKFKIFNTNNLWVNLK
AIKRLVEADALKMEIIPNPKEVDGVKVLQLETAAGAAIRFFDHAIGINVPRSRFLPVKATS
DLQLVQSDLYTLVDGFVTRNSARTDPSNPSIELGPEFKKVGSFLGRFKSIPSIVELDSLKV
SGDVWFGSGIVLKGKVTITAKPGVKLEIPDGAVLENKDINGAEDL
526 7 MAFEKIKVANPIVEMGDEMTRVFWQSIKEKLIFPFLDLDIKYYDLGVLHRDATDDKVTVE
AAEATLKYNVAIKCATITPDEDRVKEFNLKQMWRSPNGTIRNIINGTVFREPIICKNVPKL
VPGWTKPICIGRHAFGDQYRATDAVLKGPGKLRLVFEGKDETVDLEVFNFTGAGGVALA
MYNTDESIQGFAEASMAIAYEKKWPLYLSTKNTILKKYDGRFKDIFQAVYEADWKSKYEA
AGIWYEHRLIDDMVAYALKSEGGYVWACKNYDGDVQSDFLAQGFGSLGLMTSVLMCP
DGKTIEAEAAHGTVTRHFRVHQKGGETSTNSIASIFAWTRGLAHRAKLDDNARLLDFTQ
KLEDACVGTVESGKMTKDLALLVHGSSKVTRGDYLNTEEFIDAVAAELQSRLAAN
527 8 FEGCLAKSYKSEKSDKSATYDYSANIECEKEPPKPLYGGGILTGAEAPAPVSAGGKKLLM
AKSKSAPAKGSTLKVELEKDTHYTLSAWLQLSKSTGDVKAILVTPDGNFNTAGMLVVQS SEQ NTGA Phi p wild type sequence (SEQ ID NO 520-565)
ID No
No
GCWTMLKGGATSFAAGKGELFFETNVTAELMVDSMSLQPFSFEEWKSHRHESIAKERK
KKVKITVHGSDGKVLPDAELSLERVAKGFPLGNAMTKEILDIPEYEKWFTSRFTVATMEN
EMKWYSTEYDQNQELYEIPDKMLALAEKYNISVRGHNVFWDDQSKQMDWVSKLSAPQ
LKKAMEKRMKNVVSRYAGKLIHWDVLNENLHYSFFEDKLGKDASAEVFKEVAKLDDKPI
LFMNEYNTIEEPNDAAPLPTKYLAKLKQIQSYPGNSKLKYGIGLESHFDTPNIPYVRGSLD
TLAQAKVPIWLTEIDVKKGPKQVEYLEEVMREGFAHPGVKGIVLWGAWHAKECYVMCLT
DKNFKNLPVGDVVDKLITEWKAVPEDAKTDDKGVFEAELFHGEYNVTVKHKS
528 9 MAQLQETYACSPATERGRGILLGGDAKTDTIVYCAGRTVFFRRLDAPLDAWTYTEHAYPT
TVARISPNGEWVASADVSGCVRVWGRNGDRALKAEFRPISGRVDDLRWSPDGLRIVVS
GDGKGKSLVRAFMWDSGSTVGDFDGHSKRVLSCDFKPTRPFRIVTCGEDFLANYYEGP
PFKFKHSIRDHSNFVNCIRYSPDGSKFITVSSDKRGLIYDGKTGDKIGELSSEDSHTGSI
YAVSWSADSKQVLTVSADKTAKVWDIMEDASGKVNRTLVCTGIGGVDDMLVGCLWQN
DHLVTVSLGGTFNVFSASNPDKEPVSFAGHLKTVSSLTYFPQSNPRTMLSTSYDGVIIRW
IQGVGYGGRLIRKN NTQIKCFVAAEEELITSGYDNMVFRIPLNGDQCGDAESVDVGGQP
NALNIAVQQPEFALITTDSAIVLLHKSTVTSTTKVSYTITSSAVSPDGTEAIVGAQDGKLRI
YSISGDTLTEEAVLERHRGAITSIHYSPDVSMFASADANREAVAWDRATREIKLKNMLFH
TARINCLAWSPDSRLVATGSIDTCAIIYDVDKPASSRITIKGAHLGGVHGLTFADNDTLV
TAGEDACVRVWKLV
529 10 MVFSVTKKDTKPFDGQKPGTSGLRKKVTVFQQPHYLANFVQSTFNALPADQVKGATIVV
SGDGRYFSKDAVQIITKMAAANGVRRVWVGQDSLLSTPAVSAIIRERIAADGSKATGAF
ILTASHNPGGPTEDFGIKYN MGNGGPAPESVTDKIFSNTTTITEYLIAEDLPDVDISALGV
TTFTGPEGPFDVDVFDSATDYIKLMKTIFDFESIKKLLASPKFSFCFDGLHGVAGAYAKRM
FVDELGASESSLLNCVPKEDFGGGHPDPNLTYAKELVERMGLGKSSSNVEPPEFGAAAD
GDADRNMVLGKRFFVTPSDSVAIIAANAVQSIPYFASGLKGVARSMPTSAALDVVAKNL
NLKFFEVPTGWKFFGNLMDAGMCSVCGEESFGTGSDHIREKDGIWAVLAWLSIIAYKN
KDNLGGDKLVSVEDIVLQHWATYGRHYYTRYDYENVDAEAAKELMANLVKMQSALSDV
NKLIKEIQPDVAEVVSADEFEYKDPVDGSVSKHQGIRYLFGDGSRLVFRLSGTGSVGATI
RIYIEQYEKDSSKTGRESSDALSPLVDVALKLSKIKEYTGRSAPTVIT
530 11 MATSWTLPDHPTLPKGKTVAVIVLDGWGEASADQYNCIHRAETPVMDSLKNGAPEKWT
LVKAHGTAVGLPSDDDMGNSEVGHNALGAGRIFAQGAKLVDAALASGKIWEAEGFNYI
KESFAEGTLHLIGLLSDGGVHSRLDQVQLLVKGASERGAKRIRLHILTDGRDVLDGSSV
GFVETLENDLAQLREKGVDAQVASGGGRMYVTMDRYENDWDVVKRGWDAQVLGEAP
YKFKSALEAVKTLRAEPKANDQYLPAFVIVDESGKSVGPIVDGDAVVTFNFRADRMVMLA
KALEFADFDKFDRVRVPKIKYAGMLQYDGELKLPNKFLVSPPLIERTSGEYLVKNGVRTFA
CSETVKFGHVTFFWNGNRSGYFDETKEEYIEIPSDSGITFNEQPKMKALEIAEKTRDAILS
GKFDQVRINLPNGDMVGHTGDIEATVVACKAADEAVKIVLDAVEQVGGIYLVTADHGNA
EDMVKRNKSGQPALDKSGSIQILTSHTLQPVPVAIGGPGLHPGVKFRSDINTPGLANVA
ATVM N LHG FQAPDDYETTLI EVAD
531 13 MDEEYDVIVLGTGLKECILSGLLSVDGLKVLHMDRNDYYGGESTSLNLTKIWKRFKGSE
ATPDHLGVSKEYNVDMVPKFMMANGALVRVLIRTSVTKYLNFKAVDGSFVYNNGKIHKV
PATDVEALKSNLMGLFEKRRARKFFIYVQDYEEEDPKSHEGLDLHKVTTREVISKYGLED
DTVDFIGHALALHRDDNYLDEPAIDTVKRMKLYAESLARFQGGSPYIYPLYGLGELPQAFA
RLSAVYGGTYMLNKPECKVEFDESGKAFGVTSEGETAKCKKVVCDPSYLPDKVTKVGRV
ARAICIMKHPIPDTKDSHSVQIILPKKQLKRKSDMYVFCCSYAHNVAPKGKFIAFVSTEAE
TDKPEIELKPGIDLLGPVEETFFDIYDRYEPANAPEEDNCFVTNSYDATTHFETTVKDVLAL
YSKITGKELDLSVDLNAASAGESE
532 19 GVVATTDAVEACTGVNVAVMVGGFPRKEGMERKDVMSKNVSIYKSQASALEAHAAPNC
KVLVVANPANTNALILKEFAPSIPEKNISCLTRLDHNRALGQVSERLNVQVSDVKNVLIW
GNHSSSQYPDVNHATVKTSSGEKPVRELVQDDEWLNGPFIATVQQRGAAIIKARKLSSA
LSAASSACDHIRDWVLGTPEGTFVSMGVYSDGSYGVPAGLIYSFPVTCSGGEWTIVQGL
PIDEFSRKKMDATAQELSEEKALAYSCL
533 20 MAASSRRASQLLGSAASRFLHSRGYAAAAAAPSPAVFVDKSTRVICQGITGKNGTFHTE
QAIEYGTNMVGGVTPKKGGTEHLGLPVFNSVAEAKAETKANASVIYVPPPFAAAAIMEAL
EAELDLVVCITEGIPQHDMVKVKAALNRQSKTRLIGPNCPGIIKPGECKIGIMPGYIHKPG
RIGIVSRSGTLTYEAVFQTTAVGLGQSTCVGMGGDPFNGTNFVDCLEKFVADPQTEGIVL
IGEIGGTAEEDAAAFIQASKTDKPVVAFIAGLTAPPGRRMGHAGAIVSGGKGTAQDKIKA
LREAGVTVVESPAKIGSTMFEIFKQRGMVE SEQ NTGA Phi p wild type sequence (SEQ ID NO 520-565)
ID No
No
534 22 MALPNQGTVDYPSFKLVIVGDGGTGKTTFVKRHLTGEFEKKYEPTIGVEVHPLDFTTNCG
KIRFYCWDTAGQEKFGGLRDGYYIHGQCAIIMFDVTSRLTYKNVPTWHRDLCRVCENIPI VLCGNKVDVKNRQVKAKQVTFHRKKNLQYYEISAKSNYNFEKPFLYLARKLAGDANIHFV EAVALKPPEVTFDLAMQQQH
535 24 MATKRSVGTLGEADLKGKKVFLRADLNVPLDDAQKITDDTRIRASIPTIKFLLEKGAKVIL
ASHLGRPKGVTPKFSLKPLVPRLSELLGVEVVMANDCIGEEVEKLAAALPEGGVLLLENVR
FYKEEEKNDPEFAKKLASVADLYVNDAFGTAHRAHASTEGVTKFLRPSVAGFLMQKELDY
LVGAVANPKKPFAAIVGGSKVSSKIGVIESLLAKVDILILGGGMIFTFYKAQGKAVGKSLV
EEDKLELATSLIETAKAKGVSLLLPTDVVVADKFAPDAESKTVSADAIPDGWMGLDVGPD
SIKTFSEALDTTKTVIWNGPMGVFEFEKFAAGTDAIAKQLADLTGKGVTTIIGGGDSVAA
VEKAGLADKMSHISTGGGASLELLEGKPLPGVLALDEA
536 26 GVFTD KD KAAAH M KGGAKKVVISAPSKDAPMFVVGVN ED KYTSDVN IVSN ASCTTN CL
APLAKIINDNFGIVEGLMTTVHSITATQKTVDGPSSKDWRGGRAASFNIIPSSTGAAKAV GKVLPELNGKLTGMSFRVPTVDVSVVDLTVRIEKAASYEDIKKAIKAASEGNLKGIMGYV EEDLVSTDFIGDSRSSIFDAKAGIALNDNFVKLVSWYDNEWGY
537 27 MSAYCGKYKDELIKNAAYIGTPGKGILAADESTGTIGKRFASINVENVEDNRRALRELLFT
TPGALQHISGVILFEETLYQSSKAGKPFVDILKENNVLPGIKVDKGTVELAGTDKb 1 1 I QG
HDDLGKRCAKYYEAGARFAKWRAVLKIGPNEPSQLSIDQNAQGLARYAIICQENGLVPIV
EPEILVDGPHDIERCAYVTEVVLAACYKALNDQHVLLEGSLLKPNMVTPGSDAKKVAPEV
IAEYTVRTLQRTVPPAVPAIVFLSGGQSEEEATVNLNAMNKLQTKKPWFLSFSFGRALQQ
STLKAWSGKEENVEKAQKAFLVRCKANSEATLGTYKGDATLGEGASESLHVKDYKY
538 29 MASEKHFKYVILGGGVAAGYAAREFAKQGVQPGELAIISKESVAPYERPALSKGYLFPQN
AARLPGFHTCVGSGGEKLLPEWYTEKGIELILSTEIVKADLASKTLTSAAGATFTYETLLIA
TGSSTIKLTDFGVQGAEANNILYLRDINDADKLVAAMQAKKDGKAVVVGGGYIGLELSA
ALKLNNFDVTMVYPEPWCMPRLFTAGIAHFYEGYYASKGINIVKGTVASGFDADANGDV
AVVKLKDGRVLDANIVIVGVGGRPLTGLFKGQVDEEKGGLKTDTFFETSVAGVYAIGDV
ASFPMKLYNEPRRVEHVDHARKSAEQAVKAIKAKESGETVAEYDYLPYFYSRSFDIAWQF
YGDNVGESVLFGDNDPAAAKAKFGTYWVKDGKVVGVFLEGGSADENQAIAKVARAQPL
VAANLGELGKEGLDFAAKI
539 30 MAGGGVEDVYGEDRATEEQFVTPWSFSVASGHSLLRDPRHNKGLAFSEAERDAHYLRG
LLPPAIVSQEHQEKKIMHNLRQYTVPLQRYIAMMDLQERNERLFYKLLIDNVEELLPVVYT
PVVGEACQKYGSIYRRPQGLYISLKDKGKVLEVLKNWPERSIQVIVVTDGERILGLGDLG
CQGMGIPVGKLSLYTALGGVRPSACLPITIDVGTNNQTLLDDEYYIGLKQRRATGEEYHE
LLQEFMNAVKQNYGEKVLVQFEDFANHNAFDLLAKYSKSHLVFNDDIQGTASVVLAGLL
AALKVIGGGLADQTYLFLGAGEAGTGIAELIALEMSKHTDLPLDDCRKKIWLVDSKGLLV
ESRKESLQHFKKPFAHEHEPLTTLLEAVQSLKPTVLIGTSGVGKTFTQEVVEAMASFNEKP
VIFSLSNPTSHSECTAEEAYTWTKGTAVFASGSPFDPVEYEGKTYVPGQSNNAYVFPGFG
LGVVISGAIRVHDDMLLAASEALAEQVSQENFDKGLIFPPFTNIRKISANIAAKVAAKAYD
LGLASRLPRPDDLVKYAESCMYTPLYRSYR
540 32 MAPIKIGINGFGRIGRLVARVALQCPDVELVAVNDPFITTDYMTYMFKYDTVHGQWKHH
DVKVKDAKTLLFGEKEVAVFGCRNPEEIPWGAAGADYVVESTGVFTDKDKAAAHIKGGA
KKVIISAPSKDAPMFVCGVNEKEYTSDITIVSNASCTTNCLAPLAKVINDRFGIVEGLMTT
VHAMTATQKTVDGPSSKDWRGGRAASFNIIPSSTGAAKAVGKVLPVLNGKLTGMAFRV
PTVDVSVVDLTVRLEKAATYEQIKAAIKEESEGNLKGILGYVDEDLVSTDFQGDSRSSIF
DAKAGIALNDNFVKLVSWYDNEWGYSTRVVDLIRHIHATK
541 34 MSFSWICACVRAAAVAWEAGKPLSIEEVEVAPPQAMEVRVKILFTALCHTDVYFWEAKG
QTPVFPRIFGHEAGGIVESVGEGVTDVAPGDHVLPVFTGECKECRHCKSAESNMCDLLR
INTDRGVMISDGKSRFSIDGKPIFHFVGTSTFSEYTVMHVGCVAKINPEAPLDKVCVLSC
GISTGLGASINVAKPPKGSTVAIFGLGAVGLAAAEGARIAGASRIIGIDLNANRFEEARKF
GCTEFVNPKDHTKPVQEVLAEMTDGGVDRSVECTGNINAMIQAFECVHDGWGVAVLVG
VPHKDAEFKTHPMNFLNERTLKGTFFGNFKPRTDLPNVVEMYMKKELEVEKFITHSVTFS
EINKAFDLMAKGEGIRCIIRMEH
542 39 MAPRKFFVGGNWKCNGASDDVKKIVTVLNEAEVPSEDAVEVVVSPPFVFLQQAKALLRP _59 DFAVAAQNCWVRKGGAFTGEISAEMLVNLQVPWVILGHSERRALLSESNDFVADKVAY
ALAQGLKVIACIGETLEQREAGTTMEVVAAQTKAIAEKISDWTNVVLAYEPVWAIGTGKV
ASRAQAQEVHDGLRKWLHANVGPAVAESTRIIYGGSVNGANCKELAAQPDLDGFLVGG
ASLKPEFVDIIKSATVKSSS SEQ NTGA Phi p wild type sequence (SEQ ID NO 520-565)
ID No
No
543 43 GCRAGGNSATEPYIAGHHLLLAHAAAVKIYRDKYQPAQQGKIGILLDFVWYEPLTYNTED
EFAAHRAREFTLGWFMHPITYGHYPETMQRLVADRLPNFTDEQTRLLQGSADIVGVNHY
TTYYAKNHENLTHMSYANDWQVQLVYERNGIPIGKQGYSKWLYVVPWGFYKAVMHVK
DKYRNPLMIIGENGIDQSGSDTLPHALYDKFRIDYFDQYLHELKRATDDGARVTGYFAW
SLLDNFEWRMGFTSKFGIVYVDRKTFTRYPKDSTRWFRKV
544 43 KTNKDGVDYYHRLINYMLAN KITPYVVLYHYDLPEVLNNQYNGWLSPRVVPDFAYFADFC
FK
545 43 LTRHSFPKGFVFGTASSAYQVEGNALQYGRGPCIWDTFLKFPGATPDNATANVTVDEYH
RYM
546 47 MATDAAAPAAASKWNLLTFDTEEDVAVSLAKYTAELSGKFAAERGAFTVVLSGGTLIDTL
RKLAEPPYLETVQWSKWHVFWVDERVVPKDHVDSNYKLAVDGLLSKVPIPTDQVYAIN DTLSAEGAAADYETVLKQLVKNGVLAMSTATGFPRFDLMLLGMGPDGHLASLFPGHPLL NENQKWVTHIMDSPKPPPQRITFTFPVIKSSAYVAMVVTGPGEASAVKKVLSDDKTLP
547 47 DGHLASLFPGHPLLNENQKWVTHIMDSPKPPPQRITFTFPVIKSSAYVAMVVTGPGEASA
VKKVLSDDKTLPLLPTEMAILQDGEFTWFTDKQAVSMLQN K
548 49/54 STNVARAEDPYVFFEWHVTYGTKTVLGVPQKVILINGEFPGPRINCSSNNNIVVNVFNQL
DQPLLFTWNGIQHRKNSWQDGLPGTNCPVAPGTNFTYKWQPKDQIGSFFYFPSIGMQR
TVGGYGLISVVSRLLIPVPFDPPADDLQVIIGDWYTKDHAVMASLLDAGKSFGRPAGVLI
NGRGGKDATNPPMFTFEAGKTYRLRVCNVGIKSSLNFRIQGHDMRLVEMDGSHTLQDS
YDSLDVHVGHCFSVLVDADQKPADYLMVASTRFIADGSSASAVIRYAGSNTPPAANVPE
PPAGWAWSLNQWRSFRWNLTASAARPNPQGSYHYGQINITRTIKLMITRGHLDGKLKY
GFNGVSHVDADTPLKLAEYFNVSDQVFKYNQMGDAPPGVNGPMHVTPNVITAEFRTFIE
VVFENPEKSMDSLHIDGYAFFAVGMGPGKWKPELRKTYNLLDAVSRHSIQVYPRSWSAV
MLTFDNAGMWNVRSNVWERHYLGEQLYISVISPARSLRDEYNFPENALRCGKVVGLPLP
PSYLPA
549 49/54 MA I 1 1 1 RG 1 AAAGGVLLLALLLLS 1 NVARAbDPYVhhbWHV 1 YG 1 K 1 VLGVPQKVlLlNGb
FPGPRINCSSNNNIVVNVFNQLDQPLLFTWNGIQHRKNSWQDGLPGTNCPVAPGTNFTY
KWQPKDQIGSFFYFPSIGMQRTVGGYGLISVVSRLLIPVPFDPPADDLQVIIGDWYTKDH
AVMASLLDAGKSFGRPAGVLINGRGGKDATNPPMFTFEAGKTYRLRVCNVGIKSSLNFRI
QGHDMRLVEMDGSHTLQDSYDSLDVHVGHCFSVLVDADQKPADYLMVASTRFIADGS
SASAVIRYAGSNTPPAANVPEPPAGWAWSLNQWRSFRWNLTASAARPNPQGSYHYGQI
NITRTIKLMITRGHLDGKLKYGFNGVSHVDADTPLKLAEYFNVSDQVFKYNQMGDAPPG
VNGPMHVTPNVITAEFRTFIEVVFENPEKSMDSLHIDGYAFFAVGMGPGKWKPELRKTY
NLLDAVSRHSIQVYPRSWSAVMLTFDNAGMWNVRSNVWERHYLGEQLYISVISPARSL
RDEYNFPENALRCGKVVGLPLPPSYLPA
550 49/54 TKDHAVMASLLDAGKSFGRPAGVLINGRGGKDATNPPMFTFEAGKTYRLRVCNVGIKSS Fragm LNFRIQGHDMRLVEMDGSHTLQDSYDSLDVHVGHCFSVLVDADQKPADYLMVASTRFI ent ADGSSASAVIRYAGSNTPPAANVPEPPAGWAWSLNQWRSFRWNLTASAARPNPQGSY
HYGQINITRTIKLMITRGHLDGKLKYGFNGVSHVDADTPLKLAEYFNVSDQVFKYNQMG
DAPPGVNGPMHVTPNVITAEFRTFIEVVFENPEKSMDSLHIDGYAFFAVGMGPGKWKPE
LRK
551 51 MSPAEPTREESVYMAKLAEQAERYEEMVEFMERVAKATGGAGPGEELSVEERNLLSVAY
KNVIGARRASWRIISSIEQKEEGRGNDAHATTIRSYRSKIEAELAKICDGILALLDSHLVP
SAGAAESKVFYLKMKGDYHRYLAEFKSGAERKEAAESTMNSYKAAQDIALADLAPTHPIR
LGLALNFSVFYYEILNSPDRACNLAKQAFDEAISELDSLGEESYKDSTLIMQLLRDNLTLW
TSDTNEDGGDEIKEAPAPKESGD
552 52 MDACRLLLLLLLGLLGLLAPLASAQLSREFYKASCPDAEKIVAAVIEKKLKEDPGTAAGLLR
LLFHDCFANGCDASILIDPLSNQSAEKEAGPNISVRGFEVIDDIKKELEAKCPKTVSCADI
VALGTRDAVRISGGPAYEVPTGRRDSLVSNREEADNNLPGPDIPIPKLTSEFLSRGFTPEE
MVVLLAGGHSIGKVRCIFIEPDATPMDPGYQASISKLCDGPNRDTGFVNMDEHNPNVID
SSYFANVLAKKMPLTVDRLLGLDSKTTPIIKNMLNKPNDFMPTFAKAMEKLSVLKVITGK
DG
553 53 MDRNPVAKNAGKFMTLAGVLDYAKASNISGILIGIEHAAYLATRGLDVVDAVSNALIKSG
YDKETKQQVFIQSEDPPVLSAFKKFPKFNRVFEIEFDIRDVSKPSVVEIKEFANAVKLRRS SAAQVDGFYLTGFNAVVERLRDADIQVHVGVLKNEFMSLAFDYWADPMVEIATDTWSV LADGLVTEFPSTAAAYFRSPCSDIKRNMSYTIKPGEPGALVDMAAYGALPPAPPPAPVLEP ADVHRQPLPLCPTEPMFRTFRCRLPPKETGKNAEYTANLAADG SEQ NTGA Phi p wild type sequence (SEQ ID NO 520-565)
ID No
No
554 56 MARLLFPLPIAAAAVSASSIHLAASRFRLPVVSAARRGTLFGGRVAVRAPARLATRGVSA
GAEAGGSAARAGTVIGPEEALEWVKNDRRRLLHVVYRVGDLDKTIKFYTECLGMKLLRK
RDIPEERYTNAFLGYGPEDSHFVVELTYNYGVESYDIGSGFGHFGIAVEDVEKTVELIKAK
GGTVTREPGPVKGGKSVIAFIEDPDGYKFELIERGPTPEPLCQVMLRVGDLDRAIKFYEKA
FGMELLRRKDNPQYKYTIAM MGYGPEDKNAVLELTYNYGVKEYDKGNAYAQIAIGTDDV
YKTAEVVRQNGGQITREPGPLPGISTKITACTDPDGWKSVFVDNLDFLKEL.EE
555 62 MRRLSLILLAAAALLAAAVSAEPGPAPKLSPDFYSQTCPRAERIIAEVVQSKQMANPTTAA
GVLRVFFHDCFVSGCDASVLIAPTHYAKSEKDADIN HSLPGDAFDAVVRSKLALELECPG
VVSCADILAIASRVLVTMTGGPRYPVPLGRKDSLSSNPAAPDVELPHSNFTVGRIIELFTA
KGFTVQEMVALSGAHTLGFSHCQEFASRIYNYRDKGGKPAPFDPSMNPTYAKGLQAAC
QDYQKDPTIAAFNDIMTPGKFDN MYYVNIERGLGLLSTDEDMWSDMRTKPFVQRYAAN
NTDFFEDFAKAIEKLSMYGVKTGADGEIRRRCDAFNSGPNIQ
556 65 AMAVDLTPRQPTKAYGGDGGAYYEWSPAELPMLGVASIGAAKLSLAAGGMSLPSYSDS
AKVAYVLQGKGTCGIVLPEATKEKVVAIKEGDALALPFGVVTWWHNTPESSTELVVLFLG
DTSKGHTPGKFTNFQLTGATGIFTGFSTEFVARAWDLDQDAAASLVSTQPGTGIVKLAP
GHKMPVARAEDRKGMALNCLEAKLDVDIPNGGRVVVLNTVNLPLVKEVGLGADLVRIDA
HSMCSPGFSCDSAYQVTYIVRGSGRVQVVGPDGKRVLETRIEGGSLFIVPRFHVVSKIA
DASGMEWFSIITTPNPIFSHLAGKTSVWKAISPEVLEAAFNTTPEMEKLFRSKRLDSEIFF
APS
557 73 MSSAKQVLEPAFQGAGQKPGTEIWRIENFNPVPLPKSDYGKFYCGDSYIVLQTTCN KGG
AYLFDIHFWIGKDSSQDEAGTSAIKTVELDTMLGGRAVQHREPQGYESDKFLSYFKPCII
PLEGGFASGFKTPEEEKFETRLYICKGKRAIRVKEVPFARSSLNHDDVFILDTEKKIYQFN
GANSNIQERAKALEVIQHLKDKYHEGVCDVAIVDDGKLQAESDSGEFWVVFGGFAPIGK
KTVSDDDVILETSPTKLYSIN NGKLKLEDIVLTKSILENTKCFLLDCGSELFVWVGRVTQV
DDRKAASAAVEEFIVKQNRPKTTRVTQVIQGYENHTFKSLFESWPVSSTGNASTEEGRG
KVAALLKKKGDVKGASKNSTPVNEEVPPLLEGSGKLEVWCVDGSAKTALPKEDLGKFHS
GDCYIVLYTYHSGEKREEFYLTYWIGKDSVLEDQHMALQIATTIWNSM KGRPVLGRIYQ
GKEPPQFIALFQPMVILKGGISSGYKKSIEENGLKDETYSGTGIALVHIHGTSIHN N KTLQ
VDAVSISLSSTDCFVLQSGNSMFTWIGNTSSYEQQQWAAKVAEFLKPGASVKHCKEGT
ESSAFWSALGGKQNYTSKNATQDVLREPHLYTFSFRNGKLEVTEVFNFSQDDLLTEDVM
ILDTHAEVFVWMGQCVDTKEKQTAFETGQKYVEHAVNFEGLSPDVPLYKVSEGNEPCFF
RTYFSWDNTRSVIHGNSFQKKLSLLFGMRSESGSKGSGDGGPTQRASALAALSSAFNP
SSQDKQSNDRPKSSGDGGPTQRASALAALSSSLNPSSKPKSPHSQSRSGQGSQRAAA
VAALSNVLTAEGSTLSPRNDAEKTELAPSEFHTDQDAPGDEVPSEGERTEPDVSQEETA
NENGGETTFSYDRLISKSTDPVRGIDYKRRETYLSDSEFETVFGVTKEEFYQQPRWKQEL
QKRKADLF
558 76 MASHIVGYPRMGPKRELKFALESFWDGKSSAEDLEKVATDLRASIWKQMSEAGIKYIPS
NTFSYYDQVLDTTAMLGAVPDRYSWTGGEIGHSTYFSMARGNATVPAMEMTKWFDTNY HFIVPELGPETKFSYASHKAVSEYKEAKALGVDTVPVLVGPVSYLLLSKAAKGVEKSFSLL SLLGGILPIYKEVVAELKAAGASWIQFDEPTLVKDLAAHELAAFSSAYAELESSLSGLNVLI ETYFADVPAESYKTLTSLSGVTAYGFDLVRGTKTLDL-
LKSVGIPSGKYLFAGVVDGRNIWADDLAASLSTLESLEAIVGKDKLVVSTSCSLMHTAVD
LVNETKLDSEIKSWLAFAAQKVVEVNALGKALVGLKDEAYFAANAAAQASRRSSPRVN N
EEVQKAAAALKGSDHRRATTVSARLDAQQKKLNLPVLPTTTIGSFPQTMDLRRVRREYK
AKKISEEAYVSAIKEEISKVVKIQEELDIDVLVHGEPERNDMVEYFGEQLSGFAFTANGW
VQSYGSRCVKPPIIYGDVSRPNPMTVFWSKMAQSMTPRPMKGMLTGPV
559 77 QEVAGDVRMTDTRADEAERGITIKSTGISLYYEMSEESLASYKGDRDGNDYLINLIDSPG
HVDFSSEVTAALRITDGALVVVDCIEGVCVQTETVLRQALGERIRPVLTVN KMDRCFLEL
QVDGEEAYQTFSRVIENANVIMATYEDALLGDVQVYPEKGTVAFSAGLHGWAFTLTNFA
KMYASKFGVDESKM MERLWGENFFDPATKKWTSKNTGSGTCKRGFVQFCYEPIKQIIEI
CM NDQKDKLWPMLKKLGVTMKN DEKDLMGKALMKRVMQAWLPASRALLEMMVYHLP
SPSKAQRYRVENLYEGPLDDVYANAIRNCDPEGPLMLYVSKMIPASDKGRFFAFGRVFA
GRVATGM KVRIMGPNFVPGQKKDLYTKSVQRTVIWMGKKQESVEDVPCGNTVALVGLD
QFITKNATLTGEKEVDACPIRAM KFSVSPVVRVAVQCKVASDLPKLVEGLKRLAKSDPMV
LCSIEESGEHIIAGAGELHLEICLKDLQDDFMGGAEIIVSPPVVSFRETVLDKSCRTVMSK
SPN KHNRLYMEARPLEEGLPEAIDEGRIGPRDDPKVRSKILSEEFGWDKDLAKKIWCFGP
ETTGPN MVVDMCKGVQYLNEIKDSVVAGFQWASKEGALADEN MRGICFEVCDVVLHTD SEQ NTGA Phi p wild type sequence (SEQ ID NO 520-565)
ID No
No
AIHRGGGQVIPTARRVIFASQLTAKPRLLEPVYLVEIQAPEGALGGIYGVLNQKRGHVFEE MQRPGTPLYNIKAYLPVIESFGFSATLRAATSGQAFPQCVFDHWDVMNSDPLEVDSQSF NLVKEIRKRKGLKEQMTPLSDFEDKL
560 86 MSTAEATREENVYMAKLAEQAERYEEMVEFMEKVAKTADVGELTVEERNLLSVAYKNVI
GARRASWRIISSIEQKEESRGNEAYVASIKEYRTRIETELSKICDGILKLLDSHLVPSATA
AESKVFYLKMKGDYHRYI_AEFKAGAERKEAAENTLVAYKSAQDIALADLPTTHPIRLGLAL
NFSVFYYEILNSPDRACNL.AKQAFDEAIAELDSLGEESYKDSTLIMQLLRDNLTLWTSDN
ADEGGDEIKEASKPEGEGH
561 86/51 MSPAEPTREESVYMAKLAEQAERYEEMVEFMERVAKATGGAGPGEELSVEERNLLSVAY
KNVIGARRASWRIISSIEQKEEGRGNDAHATTIRSYRSKIEAELAKICDGILALLDSHLVP
SAGAAESKVFYLKMKGDYHRYLAEFKSGAERKEAAESTMNSYKAAQDIALADLAPTHPIR
LGLALNFSVFYYEILNSPDRACNLAKQAFDEAISELDSLGEESYKDSTLIMQLLRDNLTLW
TSDTNEDGGDEIKEAPAPKESGD
562 87 MAVKVYVVYYSMYGHVGKLAEEIKKGASSVEGVEVKVWQVPEILSEEVLGKMGAPPKTD
VPIISPQELAEADGILFGFPTRFGMMASQMKAFFDATGGLWREQSLAGKPAGVFFSTGT QGGGQETTPLTAVTQLTHHGMVFVPVGYTFGAKMFDMEKVQGGSPYGAGTFAGDGSR WPSEMELEHAFHQGKYFAGIAKKLKGS
563 89 MSAADKVKPAASPAAEDPAAIAGNISYHAHYSPHFSPLAFGPEPAYFATAESVRDHLLQR
WNDTYLHFHKTDPKQTYYLSMEYLQGRALTNAVGNLGITGAYAEAVKKFGYELEALAGQ
ERDMALGNGGLGRLAACFLDSMATLNLPAWGYGLRYRYGLFKQRIAKEGQEEIAEDWLE
KFSPWEIVRHDVVYPVRFFGHVEILPDGRRKSAGGEVLNALAYDVPIPGYKTKNAISLRL
WDAKASAEDFNLFQFNDGQYESAAQLHSRAQQICAVLYPGDATEEGKLLRLKQQFFLCS
ASLQDIIFRFKERKSDRVSGKWSEFPSKVAVQMNDTHPTLAIPELMRLLMDEEGLGWDE
AWDVTNKTVAYTNHTVLPEALEKWSQSVMRKLLPRQMEIIEEIDKRFREMVISTRKDME
GKLDSMSVLDNSPQKPVVRMANLCVVSAHTVNGVAELHSNILKEELFADYVSIWPKKFQ
NKTNGITPRRWLRFCNPELSEIVTKWLKTDQWTSNLDLLTGLRKFADDEKLHAEWAAAK
LASKKRLAKHVLDATGVTIDPTSLFDIQIKRIHEYKRQLMNILGAVYRYKKLKEMSAEEKQ
KVTPRTVMVGGKAFATYTNAKRIVKLVNDVGAVVNNDPDVNKYLKVVFIPNYNVSVAEV
LIPGSELSQHISTAGMEASGTSNMKFSLNGCVIIGTLDGANVEIREEVGEDNFFLFGAKA
DQVAGLRKDRENGLFKPDPRFEEAKQYIRSGTFGTYDYTPLLDSLEGNSGFGRGDYFLV
GYDFPSYIDAQARVDEAYKDKKRWIKMSILNTAGSGKFSSDRTIDQYAKEIWGITANPV
P
564 91 MAANPRVFFDVTIGGAPAGRIVMELYADVVPKTAENFRALCTGEKGVGKMGKPLHYKGS
SFHRVIPGFMCQGGDFTAGNGTGGESIYGAKFADENFVKKHTGPGVLSMANAGPGTNG SQFFLCTAKTAWLDGKHVVFGQVVEG
565 91 AANPRVFFDVTIGGAPAGRIVMELYADVVPKTAENFRALCTGEKGVGKMGKPLHYKGSS
FHRVIPGFMCQGGDFTAGNGTGGESIYGAKFADENFVKKHTGPGVLSMANAGPGTNGS QFFLCTAKTAWLDGKHVVFGQVVEGMDVVKAVEKVGSQSGRCSKPVVIADCGQL
Table 3
Table 3 shows full length sequences of homologs to NTGA's of Table 2 found in other grass pollen species.
Figure imgf000062_0001
KEKSCNALLLKVNQIGSVTESIEAVKMSKHAGWGVMTSHRSGETEDTFIADLAVGLA TGQIKTGAPCRSERLAKYNQLLRIEEELGAAAVYAGAKFRAPVEPY
TKSGDGDQTISGPQKRSSRAVRAPSSFLPVCLLRPLPPRDGRPPSSGSLPPKLPRGAG
PGTKSSAPMASPALISDTDQWKALQAHVGAIHKTHLRDLMADADRCKALTAEFEGVF
LDYSRQQATTETVDKLFKLAEAAKLKEKIAKMFNGDKINSTENRSVLHVALRAPRDAV
INSDGVNVVPEVWAVIDKIKQFSETFRSGSWVGATGKPLTNVVSVGIGGSFLGPLFV
HTALQTDPVAAESAKGRQLRFLANVDPVDVARSIKDLDPETTLVVVVSKTFTTAETML
567 2 Ant_o NARTIKEWIVSSLGPQAVSKHMIAVSTNLKLVKEFGIDPNNAFAFWDWVGGRYSVC
SAVGVLPLSLQYGFPVVQKFLEGASSIDNHFRTSSFEKNIPVLLGLLSVWNVSFLGYPA
RAILPYSQALEKLAPHIQQLSMESNGKGVSIDGVRLPYEAGEIDFGEPGTNGQHSFYQ
LIHQGRVIPCDFIGVIKSQQPVYLKGETVSNHDELMSNFFAQPDALAYGKTPEQLRSE
NVSENLIPHKTFQGNRPSLSFLLSSLSAYEIGQLLAIYEHRIAVQGFIWGINSFDQWG
VELGKSLASQVRKQLHASRMEGKPIEGFNPSSASLLARYLAVEPSTPYDTTVLPKV
TKSGDGDQTISGPQKRSSRAVRAPSSFLPVCLLRPLPPRDGRPPSSGSLPPKLPRGAG
PGTKSSAPMASPALISDTDQWKALQAHVGAIHKTHLRDLMADADRCKALTAEFEGVF
LDYSRQQATTETVDKLFKLAEAAKLKEKIAKMFNGDKINSTENRSVLHVALRAPRDAV
INSDGVNVVPEVWAVIDKIKQFSETFRSGSWVGATGKPLTNVVSVGIGGSFLGPLFV
HTALQTDPVAAESAKGRQLRFLANVDPVDVARSIKDLDPETTLVVVVSKTFTTAETML
568 2 Ant_o NARTIKEWIVSSLGPQAVSKHMIAVSTNLKLVKEFGIDPNNAFAFWDWVGGRYSVC
SAVGVLPLSLQYGFPVVQKFLEGASSIDNHFRTSSFEKNIPVLLGLLSVWNVSFLGYPA
RAILPYSQALEKLAPHIQQLSMESNGKGVSIDGVRLPYEAGEIDFGEPGTNGQHSFYQ
LIHQGRVIPCDFIGVIKSQQPVYLKGETVSNHDELMSNFFAQPDALAYGKTPEQLRSE
NVSENLIPHKTFQGNRPSLSFLLSSLSAYEIGQLLAIYEHRIAVQGFIWGINSFDQWG
VELGKSLASQVRKQLHASRMEGKPIEGFNPSSASLLARYLSVEPSTPFDTTVLPKV
AGVRTHFYRAAVRSAYAGRGCPHRPHQPNIQFKGRGVYVYHHHHYRRLPTGTRRKE
AIQNPRKLAGGEEQIRFLFQRSTLHPRRPADEAMASPALICDTEQWKALQAHVSAIQK
THLRDLMADADRCKAMTAEFEGIFLDYSRQQATGETMEKLLKLAEAAKLKEKIEKMFK
GDKINSTENRSVLHVALRAPRDAVINSDGVNVVPEVWGVKDKIKQFSETFRSGSWV
GATGKALTNVVSVGIGGSFLGPLFVHTALQTDPEAAECAKGRQLRFLANVDPVDVAR
SIKDLDPETTLVVVVSKTFTTAETMLNARTLKEWIVSSLGPQAVSKHMIAVSTNLKLV
569 2 Cyn_d
KEFGIDPNNAFAFWDWVGGRYSVCSAVGVLPLSLQYGFPIVQKFLEGASSIDNHFYS
CSFEKNIPVLLGLLSVWNVSFLGYPARAILPYAQALEKFAPHIQQLSMESNGKGVSID
GVKLSFETGEIDFGEPGTNGQHSFYQLIHQGRVIPCDFIGVVQSQRPVYLKGETVSNH
DELMSNFFAQPDALAYGKTPEQLHSEKVPENLIPHKTFQGNRPSLSLLLPTLSAYEIGQ
LLAIYEHRIAVQGFVWGINSFDQWGVELGKSLASQVRKQLHGSRMEGKPVEGFNPS
TSSLLARYLAVKPSTPYDSTVLPKV
MASPALICDTEQWKALQAHVSAIQKTHLRDLMADADRCKAMTAEFEGIFLDYSRQQ
ATGETMEKLLKLAEAAKLKEKIEKMFKGDKINSTENRSVLHVALRAPRDAVINSDGVN
VVPEVWGVKDKIKQFSETFRSGSWVGATGKALTNVVSVGIGGSFLGPLFVHTALQTD
PEAAECAKGRQLRFLANVDPVDVARSIKDLDPETTLVVVVSKTFTTAETMLNARTLKE
WIVSSLGPQAVSKHMIAVSTNLKLVKEFGIDPNNAFAFWDWVGGRYSVCSAVGVLP
570 2 Cyn_d
LSLQYGFPIVQKFLEGASSIDNHFYSCSFEKNIPVLLGLLSVWNVSFLGYPARAILPYA
QALEKFAPHIQQLSMESNGKGVSIDGVKLSFETGEIDFGEPGTNGQHSFYQLIHQGR
VIPCDFIGVVQSQRPVYLKGETVSNHDELMSNFFAQPDALAYGKTPEQLHSEKVPENL
IPHKTFQGNRPSLSLLLPTLSAYEIGQLLAIYEHRIAVQGFVWGINSFDQWGVELGKS
LASQVRKQLHGSRMEGKPVEGFNPSTSSLLARYLAVKPSTPYDSTVLPKV
LLRRSSPFHRHRSPAARRRHPPLARPTSPRRSAMASPALISDTDQWKALQAHVGAIH
KTHLRDLMADADRCKAMTAEFEGIHLDYSRQQATTETVDKLFKLAEAAKLKEKIEKMF
SGDKINTTENRSVLHVALRAPRDAVINSDGVNVVPEVWAVIDKIKQFSETFRSGSWV
GATGKPLTNVVSVGIGGSFLGPLFVHTALQTDPAAAESAKGRQLRFLANVDPVDVAR
SIKDLDPATTLVVVVSKTFTTAETMLNARTIKEWIVSSLGPQAVSKHMIAVSTNLKLV
571 2 LoLp KEFGIDPNNAFAFWDWVGGRYSVCSAVGVLPLSLQYGFPIVQKFLEGASSIDNHFRT
SSFEKNIPVLLGLLSVWNVSFLGYPARAILPYTQALEKLAPHIQQLSMESNGKGVSID
GVRLPYEAGEIDFGEPGTNGQHSFYQLIHQGRVIPCDFIGVIKSQQPVYLKGETVSNH
DELMSNFFAQPDALAYGKTPEQLRSENVSENLIPHKTFQGNRPSLSFLLSSLSAYEIG
QLLSIYEHRIAVQGFIWGINSFDQWGVELGKSLASQVRKQLHASRMEGKPVEGFNPS
SASLLARYLAVEPSIPYDTTVLPKV
QIRHGHSPVRSSPIHIPPPPPVSFSASSLLLSPSAPINPLPPPPIRRQPAPRHPRRHILA
572 2 Poa_p GPLRGSMASPALISDTDQWKALQAHVGAIHKTHLRDLMADADRCKAMTVEFEGVFL
DYARQQATTETVDKLFKLAEAAKLKEKIEKMFSGEKINSTENRSVLHVALRAPRDAVI NSDGVNVVPEVWSVKDKIKQFSETFRSGSWVGATGKPLTNVVSVGIGGSFLGPLFV
HTALQTDPEAAESAKGRQLRFLANVDPVDVARSIKDLDPETTLVVVVSKTFTTAETML
NARTIKEWIVSSLGPQAVSKHMIAVSTNLKLVKEFGIDPN NAFAFWDWVGGRYSVC
SAVGVLPLSLQYGFPIVQKFLEGASSIDNHFRTASFEKNIPVLLGLLSVWNVSFLGYPA
RAILPYSQALEKLAPHIQQVSMESNGKGVSIDGVPLPYEAGEIDFGEPGTNGQHSFYQ
LIHQGRVIPCDFIGVIKSQQPVYLKGETVSNHDELMSNFFAQPDALAYGKTPEQLRSE
NVSENLIPHKTFKGNRPSLSFLLSSLSAYEIGQLLAIYENRIAVQGFIWGINSFDQWGV
ELGKSLASQVRKQLHASRMEGKPIEGFNPSSASLLARYLAVEPSTPYDTTVLPKV
KRTVVLGPQVPLKEQLELDKDDESLRRWKEQLLGQVDTEQLGETAEPEVKVLNLTILS PGRPDLVLPIPFQPDEKGYAFALKDGSPYSFRFSFIVSN NIVSGLKYTNTVWKTGVRV
573 3 Cyn_d
ENQKMMLGTFSPQLEPYVYEGEEETTPAGMFARGSYSAKLKFVDDDGKVYLEMSYYF EIRKEWPAA
MVLFTVTKKATTPFEGQKPGTSGLRKKVTVFQQPNYLQNFVQATFNALPADQVKGATI
VVSGDGRYFSKDAVQIITKMAAANGVRRVWVGQNSLMSTPAVSCVIRDRVGSDGS
KATGAFILTASHNPGGPTEDFGIKYN MGNGGPAPESVTDKIFSNTKTISEYLISEDLPD
VDISVVGVTSFSGPEGPFDVDVFDSSVDYIKLM KSIFDFEAIKNLVTSPKFTFCYDALH
GVAGAYAKQIFVEELGADESSLLNCVPKEDFGGGHPDPNLTYAKELVERMGLGKSTS
574 4 Cyn_d NVEPPEFGAAADGDADRN MILGKRFFVTPSDSVAIIAANAVQSIPYFSSGLKGVARSM
PTSAALDVVAKNLNLKFFEVPTGWKFFGNLMDAGMCSICGEESFGTGSDHIREKDGI
WAVLAWLSILAFKN KDNLRGDKLVSVEDIVRQHWATYGRHYYTRYDYENVDAGAAK
ELMANLVSMQSSLSDVN KLIKEIRSDVSDVVAADEFEYKDPVDGSVSKHQGIRYLFG
DGSRLVFRLSGTGSVGATIRVYIEQYEKDSSKIGRESQDALAPLVDVALKLSKMQEYT
GRSAPTVIT
KSYPAVSEDYLKAVDKAKRKLRGLIAEKNCAPLILRLAWHSAGTFDVATKSGGPYGT
M KN PSEQAHAANAGLDIAVRLLEPIKEQFPILSYADFYQLAGVVAVEVTGGPDVPFHP
575 5/64 Cyn_d GREDKPEPPPEGRLPDATKGSDHLRQVFATQMGLSDQDIVALSGGHTLGRCHKDRS
GFEGAWTSNPLIFDNSYFKELLSGEKEGLLQLPSDKALLSDPSFRPLVEKYAADEDAFF
ADYAEAHLKLSELGFAE
MAKNYPTVSAEYQEAVEKARRKLRALIAEKSCAPLMLRLAWHSAGTFDVSTKTGGPF GTM KN PAEQAHGANAGLDIAVRMLEPVKEEFPILSYADLYQLAGVVAVEVTGGPEIPF
576 5/64 Cyn_d HPGREDKPQPPPEGRLPDATKGTDHLRQVFGKQMGLSDQDIVALSGGHTLGRCHKE
RSGFEGPWTRNPLCFDNSYFTELLTGDKEGLLQLPSDKALLNDPVFRPLVEKYAADEK AFFEDYKEAHLRLSELGFADA
PHPTSDRPSSILSSPSARTTHLATMADEKLAKLREAVAGLGQISDNEKSGFISLVSRYL
SGDEEHIEWPKIHTPTDEVVVPYDTIDAPPEDLEATKALLN KLAVLKLNGGLGTTMGC
TGPKSVIEVRNGFTFLDLIVLQIESLN KKYGSNVPLLLM NSFNTHDDTLKIVEKYANSS
IDIHTFNQSQYPRVVADEFLPWPSKGKTDKDGWYPPGHGDIFPSLM NSGKLDLLLSQ
577 6 Ant_o GKEYVFIANSDNLGAIVDM KILNHLIHKQNEYCMEVTPKTLADVKGGTLISYEGRVQL
LEIAQVPDAHVDEFKSIEKFKIFNTN NLWVNLKAIKRLVEADALKMEIIPNPKEVEGVK
VLQLETAAGAAIRFFDHAIGINVPRSRFLPVKATSDLQLVQSDLYTLVDGFVTRNSART
DPSNPSIELGPEFKKVGSFLGRFKSIPSIVELDSLKVSGDVWFGSGIVLKGKVTITAKP
GVKLEIPDGAVLEN KDIKGAEDL
PTPSSSSHLPVSSPLPDLSAHLAMADEKLAKLSEAVAGLAEISENEKSGFLSLVSRYLS
GDEEHIEWAKIHTPTDEVVVPYDALETPPEDIEETKKLLDKLAVLKLNGGLGTTMGCT
GPKSVIEVRNGFTFLDLIVLQIEALN KKYGSNVPLLLM NSFSTHDDTLKIVEKYANSNI
DIHTFNQSKYPRVVADEFLPWPSKGKTCKDGWYPPGHGDIFPSLM NSGKLDLLLSQG
578 6 Cyn_d KEYVFIANSDNLGAIVDM KILNHLIHKQNEYCMEVTPKTLADVKGGTLISYEGRVQLL
EIAQVPDAHVHEFKSIEKFKIFNTN NLWVNLKAIKRLVEADALKMEIIPNPKEVDGVKV
LQLETAAGAAIRFFDHAIGINVPRSRFLPVKATSDLQLVQSDLYTLVDGLVTRNEARTN
PSNPSIELGPEFKKVGNFLGRFKSIPSIVELDSLKVSGDVWFGSGIVLKGKVSITAKPG
VKLEIPDGAVIEN KDISGPEDL
MADEKLAKLSEAVAGLAEISENEKSGFLSLVSRYLSGDEEHIEWAKIHTPTDEVVVPY
DALETPPEDIEETKKLLDKLAVLKLNGGLGTTMGCTGPKSVIEVRNGFTFLDLIVLQIE
ALN KKYGSNVPLLLMNSFSTHDDTLKIVEKYANSNIDIHTFNQSKYPRVVADEFLPWP
SKG KTCKDGWYPPGHGDIFPSLMNSGKLDLLLSQGKEYVFIANSDNLGAIVDM KILN
579 6 Cyn_d
HLIHKQNEYCMEVTPKTLADVKGGTLISYEGRVQLLEIAQVPDAHVHEFKSIEKFKIFN
TN NLWVNLKAIKRLVEADALKMEIIPNPKEVDGVKVLQLETAAGAAIRFFDHAIGINVP
RSRFLPVKATSDLQLVQSDLYTLVDGLVTRNEARTNPSNPSIELGPEFKKVGN FLGRF
KSIPSIVELDSLKVSGDVWFGSGIVLKGKVSITAKPGVKLEIPDGAVIEN KDISGPEDL LISYEGKVQLLEIAQVPDEHVNEFKSIEKFKIFNTNNLWVNLNAIKRLVQADALKMEII
580 6 LoLp PNPKEVDGIKVLQLETAAGAAIKFFDRAIGINVPRSRFLPVKATSDLLLVQSDLYTLSD
GFVTRNPARTNPANPSIELGPE
SPSPTSDDPPLPFPQKHLPPHVHATMADEKLAKLREAVAGLGQISDNEKSGFISLVSR
YLSGDEEHIEWPKIHTPTDEVVVPYDTIDAPPEDLEATKALLNKLAVLKLNGGLGTTM
GCTGPKSVIEVRNGFTFLDLIVLQIESLNKKYGSNVPLLLMNSFNTHDDTLKIVEKYAN
SSIDIHTFNQSQYPRVVADEFLPWPSKGKTDKDGWYPPGHGDIFPSLMNSGKLDLLL
581 6 LoLp SQGKEYVFIAN SDN LGAIVDM KILN HLIHKQNEYCMEVTPKTLADVKGGTLISYEGRV
QLLEIAQVPDAHVDEFKSIEKFKIFNTNNLWVNLKAIKRLVEADALKMEIIPNPKEVEG
VKVLQLETAAGAAIRFFDHAIGMNVPRSRFLPVKATSDLQLVQSDLYTLVDGFVTRNS
ARTDPSNPSIELGPEFKKVGSFLGRFKSIPSIVELDSLKVSGDVWFGSGIVLKGKVTIT
AKPGVKLEIPDGKVIENKDINGVEDL
THHHHHLTTSSHLKSPPVLSSSSASRSLLCLPARIAMAATAVAAGPDAKIEKFRDAVA
KLDEISENEKAGCISLVSRYLSGEAEQIEWSKIQTPTDEVVVPYDTLAPAPEDLDAMK
ALLDKLVVLKLNGGLGTTMGCTGPKSVIEVRNGFTFLDLIVIQIESLNKKYGCDVPLLL
MNSFNTHDDTQKIVEKYSNSNINIHTFNQSQYPRIVTEDFLPLPSKGKSGKDGWYPP
582 6 LoLp GHGDVFPSLNNSGKLDTLLSQGKEYVFVANSDNLGAIVDIKILNHLINNQNEYCMEV
TPKTLADVKGGTLISYEGRVQLLEIAQVPDEHVNEFKSIEKFKIFNTNNLWVNLKAIKR
LVEADALKMEIIPNPKEVDGVKVLQLETAAGAAIRFFEKAIGINGPRSRFLPVKATSDL
LLVQSDLYTLVDGYVIRNPARVKPSNPSIELGPEFKKVASFLARFKSIPSIVELDSLKVS
GDVSFGSGVVLKGNVTIAAKSGVKLEIPDGSVLENKDINGPEDL
THHHHHLTTSSHLKSPPVLSSSSASRSLLCLPARIAMAATAVAAGPDAKIEKFRDAVA
KLDEISENEKAGCISLVSRYLSGEAEQIEWSKIQTPTDEVVVPYDTLAPAPEDLDAMK
ALLDKLVVLKLNGGLGTTMGCTGPKSVIEVRNGFTFLDLIVIQIESLNKKYGCDVPLLL
MNSFNTHDDTQKIVEKYSNSNINIHTFNQSQYPRIVTEDFLPLPSKGKSGKDGWYPP
583 6 LoLp GHGDVFPSLNNSGKLDTLLSQGKEYVFVANSDNLGAIVDIKILNHLINNQNEYCMEV
TPKTLADVKGGTLISYEGRVQLLEIAQVPDEHVNEFKSIEKFKIFNTNNLWVNLKAIKR
LVEADALKMEIIPNPKEVDGVKVLQLETAAGAAIRFFEKAIGINGPRSRFLPVKATSDL
LLVQSDLYTLVDGYVIRNPARVKPSNPSIELGPEFKKVASFLARFKSIPSIVELDSLKVS
GDVTFGSGVVLKGNVTIAAKSGVKLEIPDGAVLENKDINGPEDL
DLQLVQSDLYTLVDGLVTRNEARTNPSNPSIELGPEFKKVGNFLGRFKSIPSIVELDSL
584 6 Poa_p
KVSGDVWFGSGIILKGKVTIT
VNVAAFPHFPPATCSSLFSGINSQRHLLLLPPSTLLFPHIYLPLPSVRTRTHLAATMADE
KLAKLGEAVTGLPQISDNEKSGFISLVSRYLSGDEEHIEWPKIHTPTDEVVVPYDAIDA
PPEDLEATKALLDKLAVLKLNGGLGTTMGCTGPKSVIEVRNGFTFLDLIVLQIESLNKK
YGSNVPLLLMNSFNTHDDTLKIVEKYANSSIDIHTFNQSQYPRVVADEFLPWPSKGKT
585 6 Poa_p DKDGWYPPGHGDIFPSLMNSGKLDLLLSQGKEYVFIANSDN LGAIVDM KILN HLIHK
QNEYCMEVTPKTLADVKGGTLISYEGRVQLLEIAQVPDAHVDEFKSIEKFKIFNTNNL
WVNLKAIKRLVEADALKMEIIPNPKEIDGVKVLQLETAAGAAIRFFDHAIGINVPRSRF
LPVKATSDLQLVQSDLYTLVDGFVTRNSARTDPSNPSIELGPEFKKVGSFLGRFKSIPS
IVELESLKVSGDVWFGSGIVLKGKVTITAKPGVKLEIPDGAVLENKDINGAEDL
CRRPPTHLPRLAPLRSRSPRQAAPAEAAMAFEKIKVANPIVEMDGDEMTRVFWQSIK
DKLIFPFLDLDIKYYDLGVLHRDATDDKVTVEAAEATLKYNVAIKCATITPDEDRVKEF
NLKQMWRSPNGTIRNIINGTVFREPIICKNVPKLVPGWTKPICIGRHAFGDQYRATDA
VLKGPGKLRLVFEGKDETVDLEVFNFTGAGGVALAMYNTDESIQGFAEASMAIAYEKK
586 7 Ant_o
WPLYLSTKNTILKKYDGRFKDIFQAVYEAGWKSKYEAAGIWYEHRLIDDMVAYALKS
EGGYVWACKNYDGDVQSDFLAQGFGSLGLMTSVLMCPDGKTIEAEAAHGTVTRHYR
VHQKGGETSTNSIASIFAWTRGLAHRAKLDDNARLLDFTQKLEDACVGTVESGKMT
KDLALLVHGSSKVTRGDYLNTEEFIDAVAAELQSRLAAN
PTPFHRRRRLPTRLAARPFPISEASCAVTAAMAFEKIKVANPIVEMDGDEMTRVFWKS
IKDKLIFPFLDLDIKYYDLGILHRDATDDKVTVEAAEATLKYNVAIKCATITPDETRVKE
FNLKHMWRSPNGTIRNIINGTVFREPIICKNVPRLVPGWTKPICIGRHAFGDQYRATD
AVLKGPGKLKLVFEGKEEQIDLEVFNFTGAGGVALSMYNTDESVRAFAAASMTMAYE
587 7 Cyn_d
KKWPLYLSTKNTILKKYDGRFKDIFQEVYEADWKSKFEAAGIWYEHRLIDDMVAYAL
KSEGGYVWACKNYDGDVQSDFLAQGFGSLGLMTSVLVCPDGKTIEAEAAHGTVTRH
FRVHQKGGETSTNSIASIFAWTRGLAHRAKLDDNARLLDFAQKLEAACVGTVESGKM
TKDLALLVHGSSKVTRSDYLNTEEFIDAVAAELQSRLAAN
RLASPLARLPLPAARVFRGVSLRCYAAAAAVAEQHRIKVDNPIVEMDGDEMTRVIWK
588 7 Cyn_d MIKDKLILPYLDVDLKYYDLGILNRDATDDRVTVESAEATREYNVAVKCATITPDETRV
KEFNLKSMWRSPNGTIRNILNGTVFREPILCKNIPRILSGWKHPICIGRHAFGDQYRA TDMIIDGPGKLKMVFVPDGGAEPVELDVYDFKGPGVALSMYNVDESIRAFAESSMAM
AFSKKWPLYLSTKNTILKTYDGRFKDIFQEVYEENWRGKFEENSIWYEHRLIDDMVAY
AVKSEGGYVWACKNYDGDVQSDFLAQGFGSLGLMTSVLLSSDGKTLESEAAHGTV
MRHFRLHQKGQETSTNSIASIFAWTRGLEHRAKLDKNERLLDFTRKLESACVETVES
GKMTKDLALLIYGPKVTREFYLNTEEFIDAVAHQLREKIQIPAAV
SPTQSRPAMAFNKIKVANPVVEMDGDEMTRVFWKSIKDKLIFPFVDLDIKYFDLGLPH
RDATDDKVTVEAAEATLKYNVAIKCATITPDEARVKEFNLKSMWRSPNGTIRNILNGT
VFREPIICQNIPRLVPGWTKPICIGRHAFGDQYRATDAVIKGPGKLKLVYEGKEEQVEL
EVFNFTGAGGVALAMYNTDESIRSFAEASMATAYEKKWPLYLSTKNTILKKYDGRFKD
589 7 Cyn_d
IFQEVYEAEWRSKYEAAGIWYEHRLIDDMVAYALKSEGGYVWACKNYDGDVQSDFL
AQGFGSLGLMTSVLVCPDGKTMEAEAAHGTVTRHYRVHQKGGETSTNSIASIFAWT
RGLAHRAKLDDNARLLDFTQKLEAACIGAVESGKMTKDLALLVHGSSNVTRSHYLNT
EEFIDAVAEELRSRLGANSNL
GDEMTRVFWKSIKDKLIFPFLDLDIKYYDLGILHRDATDDKVTVEAAEATLKYNVAIKC
ATITPDETRVKEFNLKHMWRSPNGTIRNIINGTVFREPIICKNVPRLVPGWTKPICIGR
HAFGDQYRATDAVLKGPGKLKLVFEGKEEQIDLEVFNFTGAGGVALSMYNTDESVRA
590 7 Cyn_d FAAASMTMAYEKKWPLYLSTKNTILKKYDGRFKDIFQEVYEADWKSKFEAAGIWYEH
RLIDDMVAYALKSEGGYVWACKNYDGDVQSDFLAQGFGSLGLMTSVLVCPDGKTIE
AEAAHGTVTRHFRVHQKGGETSTNSIASIFAWTRGLAHRAKLDDNARLLDFAQKLEA
ACVGTVESGKMTKDLALLVHGSSKVTRSDYLNTEEFIDAVAAELQSRLAAN
KWIKDKLIFPFLDLDIKYYDLGLPNRDATGDKVTIESAEATLKYNVAIKCATVTPDEGR VKEFNLKAMWRSPNGTIRNILNGTVFREPIICKNVPRLVPGWTKPICIGRHAFGDQYR ATDVIIRGPGKLKLVFDGVEEQIELDVFNFNGAGGVALSMYNTDESIRAFAESSMNVA
591 7 LoLp YQKRWPLYLSTKNTILKKYDGRFKDIFQENYEKNWRGKFEKAGIWYEHRLIDDMVAY
ALKSEGGYVWACKNYDGDVQSDLIAQGFGSLGLMTSVLVCPDGRTVEAEAAHGTVT RHYRVHQKGGETSTNSIASIFAWSTGLAHRAKLDDNKRLLDFTQKLEAACVGTVESG KMTKDLALLIHGPTVSRDKYLNTVEFIDAVADELKTSLSVKSKL
LNALAKLVTPFSLLPVPPSPAPPAPFPISQASSSAVAAMAFEKIKVANPIVEMDGDEMT
RVFWQSIKDKLIFPFLDLDIKYYDLGVLHRDATDDKVTVEAAEATLKYNVAIKCATITP
DEDRVKEFNLKQMWRSPNGTIRNIINGTVFREPIICKNVPKLVPGWTKPICIGRHAFG
DQYRATDAVLKGPGKLRLVFEGKDETVDLEVFNFTGAGGVALAMYNTDESIQGFAAA
592 7 LoLp
SMAIAYEKKWPLYLSTKNTILKKYDGRFKDIFQAVYEADWKSKYEAAGIWYEHRLIDD
MVAYALKSEGGYVWACKNYDGDVQSDFLAQGFGSLGLMTSVLMCPDGKTIEAEAAH
GTVTRHFRVHQKGGETSTNSIASIFAWTRGLAHRAKLDDNARLHDFTLKLEEACVGT
VESGKMTKDLALLVHGSSKVTRGDYLNTEEFIDAVAAELKSRLAAN
RRPPHLPRLAAFPISEASIAAADAMAFEKIKVANPIVEMDGDEMTRVFWQSIKEKLIFP
FLDLDIKYYDLGVLHRDATDDKVTVEAAEATLKYNVAIKCATITPDEDRVKEFNLKQM
WRSPNGTIRNIINGTVFREPIICKNVPKLVPGWTKPICIGRHAFGDQYRATDAVLKGP
GKLRLVFEGKDETVDLEVFNFTGAGGVALAMYNTDESIQGFAEASMAIAYEKKWPLYL
593 7 Poa_p
STKNTILKKYDGRFKDIFQAVYEADWKSKYEAAGIWYEHRLIDDMVAYALKSEGGYV
WACKNYDGDVQSDFLAQGFGSLGLMTSVLMCPDGKTIEAEAAHGTVTRHFRVHQK
GGETSTNSIASIFAWTRGLAHRAKLDDNARLLDFTQKLEDACVGTVESGKMTKDLAL
LVHGSSKVTRGDYLNTEEFIDAVAAELQSRLAAN
FSFDEWDAHTRRSGDKTRRRTVRLVAKGADAKPMANANVSIELLRLGFPFGNTMTAE
ILSLPAYEKWFTSRFTHATFENEMKWYSTEWSQNQENYDVPDRMLKMAQKYGIKVR
GHNVFWDDQNSQMRWVKPLNLDQLKSAMQKRLKNVVTRYAGKVIHWDVVNENLH
594 8 Cyn_d FNFFESKLGSSASAQIYNQVGQIDRNAILFMNEFNVLEQPGDPNAVPSKYIAKMNQIR
SYPGNSGLKMGVGLESHFSTPNIPYMRSTLDTLAKLKLPMWLTEVDVVKNPNQVKYL
EQVLREGYAHPNVDGIIMWAAWHAKGCYVMCLTDNNFKNLPVGDLVDKLITEWKTH
RTVATTDENGAVVLDLPLGEYKFTVHHPSLSGTTVDLMTVDGASS
MAQLAETYACSPATERGRGILLAGDPKTDTIAYCTGRSVIIRRLDAPLDAWAYQDHAY
PTTVARFSPNGEWVASADASGCVRVWGRYGDRALKAEFRPLSGRVDDLRWSPDGL
RIVVSGDGKG KSFVRAFVWDSGSTVGEFDGHSKRVLSCDFKPTRPFRIVTCGEDFLA
NFYEGPPFKFKHSIRDHSNFVNCIRYSPDGSKFITVSSDKKGLIYDGKTGEKIGELSSE
GSHTGSIYAVSWSPDSKQVLTVSADKTAKVWDIMEDATGKLNRTLVCTGIGGVDDM
595 9 Cyn_d
LVGCLWQNDHLVTVSLGGTFNVFSASNPDQEPVTFAGHLKTISSLVLFPQSNPRTILS
TSYDGVIMRWIQGVGYGGRLMRKNNTQIKCFAAVEEELVTSGYDNKIFRIPLNGDQC
GDAESVDVGGQPNAVNLAIQKPEFALVTTDSGIILLHNSKVISTTKVDYTITSSSVSP
DGSEAVVGAQDGKLRIYSISGDTLTEEAVLEKHRGAITSIHYSPDVSMFASADANREA
VVWDRATREVKLKNMLYHTARINCLAWSPDSRLVATGSLDTCAIVYEIDKPAASRITI KGAHLGGVRGLTFVDNDTLVTAGEDACIRDWKLVQQ
MVLFTVTKKATTPFEGQKPGTSGLRKKVTVFQQPNYLQNFVQATFNALPADQVKGATI
VVSGDGRYFSKDAVQIITKMAAANGVRRVWVGQNSLMSTPAVSCVIRDRVGSDGS
KATGAFILTASHNPGGPTEDFGIKYNMGNGGPAPESVTDKIFSNTKTISEYLISEDLPD
VDISVVGVTSFSGPEGPFDVDVFDSSVDYIKLMKSIFDFEAIKNLVTSPKFTFCYDALH
GVAGAYAKQIFVEELGADESSLLNCVPKEDFGGGHPDPNLTYAKELVERMGLGKSTS
596 10 Cyn_d NVEPPEFGAAADGDADRNMILGKRFFVTPSDSVAIIAANAVQSIPYFSSGLKGVARSM
PTSAALDVVAKNLNLKFFEVPTGWKFFGNLMDAGMCSICGEESFGTGSDHIREKDGI
WAVLAWLSILAFKNKDNLRGDKLVSVEDIVRQHWATYGRHYYTRYDYENVDAGAAK
ELMANLVSMQSSLSDVNKLIKEIRSDVSDVVAADEFEYKDPVDGSVSKHQGIRYLFG
DGSRLVFRLSGTGSVGATIRVYIEQYEKDSSKIGRESQDALAPLVDVALKLSKMQEYT
GRSAPTVIT
SAMATAWTLPDHPKLPKGKTVAVVVLDGWGEANPDQYNCIHVAQTPVMDSLKNGA
PERWRLVKAHGTAVGLPSDDDMGNSEVGHNALGAGRIFAQGAKLVDSALASGKIYD
GEGFNYIKESFENGTLHLIGLLSDGGVHSRLDQVQLLLKGASERGAKRIRVHILTDGR
DVLDGSSVGFVETLENDLSELREKGIDAQIASGGGRMNVTMDRYENDWGVVKRGW
DAQVLGEAPHKFKSAVEAVKTLRAVPDANDQYLPPFVIVDESGKAVGPIVDGDAVVT
597 11 Cyn_d
FNFRADRMVMLAKALEYADFDKFDRVRVPKIRYAGMLQYDGELLLPKRYLVSPPEIDR
TSGEYLVKNGVRTFACSETVKFGHVTFFWNGNRSGYFDESKEEYVEVPSDSGITFNV
KPKMKAVEIAEKARDAILSGKFDQIRVNLPNGDMVGHTGDIEATVVACKAADEAVKII
LDAVEQVGGIYLVTADHGNAEDMVKRNKAGKPLLDKSGAIQILTSHTLQPVPVAIGG
PGLHPGVKFRSDIETPGLANVAATVMNLHGFEAPADYEPTLIEVAD
M DEEYDVIVLGTGLKECILSGLLSVDGLKVLH MDRN DYYGGESTSLN LTKLWKRFKG
NDNPPEHLGISKQYNVDMIPKFMMANGALVRVLIHTSVTKYLNFKAVDGSFVYNNGK
IHKVPATDVEALKSNLMGLFEKRRARKFFIYVQDYEEEDPKSHEGLDLHKVTTREVIS
KYGLEDDTVDFIGHALALHRDDNYLDEPAIHTVKRMKLYAESLARFQSASPYIYPLYGL
598 13 Cyn_d
GELPQAFARLSAVYGGTYMLNKPECKVEFDENGKAYGVTSEGVTAKCKKVVCDPSYL
PEKVKKVGKVARAICIMKHPIPHTKDSHSVQIILPKKQLKRKSDMYVFCCSYAHNVAP
NGKFIAFVSTEAETDKPEIELKPGIDLLGPVEETFFDIYDRYEPTNNPEEDSCFLTNSYD
ATTHFETTVQDVLSMYNKITGKELDLSVDLNAASATEQE
MAKEPMRVLVTGAAGQIGYALVPMIARGIMLGADQPVILHMLDIPPAAEALNGVKMEL
VDAAFPLLKGVVATTDVVEACTGVNVAVMVGGFPRKEGMERKDVMSKNVSIYKAQA
SALEAHAAPNCKVLVVANPANTNALILKEFAPSIPEKNITCLTRLDHNRALGQISERLN
599 19 Cyn_d
VQVSDVKNVIIWGNHSSTQYPDVNHATVKTPSGEKPVRELVADDEWLNGEFVKTVQ
QRGAAIIKARKLSSALSAASSACDHIRDWVLGTPEGTYVSMGVYSDGSYGVPAGLIY
SYPVTCSGGEWKIVQGLPIDDLSRQKMDATAQELSEEKTLAYSCL
AATRRASHLLGSTASRLLHARGFAAAAAAAPSPAVFVDKSTRVICQGITGKNGTFHTE
QAIEYGTNMVGGVTPKKGGTEHLGLPVFNSVAEAKAETKANASVIYVPPPFAAAAIME
AMDAELDLVVCITEGIPQHDMVKVKAALNRQSKTRLIGPNCPGIIKPGECKIGIMPGYI
600 20 Cyn_d
HKPGRVGIVSRSGTLTYEAVFQTTAVGLGQSTCVGIGGDPFNGTNFVDCLEKFVNDP
QTEGIVLIGEIGGTAEEDAAAFIQESKTEKPVVAFIAGLTAPPGRRMGHAGAIVSGGK
GTAQDKIKALREAGVTVVESPAKIGSKMFEIFKERGMVE
MALPNQQVVDYPSFKLVIVGDGGTGKTTFVKRHLTGEFEKKYEPTIGVEVHPLDFSTN CGKIRFYCWDTAGQEKFGGLRDGYYIHGQCAIIMFDVTSRLTYKNVPTWHRDLCRVC
601 22 Cyn_d
ENIPIVLCGNKVDVKNRQVKAKQVTFHRKKNLQYYEISAKSNYNFEKPFLYLARKLAG DQNLHFVEAVALKPPEVQIDMAMQQQHEAELVAAAAQ
MATKRSVGTLGEADLKGKKVFVRADLNVPLDDAQKITDDTRIRASVPTIKFLLEKGAK VILASHLGRPKGVTPKYSLKPLVPRLSELLGIDVVMANDCIGEEVEKLAAALPEGGVLL LENVRFYKEEEKNDPEFAKKLASVADLYVNDAFGTAHRAHASTEGVTKYLKPAVAGFL
602 24 Cyn_d MQKELDYLVGAVANPKKPFAAIVGGSKVSTKIGVIESLLAKVDILILGGGMIYTFYKAQ
GYAVGKSLVEEDKLDLATSLIEKAKAKGVSLLLPTDIVVADKFAADAESKIVPATSIPD DWMGLDVGPDATKTFNEALDTTQTIIWNGPMGVFEFDKFAAGTEAIAKKLAELTSTK GVTTIIGGGDSVAAVEKAGLADKMSHISTGGGASLELLEGKPLPGVLALDEA
MSAHVGKFADELIKNAAYIGTPGKGILAADESTGTIGKRFSSINVENIEENRRALRELL
FCAPGALQYLSGVILFEETLYQKTKDGKPFVDVLKEGGVLPGIKVDKGTIEVAGTDKE
TTTQGHDDLGKRCAKYYEAGARFAKWRAVLKIGPNEPSQLAIDLNAQGLARYAIICQ
603 27 Cyn_d
ENGLVPIVEPEILVDGPHDIERCAYVTEMVLAACYKALSEHHVLLEGTLLKPNMVTPGS
DAKKVAPEVIAEYTVRALQRTVPAAVPAIVFLSGGQSEEEATLNLNAMNKLNTKKPWS
LSFSFGRALQASTLKAWAGKEENVEKARAALLARCKANSEATLGTYKGDAAAGEGVS ESLHVKDYKY
MAKHFKYVILGGGVAAGYAAREFGKQGVKPGELAIISKEPVAPYERPALSKGYLFPQN
AARLPGFHTCVGSGGERLLPEWYSEKGIELILSTEIVKVDLASKTLTSASEATFTYEILL
IATGSSVIKLTDFGVQGAEYNNILYLRDIQDGEKLVAAMQAKKDGKAVVVGGGYIGL
ELSAALKMNNFDVTMVYPEPWCMPRLFTAGIAHFYEGYYASKGINLVKGTYAAGFDA
604 29 Cyn_d
DSNGDVTAVKLKDGRVLEADIVIVGVGGRPLTGLFKGQVAEEKGGIKTDGFFETSVP
DVYAIGDVATFPMKLYNDQRRVEHVDHARKSAEQAVRAIKAKESGESIAEYDYLPYFY
SRSFDVAWQFYGDNVGDDVLFGDNDPAAAKPKFGSYWVKDGKVVGVFLEGGSADE
YQAIARVARAQPQVADVEALRKDGLDFAIKT
MAGGGVEDAYGEDRATEEQLVTPWAFSVASGYTLLRDPRHNKGLAFSEAERDAHYL
RGLLPPAFASQELQEKKLMHNLRQYTVPLQRYIAMMDLQERNERLFYKLLIDNVEELLP
VVYTPTVGEACQKYGSIYRRPQGLYISLKDKGKILEVLKNWPERSIQVIVVTDGERILG
LGDLGCQGMGIPVGKLSLYTALGGVRPSACLPITIDVGTNNETLLNDEFYIGLRQRRA
TGEEYHELLEEFMTAVKQNYGEKVLIQFEDFANHNAFDLLAKYSKSHLVFNDDIQGTA
605 30 Cyn_d
SVVLAGLLASLKVVGGSLADHTYLFLGAGEAGTGIADLIALEMSKHNEMPIDECRKKI
WLVDSKGLIVESRKESLQHFKKPWAHEHEPLKTLLEAVESIKPTVLIGTSGVGRTFTK
EVIEAMASFNEKPVIFSLSNPTSHSECTAEEAYTWTQGRAVFASGSPFDPVEYEGKVY
VPGQSNNAYIFPGFGLGVVISGAIRVHDDMLLAASEALAEQVTEEHFGKGLIFPSFTNI
RGISARIAAKVAAKAYELGLASHLPRPDDLVKYAESCMYTPAYRSYR
MAKIKIGINGFGRIGRLVARVALQSDDVELVAVNDPFITTDYMTYMFKYDTVHGQWK
HHDVKVKDSKTLLFGEKEVTVFGCRNPEEIPWGEAGAEYVVESTGVFTDKDKAAAHL
KGGAKKVVISAPSKDAPMFVCGVNEKEYKSDIHIVSNASCTTNCLAPLAKVINDKFGI
606 32 Cyn_d
VEGLMTTVHAITATQKTVDGPSAKDWRGGRAASFNIIPSSTGAAKAVGKVLPALNGK
LTGMAFRVPTVDVSVVDLTVRLEKSATYDEIKAAIKAESEGDLKGILGYVEEDLVSTDF
QGDNRSSIFDAKAGIALNDKFVKLVSWYDNEWGYSSRVIDLIRHMHST
SSVAIWVLFPSEIVISVPVDSRGERAMATAGKVIKCKAAVAWEAGKPLSIEEVEVAPP
QAMEVRVKILFTSLCHTDVYFWEAKGQTPVFPRIFGHEAGGIVESVGEGVTDVAPGD
HVLPVFTGECKECPHCKSAESNMCDLLRINTDRGVMISDGKSRFSIDGKPIYHFVGTS
TFSEYTVMHVGCVAKINPEAPLDKVCVLSCGISTGLGASINVAKPPKGSTVAIFGLGA
607 34 Ant_o
VGLAAAEGARIAGASRIIGIDLNANRFEEARKFGCTEFVNPKDHSKPVQEVLIEMTNG
GVDRSVECTGNVNAMIQAFECVHDGWGVAVLVGVPHKDAEFKTHPMKFLNERTLKG
TFFGNFKPRTDLPNVVEMYMKKELEVEKFITHSVPFSEINKAFDLMARGEGIRCIIRME
N
HTDVYFWEAKGQTPVFPRILGHEAGGIVESVGEGVTELVPGDHVLPVFTGECKECAH
CKSEESNLCDLLRINVDRGVMIGDGQSRFTIDGKPIFHFVGTSTFSEYTVIHVGCLAKI
NPEAPLDKVCVLSCGISTGLGATLNVAKPKKDSTVAIFGLGAVGLAAMEGAKMAGAS
608 34 Ant_o
RIIGVDLNPAKYEQAKKFGCTDFVNPKDHTKPVQEVLVEMTNGGVDRAVECTGHIDA
M IAAFECVHDGWGVAVLVGVPH KEAVFKTHPM N FLN ERTLKGTFFGN YKPRTDLPEV
VEMYMRKELDVEKFITHSVPFSQINTAFDLMLKGEGLRCVMRMGE
SLEERLVDLGFLLEKQMATTGKVIKCKAAVAWEAGKPLSMEEVEVAPPQAMEVRIKIL
FTSLCHTDVYFWEAKGQNPVFPRIFGHEAGGIVESVGEGVTDVAPGDHVLPVFTGEC
KECAHCKSAESNMCDLLRINTDRGVMIGDGKSRFSINGKPIYHFVGTSTFSEYTVMH
609 34 Cyn_d VGCVAKINPEAPLDKVCVLSCGISTGLGASINVAKPPKGSTVAVFGLGAVGLAAAEGA
RIAGASRIIGVDLNPNRFEEARKFGCTEFVNPKDHKKPVQEVLAEMTNGGVDRSVEC
TGN I NAM IQAFECVHDGWGVAVLVGVPHKDAEFKTHPMN FLN ERTLKGTFFGN FKPR
TDLPNVVELYMKKELEVEKFITHTVPFSEINKAFDLMAKGEGIRCIIRMDH
MATTGKVIKCKAAVAWEAGKPLSMEEVEVAPPQAMEVRIKILFTSLCHTDVYFWEAK
GQNPVFPRIFGHEAGGIVESVGEGVTDVAPGDHVLPVFTGECKECAHCKSAESNMC
DLLRINTDRGVMIGDGKSRFSINGKPIYHFVGTSTFSEYTVMHVGCVAKINPEAPLDK
610 34 Cyn_d VCVLSCGISTGLGASINVAKPPKGSTVAVFGLGAVGLAAAEGARIAGASRIIGVDLNP
NRFEEARKFGCTEFVNPKDHKKPVQEVLAEMTNGGVDRSVECTGNINAMIQAFECVH
DGWGVAVLVGVPHKDAEFKTHPMN FLN ERTLKGTFFGN FKPRTDLPNVVELYMKKEL
EVEKFITHTVPFSEINKAFDLMAKGEGIRCIIRMDH
HTDVYFWEAKGQTPVFPRILGHEAGGIVESVGEGVTELVPGDHVLPVFTGECKECAH
CKSEESNLCDLLRINVDRGVMIGDGQSRFTINGKPIFHFVGTSTFSEYTVIHVGCLAKI
NPEAPLDKVCVLSCGISTGLGATLNVAKPKKGSTVAIFGLGAVGLAAMEGAKMAGAS
611 34 LoLp
RIIGVDLNPAKYEQAKKFGCTDFVNPKDHTKPVQEVLVEMTNGGVDSAVECTGNINA
MISAFECVHDGWGVAVLVGVPH KEAVFKTHPM N FLN ERTLKGTFFGN YKPRTDLPEV
VEMYM GEGAMATAGKVIKCKAAVAWEAGKPLSIEEVEVAPPQAMEVRVKILFTALCHTDVYF
WEAKGQTPVFPRIFGHEAGGIVESVGEGVTELAPGDHVLPVFTGECKECPHCKSAES
NMCDLLRINTDRGVMLSDGKSRFSIDGKPIYHFVGTSTFSEYTVLHVGCVAKINPEAP
612 34 LoLp LDKVCVLSCGISTGLGASINVAKPPKGSTVAIFGLGAVGLAAAEGARIAGASRIIGIDL
NANRFEEARKFGCTEFVNPKDHNKPVQEVLIEMTNGGVDRSVECTGNINAMIQAFEC
VHDGWGVAVLVGVPHKDAEFKTHPMNFLNERTLKGTFFGNFKPRTDLPNVVEMYMK
KELEVEKFITHSVPFSEINKAFDLMAKGEGIRCIIRMEN
AQRTMATAGKVIKCKAAVAWEAGKPLSIEEVEVAPPQAMEVRVKILFTSLCHTDVFF
WEPKVQKPLFPRIFGHEAGGIVESVGEGVTDVAPGDHVLPVFTGECKECRHCKSAES
NMCDLLRINTDRGVMISDGKSRFSIDGKPIYHFVGTSTFSEYTVMHVGCVAKINPEAP
613 34 Poa_p LDKVCVLSCGISTGLGASINVAKPPKGSTVAIFGLGAVGLAAAEGARIAGASRIIGVDL
NANRFEEARKFGCTEFVNPKDHTKPVQEVLAEMTDGGVDRSVECTGNINAMIQAFEC
VHDGWGVAVLVGVPHKDAEFKTHPMNFLNERTLKGTFFGNFKPRTDLPNVVEMYMK
KELEVEKFITHSVPFSEINKAFDLMAKGEGIRCIIRMEH
GGNWKCNGTGEDVKKIVTVLNEAEVPSEDVVEVVVSPPFVFLQQVKGLLRPDFSVAA
QNCWVRKGGAFTGEISAEMLVNQQLPWVILGHSERRALLGESNDFVADKVAYALSQ
39/5
614 Cyn_d GLKVIACIGETLEQREAGTTMDVVAAQTKAIAEKISDWTNVVLAYEPVWAIGTGKVA 9
SPAQAQEVHDGLRKWLQSAVSPAVAESTRIIYGGSVNGGNCKELAAQPDVDGFLVG
GASLKPEFVDIIKSATVKSSS
M GRKFFVGG N W KC N GTTEQVD KIVKTLN EGQI PSTD VVEVVVSPPYVFI PVVKTQLR
PEIQVAAQNCWVKKGGAYTGEVSAEMLANLGVPWVILGHSERRALLGESNEFVGDK
39/5
615 Cyn_d VAYALAQGLKVIACVGETLEQRESGSTMDVVAAQTKAIAERIQDWTNVVVAYEPVWA 9
IGTGKVATPAQAQEVHASLREWLKTNVSPEVSESTRIIYGGSVTAANCKELAGQPDV
DGFLVGGASLKPEFIDIINSATVKSA
TMALSAHGKVGENTNLTRESFPPGFVFGTASSAYQVEGNANKYGRGPCIWDTFLMHP
GTTPDNATANVTVDEYHRYMDDVDNMVRVGFDAYRFSISWSRIFPSGVGKINKDGV
DYYHRLIDYMLANKITPYVVLHHFDLPQVLQDQYNGWLSPRVVGDFEKFADFCFKTY
GDRVKNWFTINEPRMMAVHGYSDAFFAPARCTGCKVGGNSATEPYIAGHHLLLSHA
616 43 Cyn_d AAVKTYREKYQAQQKGKIGILLDFVWYEPLSDSMEDGYAAHRARMFTLGWFLHPITY
GHYPPSMENIVRGRLPNFTFEQSEMVKGSADYIGINHYTTYYASHYINDTEMSYRND
WSVKLSYSRNGVPIGKKAYSDWLYVVPWGIYKAVMWTKEKFNNPVIIIGENGIDQPG
NETLPGALYDTFRIDYFEQYLRELKSAVNDGANVIGYFAWSLLDTFEWRLGFTSKFGL
VYVDRQTFTRYPKDSARWFRKVIKREE
SATAAAAVAFLPPLTGRTSPPAYRVPANSRRGSVSNSRIFTSFAPSPILRAAAMATDG
AAPAASDAGSKQKLLTFDSEEELAVSLAKYTAELSAKFAAERGAFTAVLSGGSLIKALR
KLTEPPYLDSVDWSKWHVFWVDERVVPKDHEDSNYKLALDGFLSKVPIPTRQVYAIN
617 47 Cyn_d
DALSAEGAADDYETCLKQLVKNGVIAMSAATGFPRFDLQLLGMGPDGHIASLFPGHP
LVNENQKWVTYIKDSPKPPPERITFTFPVINSSAYIAMVVTGAGKAAAVQKALSDKEI
SSDKLPVEMAVLQDGEFTWFTDKEAVSLLQNK
MGPGTWSPQSRKTYNLLDTVSRHTIQVYPRSWTAVMLTFDNAGMWNVRSNLWERQ
618 49 Cyn_d
YLGEQMYISVISPARSLRDEYNMPETSLRCGKVVGLPMPPSYLPA
GVLLVLTALAVVHAEDPYLFFEWKVTYGTKSLLGVPQKVILINGEFPGPRINCSSNNNI
VVNVFNQLDQPLLFTWNGMQHRKNSWMDGLPGTNCPIAPGTNFTYKWQPKDQIGS
FFYFPSLGMQRAAGGYGPISVVSRLLIPVPFDPPADDHVVLIGDWYTKDHEVMARLLD
SGRSIGRPAGVLINGKGGKDAAAAPIFTFEAGKTYRLRVCNTGIKSSLNFRIQGHDMK
LVEMDGSHTVQDMFDSLDVHPGHCFSVLVDADQKPGDYYVVASTRFIHDPKSVSAV
619 54 Cyn_d
IRYAGSSTPPAPHVPEPPEGWAWSINQWRSFRWNLTASAARPNPQGSYHYGQINIT
RTIKLQISRGHIDGKLRYGFNGVSHVDADTPLKLAEYFNVTDGVFKYNQMGDAPPAV
NGPLRVMPSVISAEFRTFIEVIFENPEKSMDSLHLDGYAFFAVGMGPGKWSPELRKTY
NLLDAVSRHTIQVYPRSWTAIMLTFDNAGMWNVRSNIWERHYLGEQVYVSVISPERS
LRDEYNMPENALRCGKVIGLPLPPSYNPA
PPPSYSHKPGDVFHGRLLIDPPIPPQLLHYNPSRERNLFHSVRRPLILMATTMRGTAAT
AGGVLLLALLVLSTTQVARAEDPYLFFEWHVTYGTRTLLGVPQKVILINDEFPGPRINC
SSNNNIVVNVFNQLEEPLLFTWNGMQQRKNSWQDGLPGTNCPVAPGTNYTFKWQA
KDQIGSFFYFPSLGMQRAAGGYGMISVVSRLLIPVPFDPPADDFQVLVGDWYTKDHT
49/5
620 Ant_o VMASLLDAG KSPGRPAGVLINGKGGKDAASQPMFTFEAGKTYRLRVCNVGIKSSLNF 4
RIQGHDMKLVEMEGSHTLQNTYDSLDVHVGQCLSVLVDADQKPADYLMVASTRFIA
DATSVSAVIRYAGSNTPPAANVPEPPAGWAWSINQWRSFRWNLTASAARPNPQGSY
HYGQINITRTIKLKITRGHLDGKLKYGFNGVSHVDADTPLKLAEYFNVTDGVFRYNQM
GDSPPGVNGPLHAIPNVITAEFRTFIEIIFENPEKSMDSLHLDGYAFFAVGMGPGEWS PELRKTYNLLDAVSRNSIQVYPRSWTAVMLTFDNAGMWNVRSNLWERHYLGEQFYI SVVSPARSLRDEYN MPEDDLRCGKVVGLPMPPSYLPA
PPPSYSHKPGDVFHGRLLIDPPIPPQLLHYNPSRERNLFHSVRRPLILMATTMRGTAAT
AGGVLLLALLVLSTTQVARAEDPYLFFEWHVTYGTRTLLGVPQKVILINDEFPGPRINC
SSN N NIVVNVFNQLEEPLLFTWNGMQQRKNSWQDGLPGTNCPVAPGTNYTFKWQA
KDQIGSFFYFPSLGMQRAAGGYGMISVVSRLLIPVPFDPPADDFQVLVGDWYTKDHT
VMASLLDAG KSPGRPAGVLINGKGGKDAASQPMFTFEAGKTYRLRVCNVGIKSSLNF
/5
Ant_o RIQGHDMKLVEMEGSHTLQNTYDSLDVHVGQCLSVLVDADQKPADYLMVASTRFIA
DATSVSAVIRYAGSNTPPAANVPEPPAGWAWSINQWRSFRWNLTASAARPNPQGSY
HYGQINITRTIKLKITRGHLDGKLKYGFNGVSHVDADTPLKLAEYFNATKGIFEYNLIG
DTPPPEGTPIKLAPNVINTEWRTYIEVVFENPEKSIDSFHLNGYAFFAAGMGPGLWTPE
CRQTYNLLDTVSRHTIQVYPRSWTAVMLTFDNAGMWNLRSNLWERYYMGEQMYISC
VSPARSLRDEYN MPENGLRCGNVIGLPLPPSYIPG
TIAQTPHYTFHSREHHITRARPASVCLPREHFGRRPAGIMAATMRAAAAGVLLVLTAL
AVVHAEDPYLFFEWKVTYGTKSLLGVPQKVILINGEFPGPRINCSSN N NIVVNVFNQL
DQPLLFTWNGMQHRKNSWMDGLPGTNCPIAPGTNFTYKWQPKDQIGSFFYFPSIAM
QRSAGGYGLISVHSRDLIPVPFDIPADDFAVLAGDWYTKDHTVLAKHLDAGKGIGRP
AGLIINGKNDKDAASAPMYN FEAGKTYRFRVCNVGIKASLNVRVPGHNLKLVEMEGS
/5
Cyn_d HTVQN MYDSLDVHVGQCLSFLVTADQKPADYFLVVSTRFIKEVSTITALIRYKGSSTP
PSPKLPEGPSGWAWSINQWRSFRWNLTASAARPNPQGSYHYGQINITRTIKLQISRG
HIDGKLRYGFNGVSHVDADTPLKLAEYFNATDGVFQYNLISDVPPKAGTPIKLAPNVL
SAEFRTFIEVVFENPEKSIDSFHIDGYAFFAAGMGPGTWSPQSRKTYNLLDTVSRHTI
QVYPRSWTAVMLTFDNAGMWNVRSNLWERQYLGEQMYISVISPARSLRDEYN MPET
SLRCGKVVGLPMPPSYLPA
TIAQTPHYTFHSREHHITRARPASVCLPREHFGRRPAGIMAATMRAAAAGVLLVLTAL
AVVHAEDPYLFFEWKVTYGTKSLLGVPQKVILINGEFPGPRINCSSN N NIVVNVFNQL
DQPLLFTWNGMQHRKNSWMDGLPGTNCPIAPGTNFTYKWQPKDQIGSFFYFPSLGM
QRAAGGYGPISVVSRLLIPVPFDPPADDHVVLIGDWYTKDHEVMARLLDSGRSIGRP
AGVLINGKGGKDAAAAPIFTFEAGKTYRLRVCNTGIKSSLNFRIQGHDMKLVEMDGS
/5
Cyn_d HTVQDMFDSLDVHPGHCFSVLVDADQKPGDYYVVASTRFIHDPKSVSAVIRYAGSST
PPAPHVPEPPEGWAWSINQWRSFRWNLTASAARPNPQGSYHYGQINITRTIKLQISR
GHIDGKLRYGFNGVSHVDADTPLKLAEYFNVTDGVFKYNQMGDAPPAVNGPLRVMP
SVISAEFRTFIEVIFENPEKSMDSLHLDGYAFFAVGMGPGKWSPELRKTYNLLDAVSR
HTIQVYPRSWTAIMLTFDNAGMWNVRSN IWERHYLGEQVYVSVISPERSLRDEYN M
PENALRCGKVIGLPLPPSYNPAR
TIAQTPHYTFHSREHHITRARPASVCLPREHFGRRPAGIMAATMRAAAAGVLLVLTAL
AVVHAEDPYLFFEWKVTYGTKSLLGVPQKVILINGEFPGPRINCSSN N NIVVNVFNQL
DQPLLFTWNGMQHRKNSWMDGLPGTNCPIAPGTNFTYKWQPKDQIGSFFYFPSLGM
QRAAGGYGPISVVSRLLIPVPFDPPADDHVVLIGDWYTKDHEVMARLLDSGRSIGRP
AGVLINGKGGKDAAAAPIFTFEAGKTYRLRVCNTGIKSSLNFRIQGHDMKLVEMDGS
/5
Cyn_d HTVQDMFDSLDVHPGHCFSVLVDADQKPGDYYVVASTRFIHDPKSVSAVIRYAGSST
PPAPHVPEPPEGWAWSINQWRSFRWNLTASAARPNPQGSYHYGQINITRTIKLQISR
GHIDGKLRYGFNGVSHVDADTPLKLAEYFNATDGVFQYNLISDVPPKAGTPIKLAPNV
LSAEFRTFIEVVFENPEKSIDSFHIDGYAFFAAGMGPGTWSPQSRKTYNLLDTVSRHTI
QVYPRSWTAVMLTFDNAGMWNVRSNLWERQYLGEQMYISVISPARSLRDEYN MPET
SLRCGKVVGLPMPPSYLPA
TIAQTPHYTFHSREHHITRARPASVCLPREHFGRRPAGIMAATMRAAAAGVLLVLTAL
AVVHAEDPYLFFEWKVTYGTKSLLGVPQKVILINGEFPGPRINCSSN N NIVVNVFNQL
DQPLLFTWNGMQHRKNSWMDGLPGTNCPIAPGTNFTYKWQPKDQIGSFFYFPSIAM
QRSAGGYGLISVHSRDLIPVPFDIPADDFAVLAGDWYTKDHTVLAKHLDAGKGIGRP
AGLIINGKNDKDAASAPMYN FEAGKTYRFRVCNVGIKASLNVRVPGHNLKLVEMEGS
/5
Cyn_d HTVQN MYDSLDVHVGQCLSFLVTADQKPADYFLVVSTRFIKEVSTITALIRYKGSSTP
PSPKLPEGPSGWAWSINQWRSFRWNLTASAARPNPQGSYHYGQINITRTIKLQISRG
HIDGKLRYGFNGVSHVDADTPLKLAEYFNVTDGVFKYNQMGDAPPAVNGPLRVMPS
VISAEFRTFIEVIFENPEKSMDSLHLDGYAFFAVGMGPGKWSPELRKTYNLLDAVSRH
TIQVYPRSWTAIMLTFDNAGMWNVRSN IWERHYLGEQVYVSVISPERSLRDEYN MPE
NALRCGKVIGLPLPPSYNPAR
PLSHFHRPPHATHRSTAAAALIDLHTSRPEEETRRARRDMTAGSRMRACAAAAVLAL
/5
LoLp ALLAVAVRAEDPYLFFEWKVTYGTRSPMGVPQKMILINDAFPGPTINCTSN N NIIVNVF
NQIDKPLLFTWHGIQQRKNSWQDGMPGAMCPIMPGTNFTYKMQFKDQIGTFFYFPSI GMQRAAGGYGLISIHSRPLIPIPFDPPAADFSAMIGDWFTKDHTVLEKHLDTGKTIGR
PAGLLINGKNEKDASNPPMYEVEAGKTYRFRICNVGIKASLNVRVQGHITRLVEMEGS
HTVQNEYDSIDVHVGQCLSVLVTANQKPGDYFFVASTRFIKEVNTITAVIRYKGSNTP
PSPKLPEAPSGWAWSI NQWRSFRWN LTASAARPN PQGSYHYGQI N ITRTIKLM VTR
GHLEGKLKYGFNGVSHVDADTPLKLAEYFNVSDKVFKYNQMGDSPPGVNGPMHVAP
NVITAEFRTFIEVVFENPEKSMDSLHIDGYAFFAVGMGPGKWSPDLRKTYNLLDAVSR
HTIQVYPRSWSAVMLTFDNAGMWNLRSNLWERYYMGEQLYVSCTSPARSLRDEYN
MPENGLRCGKIVGLPLPAPYIIA
PLSHFHRPPHATHRSTAAAALIDLHTSRPEEETRRARRDMTAGSRMRACAAAAVLAL
ALLAVAVRAEDPYLFFEWKVTYGTRSPMGVPQKMILINDAFPGPTINCTSNNNIIVNVF
NQIDKPLLFTWHGIQQRKNSWQDGMPGAMCPIMPGTNFTYKMQFKDQIGTFFYFPSI
GMQRAAGGYGLISIHSRPLIPIPFDPPAADFSAMIGDWFTKDHTVLEKHLDTGKTIGR
PAGLLINGKNEKDASNPPMYEVEAGKTYRFRICNVGIKASLNVRVQGHITRLVEMEGS
49/5
627 LoLp HTVQNEYDSIDVHVGQCLSVLVTANQKPGDYFFVASTRFIKEVNTITAVIRYKGSNTP 4
PSPKLPEAPSGWAWSI NQWRSFRWN LTASAARPN PQGSYHYGQI N ITRTIKLM VTR
GHLEGKLKYGFNGVSHVDADTPLKLAEYFNVSDKVFKYNQMGDSPPGVNGPMHVAP
NVITAEFRTFIEVVFENPEKSMDSLHIDGYAFFAVGMGPGKWSPDLRKTYNLLDAVSR
HTIQVYPRSWSAVMLTFDNAGMWNVRSNLWERHYLGEQLYISVISPARSLRDEYNM
PETALRCGKVVGLPLPPSYLPA
IPYPAATPTLLSFKRAELDSARQVFHPARLPPILMAATTMRATAAGGVLLLALLLVTTNV
ARAEDPYVFFEWHVTYGTKSLLGVPQKVILINGEFPGPRINCSSNNNIVVNVFNQLDQ
PLLFTWNGIQHRKNSWQDGMPGTNCPVVPGTNYTFKWQAKDQIGSFFYFPSIGMQR
TVGGYGLISVVSRLLIPVPFDPPADDLQVLIGDWYNKDHTVMASLLDAGKSPGRPAG
VLINGRGAKDAANPPMFTFEAGKTYRLRICNVGIKASLNFRIQGHDMRLVEMDGSHT
49/5
628 LoLp VQDSFDSLDVHVGHCLSVLVDADQKPADYLMVASTRFMVEPSSVSAVIRYAGSNTPP 4
APNVPEPPAGWAWSLNQWRSFRWN LTASAARPN PQGSYHYGQI N ITRTIKLM VTRG
HLEGKLKYGFNGVSHVDADTPLKLAEYFNVSDKVFKYNQMGDSPPGVNGPMHVAPN
VITAEFRTFIEVVFENPEKSMDSLHIDGYAFFAVGMGPGKWSPDLRKTYNLLDAVSRH
TIQVYPRSWSAVMLTFDNAGMWNVRSNLWERHYLGEQLYISVISPARSLRDEYNMP
ETALRCGKVVGLPLPPSYLPA
IPYPAATPTLLSFKRAELDSARQVFHPARLPPILMAATTMRATAAGGVLLLALLLVTTNV
ARAEDPYVFFEWHVTYGTKSLLGVPQKVILINGEFPGPRINCSSNNNIVVNVFNQLDQ
PLLFTWNGIQHRKNSWQDGMPGTNCPVVPGTNYTFKWQAKDQIGSFFYFPSIGMQR
TVGGYGLISVVSRLLIPVPFDPPADDLQVLIGDWYNKDHTVMASLLDAGKSPGRPAG
VLINGRGAKDAANPPMFTFEAGKTYRLRICNVGIKASLNFRIQGHDMRLVEMDGSHT
49/5
629 LoLp VQDSFDSLDVHVGHCLSVLVDADQKPADYLMVASTRFMVEPSSVSAVIRYAGSNTPP 4
APNVPEPPAGWAWSLNQWRSFRWN LTASAARPN PQGSYHYGQI N ITRTIKLM VTRG
HLEGKLKYGFNGVSHVDADTPLKLAEYFNVSDKVFKYNQMGDSPPGVNGPMHVAPN
VITAEFRTFIEVVFENPEKSMDSLHIDGYAFFAVGMGPGKWSPDLRKTYNLLDAVSRH
TIQVYPRSWSAVMLTFDNAGMWNLRSNLWERYYMGEQLYVSCTSPARSLRDEYNMP
ENGLRCGKIVGLPLPAPYIIA
RSPPILMATTMRATAAAAILLLALLLLSTTNVARAEDPYVFFEWHVTYGTKNLLGVPQK
VILINGEFPGPRINCSSNNNIVVNVFNQLDQPLLFTWNGIQHRKNSWQDGLPGTNCP
VAPGTNYTYKWQPKDQIGSFFYFPSIGMQRAVGGYGLISVVSRLLIPVPFDPPADDLQ
VLIGDWYTKDHAVMASLLDAGKSFGRPAGVLINGRGGKDATNPPMFTFEAGKTYRLR
49/5 VCNVGIKASLNFRIQGHDMRLVEMDGSHTLQDSYDSLDVHVGHCLSVLVDADQKPA
630 Poa_p
4 DYLMVASTRFIVDASSVSAVIRYVGSNTPPAPNVPEPPAGWAWSLNQWRSFRWNLT
ASAARPN PQGSYHYGQI N ITRTI KLM ITRGHLDGKLKYGFNGVSHVDADTPLKLAEYF
NVSDQVFKYNQMGDSPPGVNGPMHITPNVITAEFRTFIEVVFENPEKSMDSLHLDGY
AFFAVGMGPGKWKPELRKTYNLLDAVSRHSIQVYPRSWSAVMLTFDNAGMWNVRS
NLWERHYLGEQLYISVISPARSLRDEYNFPENALRCGKVVGLPLPPSYLPA
MSPSEPTREESVYMAKLAEQAERYEEMVEFMERVARSAGGAGGGEELSVEERNLLSV
AYKNVIGARRASWRIISSIEQKEEGRGNEAHAASIRAYRSKIEAELARICDGILALLDS
631 51 Cyn_d HLVPSAGAAESKVFYLKMKGDYHRYLAEFKSGTERKEAAESTMNAYKAAQDIALADL
APTHPIRLGLALNFSVFYYEILNSPDRACNLAKQAFDEAISELDSLGEESYKDSTLIMQ
LLRDNLTLWTSDTNEDGGDEIKEAAAPKESGD
MDARMVFPLFLVAVAAAPLASGQLSPDFYKTTCPDAEKIIFGVVEKRFKEDPGTAAGL LRLVFHDCFANGCDASILIDPLSNQASEKEAGPNISVKGYDVIDDIKTELEKKCPEVVS
632 52 Cyn_d
CADIVAVSARDAVKLTGGPAYEVPTGRRDAVVSNREDADNLPGPDIAVPKLLSDFSK KGFDVEEAVALLAGGHTIGSCKCFFIEADAAPIDPEYKKNISAACDGANRDRGSVPLD QITPNVFDGNYFALALAKKMPLTVDRLMGMDPKTEPVLKAMAAKPESFVPIFAKAMEK ISALQVLTGKDGEIRKSCGEFNNPKPTSDGPSVIRISSLNPDHMGLSGPGARKVGGR ADGMKANGAED
EITLTKSYGDIAKDLSIIKPFASGIMVPKHFIQPLNKEDYLLPYTTLVKDARALGLEVFA
AGFNNDMLTSYNYSYDPAAEYLQFIDNPDFSVDGVLTDFTPTASGAVACLAHTKGNA
LLPTAKALLPTENGERPLIITHNGASGVFPGCTDLAYQQAVRDGADIIDCAVRMTKDG
VAFCLGSADLTTSTTAATTFMTKVVTVSEIQNRSGIFSFDLSWSEIQTLKPDLSGPYA
QAGLKRNPAAKNAGKFLTLSEFLELAKSSNVSGIMIEIEDAPYLATRGLGVVDAVSSA
633 53 Ant_o
LVNASYDKESNHQRVLIQSDDSSVLSVFKKFPKFERILVIEPIISDASKPSIDEIKEFAH
TVMVSRGSLVQVNGFFLTAFSDLAERIHDANLTLHVGVLKNEFMNFGFDYFADPMVEI
ATYYSLLFCDGLVTEFPATAAAYFRSPCSDTSKNLSYTILAANPGALEQMVPLGALPPA
LPPAPVLEPADVIDPPLPPVAVSSPPESTPNGDDQPSGASSNAGNCRLLVAGIAAAFLY
LMSSH
IFTKRTAVCSSRMGSRYPLLFLILLLVHGANALPPVPEWLTLTGRRPLVIARGGFSGVF
PDSSNLAFSNAVTYSLPDVVLFCDLQFSSDGVGFCLSNLNLDNSTLISKNEGFASRGS
TYQVNGQDIQGWFSLDFKAEELHNIPLIQNTLSRSQIFDGVPYLLSLDNVVKTVQPHE
IWINVQYDSFLREHGLSSEDYILGLPKEFPVTWVSSPEVALLKSLSGKLRNNTKLIFRF
LSEDLVEPTTKKTYGELLKDLKSITTFASGILVPKQFIWPMNKDMYLDPATSLVEDAHA
IGLEVYASGFANDDSCISHNYSYDPSKEYLQFIDNSDFSVDGVLTDYPPTASAAVACL
AHTKGNALAPPGTDTPGGGRPLIITHNGASGVFSDSTDLAYQQAVKDGADIIDCWVR
634 53 Ant_o
MTKDGVAFCLGSLDLNSSTTAATSFLGKMTTVNEIQNKSGIFSFDLTWNEIQTLKPNL
IGPFSEASLDRNPAAKNAGKFMTLAGFLDYAKASNISGILIGIEHAAFLETRGHDVVAT
VSNALIKSGYDKETKKCVLIQSEDPPVLSAFKKFPKFKRVFEIEFDIGDVSQPSVVQIL
EFANAVKLRRSSAARVDGFFLTGFTDALVDRLHAANIAVYVGVLKNEYMSLAFDYWA
DPMVEIATDTWAVGADGLVTEFPATAAAYFRSPCSDTSKNLSYTILAANPGALEQMVP
LGALPPALPPAPVLEPADVIDPPLPPVAVSSPPESTPNGDDQPSGASSNAGNCRLLVA
GIAAAFLYLMSSH
LKNEFMNFGFDYFADPMVEIATYYSLLFCDGLVTEFPATAAAYFRSPCSDTSKNLSYTI
635 53 Cyn_d LAANPGALEQMVPLGALPPALPPAPVLEPADVIDPPLPPVAVSSPPESTPNGDDQPSG
ASSNAGNCRLLVAGIAAAFLYLMSSH
PRWGRRKAFPSFVLGVSCEGAPPDQMGASNPHMFLILLLLLHGASAAPNAPLPKWRT
LSGRPPLVIAHGGFSGLFPDSSQFAYQFAMSTSLPDVALFCDLQFSSDGMGFCKSGL
TLDNSTIISEVFPKMEKTYKVNGEDVRGWFSLDFTADQLVQNVTLIQNIFSRPSTFDG
ALGMYMVDDVVELRPPHIWLNVEYHSFFLEHKISTEDYLKALPKEFSFSYISSPEVAFL
KSVGGLLKQSKTKFVFRLLNENVVEPSTKKTYGELAKDLKFIKEFASGILVPKTYIWPL
NKDQYLAPSTSLVKDAHALGLEVYASGFANDVGLSYNYSYDPSAEYLQFIDNPDFAV
DGLLTDFPPTASGAVACLAHSKGNPLPPPQRPRPLIISHNGASGVFPGSTDLAYQQAM
636 53 Cyn_d
KDGTDIIDCTVQMSKDGVAFCMPSADLGSCTTAGISFIN KGSTVHEIQNKSGIFSFDL
SWSEIQTLKPDLVGPFAQAGLKRNPVAKNAGKFMTLPGFLDMAKASNVSGILINIEHA
AYLATKGLGVVDAVTGALTKAGYDKETKQQVLIQSEDSSVLSAFKKSFPASKRVLSID
TEISDVAKPSVDDIKGVADGVRIHRSSVAQVTGYFLTHFTHVVDTLHAANLTVFIGVL
KNEFMNLGFDYFADPMVEIVTYSDAVMADGLITEFPATAAAYFKSPCSDMNLNLSYSI
LPAQPGALVNIAVPGALPPVGAPAPLLEPADVLDPPLPPVRAVSTAAAPAPTGAADNTT
SAASTTAGNRSSSLLVAGIVALLSLSFLQ
LVKDAHALGLEVYASGFANDDACMSHNYSYDPNAEYLNFIDNSDFSVDGFLTDYPPT ASGAIACLAHTKGNALASIGNETTDGSRPLIITHDGASGVFPGSTDLAYQQAVKDGA
637 53 LoLp DIIDCWVRMSKDGVAFCLGSSDLNGSTTAATTFLGKMTNVDEIQNKSGIFSFDLSW
NEIQTLKPNLIGPFSESAMDRNPAAKNAGKFMTLAAFLDYAKASNISGILIGIEGAAYL ATRGL
YLATRGLDVVGAVSTALTKFGYDKETKQVVLIQSEDPPVLSAFKKFPKFKRVYEIEFDI TDISKPSVVEISEMANAVKLRRSSAVQVDGFYLTGFTHALVDRLHAAKIEVYVGVLKN
638 53 LoLp EFMSLAFDYWADPMKEIATDTWAVPADGLITDFPATAAAYFRSPCSDMEQNMSYYTI
SPAEVGTLVRMASYGLPPAPPPAPVLEPEDVHHQPLPLCPKEPMFRTFRCRMPPKGEY TMATDG
QFIDNPDFAVDGLLTDFPPTASGAVACLAHSKGNPLPPPQRPRPLIISHNGASGVFPG
STDLAYQQAMKDGTDIIDCTVQMSKDGVAFCMPSADLGSCTTAGTSFINKGSTVHEI
QNKSGIFSFDLSWSEIQTLKPDLVGPFAQAGLKRNPVAKNAGKFMTLPGFLDMAKAS
639 53 LoLp
NVSGILINIEHAAYLATKGLGVVDAVTGALTKAGYDKETKQQVLIQSEDSSVLSAFKK
SFPASKRVLSIDTEISDVAKPSVDDIKGVADGVRIHRSSVAQVTGYFLTHFTHVVDTL
HAAN LTVFIGVLKN EFMN LGFDYFAD MGGRYPHMLLILILLHAANAALDEPVDKWKTLGGTPPLVIARGGFSGLFPESSPAAYQ
FAISTALPGVILHCDLQLSSDAKGFCRSGVRLDKSTLIEDIYPNRDKTYKIGPEDVHA
WFSVDFTEAELLNVTVKQTIYSRPSTFDGVMPMYRLEDVASLEPDGIWVNVEYNSFY
KEHKISTEDFLLALPKEFPITYISSPDISFLKSIGGKLKGNTKLILRSLWENATEPTLLKS
YGDIMKDLSIIKPFASGILVPRHFIWPTNKDEYLLPSTSLVKDAHALGLEVYAAGFAND
IFTSYNYSYDPAAEYLQFIDNPDFSVDGVLTDFTPTASGAIACLAHTKGNALLPIAKPLL
640 53 LoLp ATENGERPLIITHNGASGVFSGCTDLAYQQAVRDGADILDCSVRMTKDGVAFCLGSA
DLTTSTTAATTFMAKVVTVSEIQNKSGIFSFDLSWSEIQTLKPELNGPYAQAGLKRNP
AAKNAGKFWSLSEFLDFAKTSNVSGVLIEIEDAPYLATRGLGVVDAISSALVNASYDK
ESHQQRVLIQSDDSSVLSVFKKFPKFERVFVIDPVISDASKPSIDEIKEFAHTVMVSR
GALVRAHGFFLTGFNDMLVGKIHDANLTLHVGVLKNEFMNIGFDYFADPMVEIVTYY
MGLVCDGIVTEFPATAAAYFRSPCSDLTKN MSYSILAANPGGLEKMVPLGALPPALPP
APVLEPADVIDPPLPPVAVSSPPESTPEGDEDASAASSNAANCLLVAGIAAFLYLSSH
QFIDNPDFAVDGLLTDFPPTASGAVACLAHSKGNPLPPPQRPRPLIISHNGASGVFPG STDLAYQQAMKDGTDIIDCTVQMSKDGVAFCMPSADLGSCTTAGISFINKGSTVHEI QNKSGIFSFDLSWSEIQTLKPDLVGPFAQAGLKRNPVAKNAGKFMTLPGFLDMAKAS
641 53 Poa_p
NVSGILINIEHAAYLATKGLGVVDAVTGALTKAGYDKETKQQVLIQSEDSSVLSAFKK SFPASKRVLSIDTEISDVAKPSVDDIKGVADGVRIHRSSVAQVTGYFLTHFTHVVDTL HAAN LTVFIGVLKN EFMN LGFDYFAD
SEIQTLKPNLIGPFSASGLDRNPAAKNAGKFMTLAGFLDYAKASNITGILIGIEHSAYL
ATRGLDVVDAVSSALIKSAYDKETKQRVFIQSEDPPVLSAFKKIPKFMRVFEIEFDIRD
VSQPSVVEISEFANAVKLRRSSATQADGYYLTGFTTALVQRLHAANILVYVGVLKNEF
642 53 Poa_p
MSLAFDYWADPMVEIATDTWSVFADGLVTEFPATAAAYFRSPCSNMERNLSYTIRPA
SPGILLDLAAYGALPPAPPPAPVLEPADIHRQPLPLCPTEPMFRTFRCRLAPKATGKSAE
YTANLASDG
SEIQTLKPNLIGPFSASGLDRNPAAKNAGKFMTLAGFLDYAKASNITGILIGIEHSAYL
Poa_p ATRGLDVVDAVSSALIKSAYDKETKQRVFIQSEDPPVLSAFKNIPKSNRVFEIEFDIGD
643 53
VSQPSVVEITKFANVVKLRRSSAAKVDGFYLTGFTDAVKRLKDAKIEVHVGVLKNEFM SLAFDYWADPMVEIATDTWSVFADGLVTEFPATAAAYFRSPCSDMT
SEIQTLKPNLIGPFSASGLDRNPAAKNAGKFMTLAGFLDYAKASNITGILIGIEHSAYL ATRGLDVVDAVSSALIKSAYDKETKQRVFIQSEDPPVLSAFKKIPKFMRVFEIEFDIRD
Poa_p VSQPSVVEISEFANAVKLRRSSATQADGYYLTGFTTALVQRLHAANILVYVGVLKNEF
644 53
MSLAFDYWADPMVEIATDTWSVFADGLVTEFPATAAAYFRSPCSNMERNLSYTIRPA SPGILLDLAAYGALPPAPPPAPVLEPTDVHRQPLPLCPTEPIFRTFRCRLPPKETGKNPE YTGSLAANG
GVTEYSKGNAYAQVAIGTNDVYKSAEAVDLATKELGGKILRQPGPLPGINTKIASFVD
645 56 Cyn_d
PDGWKVVLVDHADFLKELQ
MRAFPATAGRGAVACAAAAPVPRRSLLLSTAAAGATLHSDSLRLATRSASGAGAIGA
SADAAKAATFAGKDEAVAWAKSDNRRLLHVVYRVGDLDRTIKFYTECLGMKLLRKRD
IPEDKYSNAFLGYGPEDSHFVVELTYNYGVDKYDIGEGFGHFGIAVDDVAKTVEFIRA
646 56 Cyn_d
KGGKVTREPGPVKGGKTVIAFVEDPDGYKFEILERPGTPEPLCQVMLRVGDLDRAISF
YEKACGMELLRKRDNPEYKYTVAMLGYGPEDKNAVLELTYNYGVTEYAKGNAYGQIAI
GTDDVYKTAEVAKLFGGQVVREPGPLPGINTKITSILDPDGWKSVFVDNIDFAKELE
EPGPVKGGKSVIAFVEDPDGYKFELIERGPTPEPLCQVMLRVGDLDRAINFYEKAFGM
647 56 Cyn_d ELLRKRDNPQYKYTIAMMGYGPEDKNAVLELTYNYGVTEYDKGNAYAQIAISTDDVYK
TAEVVRLNAGHITREPGPLPGINTKITACTDPDGWKTVFVDNIDFLKELEE
MARLLLPLPFAAAAAASSSLHLAASRLRVPSVSVTRREGLFGGRLAGVSVPARLARRG LSAGAEAGGGSAAQVVGPEEAMEWVKKDRRRLLHVVYRVGDLDKTIKFYTECLGMK
648 56 Cyn_d LLRKRDIPEERYTNAFLGYGPEDSHFVVELTYNYGVESYNIGTGFGHFGIAVEDVAKTV
DLIKAKGGTVTREPGPVKGGKSVIAFVEDPDGYKFELIERGPTPEPLCQVMLRVGDLD RAIN FYE KAFG M ELLRKQD N PQYKKEYVLLTYY
MATGSEAVLEWNKQDKKRMLHAVYRVGDLDRTIKCYTECFGMKLLRKRDVPDEKYT
649 56 Cyn_d
NAFLGFGPEDKNFALEL
ELTYNYGVDKYEIGEGFGHFAIATEDISKLAEAVKSSCCCKITREPGPVKGGSTVIAFA
650 56 Cyn_d QDPDGYMFELIQRGPTPEPLCQVMLRVGDLERSIKFYEKALGMRLLRKKDVPEYKYTI
AMLGYDDEDKTTVL
PGHSFPPFAPLHRLHENIVSNSPTLPSPSHFLDSHAPRRSSRRLLATSLQLGGTYRINP
651 62 Ant_o RASHTHAGSTYQAAAMRRQSLLLLLAAATLLAATVSAQPGPTQPGPAQPVPTLPGPGP
VPTLSPDFYSQTCPRAERIIAEVVQSKQMANPTTAAGVLRVFFHDCFVTGCDASVLIA PTRFAKSEKDAEINHSLPGDAFDAVVRAKLALELECPGVVSCADILALASRVLVTMTG
GPRYPIPLGRKDSLSSSPTAPDVELPHGNFTVGKIIELFLAKGFSIQEMVALSGAHTLG
FSHCQEFASRLYNYRDNGGKPAPFDPSMNPTYAKGLQAACQDYQKDPTIAAFNDIMT
PGKFDNMYYINLQRGLGLLSTDEELWSDLRTKPFVQRYAANNTDFFEDFSKAMEKLS
LYGVKTGAEGEIRRRCDAYNSGPITV
RHSIPSVGSRSSIALPPRTAIPSPRRISWTLTRAPRLQEGTHQEHYRISAMRLSLLLVL
VAAFSAGAASQPLPPAGGKPLLTPDYYKQTCPRAERIIAEVIQSKQMANPTTAAGVLR
VFFHDCFVGGCDASVLIASNQFAKSEHDADINQSLPGDAFDAVVRAKLALEMECPGV
652 62 Cyn_d VSCADILSLASGVLVTMTGGPRYPVPLGRKDSLSSSPTAADADLPHSNFTVDRLIQMF
GAKGFSVQELVALSGAHTLGFSHCKEFADRIFNYRDKAGKPEPFDPTMNPALAKGLQ
GACKDYLKDPTIAAFNDIMTPGKFDNMYFINLERGLGLLSTDEELWTDARTKPFVQLY
ASNSTKFFEDFGRAMEKLSLFGVKTGADGEIRRRCDTYNHGPMPK
FSAGAASQPLPPAGGKPLLTPDYYKQTCPRAERIIAEVIQSKQMANPTTAAGVLRVFF
HDCFVGGCDASVLIASNQFAKSEHDADINQSLPGDAFDAVVRAKLALEMECPGVVS
CADILSLASGVLVTMTGGPRYPVPLGRKDSLSSSPTAADADLPHSNFTVDRLIQMFGA
653 62 Cyn_d
KGFSVQELVALSGAHTLGFSHCKEFADRIFNYRDKAGKPEPFDPTMNPALAKGLQGA
CKDYLKDPTIAAFNDIMTPGKFDNMYFINLERGLGLLSTDEELWTDARTKPFVQLYAS
NSTKFFEDFGRAMEKLSLFGVKTGADGEIRRRCDTYN
EHSRPLRSRHSLPSTSSEKHPLQVPRRPLSLAFLGPPRTSPALTSPAKLEGIKLTQRAT
RAQDPRTKQQLAAMRRMSLLLLAAAAVLAAAVVAVHAGPPPPVKLSPDFYSQTCPRA
ERIIAEVVQSKQMANPTTAAGVLRVFFHDCFVSGCDASVLIAPTHYAKSEKDADINHS
LPGDAFDAVVRSKLALELECPGVVSCADILALASRVLITMTGGPRYPVPLGRKDSLSS
654 62 LoLp
NPAAPDVELPHSNFTVGRIIELFLAKGFTVQEMVALSGAHTLGFSHCQEFASRIYNYR
DKGGKPAPFDPSMNPTYAKGLQAACQNYQKDPTIAAFNDIMTPGKFDNMYYVNIQR
GLGLLSTDEDMWSDMRTKPFVQRYAANNADFFDDFSKAMEKLSMYGVKTGADGEIR
RRCDAFNSGPITQ
PKSHTRVGSTYQPAAMRRLSLLLLAAAALLAAAVSAAPGPAPKLSPDFYSQTCPRAERI
IAEVVQSKQMANPTTAAGVLRVFFHDCFVSGCDASVLIAPTHYAKSEKDADINHSLP
GDAFDAVVRSKLALELECPGVVSCADILALASRVLVTMTGGPRYPVPLGRKDSLSSNP
655 62 Poa_p TAPDVELPHSNFTVGRIIELFVAKGFTVQEMVALSGAHTLGFSHCQEFASRIYNYRDK
GGKPAPFDPSMNPTYAKGLQAACQDYQKDPTIAAFNDIMTPGKFDNMYYVNIQRGLG
LLSTDEDMWSDMRTKPFVQRYAANNTDFFDDFSKAMEKLSMYGVKTGADGEIRRRC
DAFNSGPTTQ
AKVAYVLQGAGTCGIVLPEATKEKVVAVKEGDALALPFGVVTWWHNLPESATELVVL
656 65 Cyn_d
FLGDTSKGHKPGQFTNFQLTGATGIFTGFSTEFVGRAWD
FVGRAWDLTEADAAKLVSSQPASGIIKLGAGQKLPAPSAEDREGMALNCLEAPLDVD
657 65 Cyn_d IKNGGRVVVLNTVNLPLVKEVGLGADLVRIDAHSMCSPGFSCDSAYQVTYIVRGSGR
VQVVGPDGKRVLETRVEGGYLFIVPRFHVVSKIADESGMEWFSIIT
MVNRTATAEVMSMDLSPKKPAKAYGSDGGSYYDWSPADLPMLGVASIGAAKLHLAA
GGLALPSYSDSAKVAYVLQGTGTCGVVLPEATKEKVIPVKEGDALALPFGVVTWWHN
AHAAATDLVVLFLGDTSKGHKAGQFTNFQLTGASGIFTGFSTEFVGRAWDLDQDAA
658 65 Cyn_d AKLVSTQPGSGIVMVKDGHKMPAPRDEDRAGMVLNCLEAPLDVDIKGGGRVVVLNT
QNLPLVKEVGLGADLVRIDAHSMCSPGFSCDSAYQVTYIVRGSGRVQVVGIDGTRVL
ETRAEGGCLFIVPRFFVVSKIADETGMEWFSIITTPNPIFSHLAGKTSVWKAISPAVLE
TSFNTTPEMEKLFRSKRLDSEIFFAP
MSSAKAVLEPAFQGAGHKPGTEIWRIEDFKPVPLPKSDYGKFYRGDSYIVLQTTCNK
GGAYLLDIHFWIGKDSSQDEAGTAAIKTVELDTMLGGRAVQHREPQGYESDKFLSYF
KPCIIPLEGGFASGFKKPEEDKFETRLYICKGKRAIRVKEVPFARSQLNHDDVFILDTE
KKIYQFNGANSNIQERAKALEVIQHLKEKYHDGVCGVAIVDDGKLQAESDSGEFWVL
FGGFAPIGKKTVSDDDVVLETTPPKLYSINNGQLKLEDTVLTKSILENTKCFLLDCGAE
LFVWVGRVTQVEDRKTASVAVENFILKQNRPKTTRITQVIQGYENHTFKSKFESWPV
SNAAGNASAEEGRGKVAALLKQKGDVKGVSKSNAPVQDEVPPLLESGDKLEVWCIN
659 73 Cyn_d ENGKTCLEKEELGKFYSGDCYVVLYTYHSGDKREEFYLTYWIGKDSLPEDQEMALQT
SNTIWNSLKGRPVLGRIYQGKEPPQFVALFQPMVILKGGISSGYKKFVEQKGLTDETY
SADGIALVRISGTSVHNNKTLQVDSVSTSLSSTECFVLQSGKLMFTWIGNSSSFEQQ
QWAVKVAEFLKPGIAVKHCKEGTESSAFWSAIGGKRTYTSKNVAPDVFIRDPHLYTF
SLRNGKMEVTEVFNFSQDDLLTEDMMIFDTHSEVFIWVGQCVETKDKQKAFEIGQKY
VEHAVAFEGIAPDVPLYKVIEGNEPCFFRTYFSWDNTRSVIQGNSFEKKLSVLFGMRS
EGGCKSSGDGGPTQRASALAALSSALNPSSQGKQSNERPTSSGDGGPTQRASAMA
ALTSALNPSSKPSSPQHQSRSGQGSQRAAAVAALSNVLTAEGSSHSPHAEKTEVAPF SESEAEESPESFTDQDAQGGRTEPDVSHEQTANENGGETTFSYDRLISKSTNPVGGI DYKRRETYLSDSEFETIFGMTKEEFYEQPRWKQELQKKKADLF
MASHIVGYPRMGPKRELKFALESFWDGKSSAEDLEKVATDLRASIWKQMSEAGIKYI
PSNTFSYYDQVLDTTAMLGAVPERYSWTGGEIGLSTYFSMARGNATVPAMEMTKWF
DTNYHFIVPELGPTIKFTYASHKAVSEYKEAKALGIDTVPVLIGPVSYLLLSKPAKGVDK
SFSLLSLLSSILPIYKEVVSELKAAGASWIQFDEPTLVKDLDAHELAAFTSAYAELESAF
SGLNVLIETYFADIPAENYKTLTSLSGVTAYGFDLVRGSKTLDLVRSSFPSGKYLFAGA
660 76 Cyn_d
VDGRNIWADDLATSLSTLESLEAVVGKAKLVVSTSCSLMHTAVDLVNETKLDDEIKS
WLAFAAQKVVEVNALAKALAGQKDEAYFAANAAAQASRRSSPRVTNEEVQKAAAAL
RGSDHRRATNVSARLDAQQKKLNLPVLPTTTIGSFPQTMDLRRVRREYKAKKISEEEY
TNAIKEEISKVVKIQEELDIDVLVHGEPERNDMVEYFGEQLSGFAFTANGWVQSYGS
RCVKPPIIYGDVSRPNPMTVYWSKTAQSMTSRPMKGMLTGPV
MASHIVGYPRMGPKRELKFALESFWDGKSSAEDLEKVATDLRASIWKQMSEAGIKYI
PSNTFSYYDQVLDTTAMLGAVPERYSWTGGEIGLSTYFSMARGNATVPAMEMTKWF
DTNYHFIVPELGPTIKFTYASHKAVSEYKEAKALGIDTVPVLIGPVSYLLLSKPAKGVDK
SFSLLSLLSSILPIYKEVVSELKAAGASWIQFDEPTLVKDLDAHELAAFTSAYAELESAF
SGLNVLIETYFADIPAENYKTLTSLSGVTAYGFDLVRGSKTLDLVRSSFPSGKYLFAGA
661 76 Cyn_d VDGRNIWADDLATSLSTLESLEAVVGKAKLVVSTSCSLMHTAVDLVNETKLDDEIKS
WLAFAAQKVVEVNALAKALAGQKDEAYFAANAAAQASRRSSPRVTNEEVQKAAAAL
RGSDHRRATNVSARLDAQQKKLNLPVLPTTTIGSFPQTMDLRRVRREYKAKKISEEEY
TNAIKEEISKVVKIQEELDIDVLVHGEPERNDMVEYFGEQLSGFAFTANGWVQSYGS
RCVKPPIIYGDVSRPNPMTVYWSKTAQSMTSRPMKGMLTGPVTILNWSFVRNDQPR
FETCYQIALAIKKEVEDLEAAGIQVIQIDEAA
EELRKIMDKKN NIRNMSVIAHVDHGKSTLTDSLVAAAGIIAQEVAGDVRMTDTRADE
AERGITIKSTGISLYYEMTDDSLKSFKGDRDGNEYLINLIDSPGHVDFSSEVTAALRIT
DGALVVVDCIEGVCVQTETVLRQALGERIRPVLTVNKMDRCFLELQVDGEEAYQTFS
RVIENANVIMATYEDKLLGDVQVYPEKGTVAFSAGLHGWAFTLTNFAKMYASKFGVD
ESKMMERLWGENFFDPSTKKWTTKNTGSPTCKRGFVQFCYEPIKQIINTCMNDQKD
KLWPMLQKLNVTMKSDEKELMGKALMKRVMQTWLPASTALLEMMIFHLPSPSTAQK
YRVENLYEGPLDDIYATAIRNCDPEGPLMLYVSKMIPASDKGRFFAFGRVFSGRVATG
662 77 Cyn_d MKVRIMGPNYVPGQKKDLYVKSVQRTVIWMGKKQESVEDVPCGNTVAMVGLDQFIT
KNATLTNEKEVDACPIRAMKFSVSPVVRVAVQCKVASDLPKLVEGLKRLAKSDPMVL
CTIEESGEHIIAGAGELHLEICLKDLQEDFMGGAEIIVSPPVVSFRETVLEKSCRTVMS
KSPNKHNRLYMEARPLEEGLPEAIDEGRIGPRDDPKVRSKILSEEFGWDKDLAKKIW
CFGPETTGPNMVVDMCKGVQYLNEIKDSVVAGFQWASKEGALAEENMRGICFEVCD
VVLHADAIHRGGGQVIPTARRVIYASQLTAKPRLLEPVYLVEIQAPENALGGIYGVLNQ
KRGHVFEEMQRPGTPLYNIKAYLPVIESFGFSSTLRAATSGQAFPQCVFDHWDMMSS
DPLEAGSQAAQLVLDIRKRKGLKEQMTPLSEFEDKL
PLRIRASQRATMSPAEPTREESVYMAKLAEQAERYEEMVEFMERVAKATGGAGPGEE
LSVEERNLLSVAYKNVIGARRASWRIISSIEQKEESRGNDAHAATIRSYRSKIEAELAK
663 86 Ant_o ICDGILALLDSHLVPSAAAAESKVFYLKMKGDYHRYLAEFKSGAERKEAAESTMNSYK
AAQDIALADLAPTHPIRLGLALNFSVFYYEILNSPDRACNLAKQAFDEAISELDSLGEE
SYKDSTLIMQLLRDNLTLWTSDTNEDGGDEIKEAPAPKESEGQ
QTRGKMSTAEATREENVYMAKLAEQAERYEEMVEFMEKVAKTADVGELTVEERNLLS VAYKNVIGARRASWRIISSIEQKEESRGNEAYVASIKEYRTRIETELSKICDGILKLLDS
664 86 Ant_o
HLVPSATAAESKVFYLKMKGDYHRYLAEFKAGTERKEAAENTLVAYKSAQDIALADLP TTHPIRLGLALNFSVFYYEILNSPDRACNLAKQAFDEAIAELDSLGEESYKDSTLIMQLL
SIEQKEEGRGNEDRVTLIKDYRGKIETELTKICDGILKLLESHLVPSSTAPESKVFYLK MKGDYYRYLAEFKTGTERKDAAENTMVAYKAAQDIALAELAPTHPIRLGLALNFSVFY
665 86 Cyn_d
YEILNSPDRACSLAKQAFDEAISELDTLSEESYKDSTLIMQLLRDNLTLWTSDISEDPA EEIREAAPKSGEGQ
VFYLKMKGDYHRYLAEFKTGAERKEAADATLAAYQAAQDIAIKELPPTHPIRLGLALNF
666 86 Cyn_d SVFYYEILNSPDRACSLAKQAFDEAISELDTLGEESYKDSTLIMQLLRDNLTLWTSDM
QDDGGDEMRDASKPEDEQ
PPRHPTAMRVPHPPHPGGRVLLKCPTPPVASPNRTDASHPPQEDPLRRANPVAFPVPG
SPEEIPPPAAMSPSEPTREESVYMAKLAEQAERYEEMVEFMERVARSAGGAGGGEEL
SVEERNLLSVAYKNVIGARRASWRIISSIEQKEEGRGNEAHAASIRAYRSKIEAELARI
667 86 Cyn_d
CDGILALLDSHLVPSAGAAESKVFYLKMKGDYHRYLAEFKSGTERKEAAESTMNAYK
AAQDIALADLAPTHPIRLGLALNFSVFYYEILNSPDRACNLAKQAFDEAISELDSLGEE
SYKDSTLIMQLLRDNLTLWTSDTNEDGGDEIKEAAAPKESGDAQ MAKLAEQAERYEEMVEYMEKVAKTVDVEELTVEERNLLSVAYKNVIGARRASWRIVS SIEQKEESRKNEEHVNLIKEYRGKIEAELSNICDGILKLLDSHLVPSSTAAESKVFYLK
668 86 Cyn_d MKGDYHRYLAEFKTGAERKESAESTMVAYKAAQDIALAELAPTHPIRLGLALNFSVFY
YEILNSPDKACNLAKQAFDEAISELDTLGEESYKDSTLIMQLLRDNLTLWTSDLTEEG AEDGKEASKGEAGEGQ
MAKLAEQAERYEEMVEYMEKVAKTVDVEELTVEERNLLSVAYKNVIGARRASWRIVS SIEQKEESRKNEEHVNLIKEYRGKIEAELSNICDGILKLLDSHLVPSSTAAESKVFYLK
669 86 Cyn_d MKGDYHRYLAEFKTGAERKESAESTMVAYKAAQDIALAELAPTHPIRLGLALNFSVFY
YEILNSPDKACNLAKQAFDEAISELDTLGEESYKDSTLIMQLLRDNLTLWTSDLTEEG AEEGKEAPKGDAGEGQ
670 86 LoLp QTRGRMSTAEATREENVYMAKLAEQAERYEEMVEFM
HAGPAPSAPGDLLKSPPLPAPASPTNTFTSSVPGSPQLPPYLPLAHPTMSPAEPTREES
VYMAKLAEQAERYEEMVEFMERVAKATGGAGPGEELSVEERNLLSVAYKNVIGARRA
SWRIISSIEQKEEGRGNDAHAATIRSYRTKIEAELAKICDGILALLDSHLVPSAGAAES
671 86 LoLp
KVFYLKMKGDYHRYLAEFKSGAERKEAAESTMNSYKAAQDIALADLAPTHPIRLGLAL
NFSVFYYEILNSPDRACNLAKQAFDEAISELDSLGEESYKDSTLIMQLLRDNLTLWTS
DTNEDGGDEIKEAPAPKESGEGQ
SWRIISSIEQKEESRGNEAYVASIKEYRTRIETELSKICDGILKLLDSHLVPSATAAESK VFYLKMKGDYHRYLAEFKAGAERKEAAENTLVAYKSAQDIALADLPTTHPIRLGLALNF
672 86 LoLp
SVFYYEILNSPDRACNLAKQAFDEAIAELDSLGEESYKDSTLIMQLLRDNLTLWTSDN ADEGGDEIKEASKPEGEGH
NPQKLKMAELSREENVYMAKLAEQAERYEEMVEFMEKVAKTVDSEELTVEERNLLSV
673 86 LoLp AYKNVIGARRASWRIISSIEQKEESRGNEDRVTLIKDYRGKIETELTKICDGILKLLDS
H
MAKLAEQAERYEEMVEYMEKVAKTVDVEELTVEERNLLSVAYKNVIGARRASWRIVS SIEQKEEGRGNEEHVTLIKEYRGKIEAELSKICDGILKLLDSHLVPMSTAAESKVFYLK
674 86 LoLp MKGDYHRYLAEFKASAERKEAAESTMVAYKAAQDIALAELAPTHPIRLGLALNFSVFY
YEILNSPDKACNLAKQAFDEAISELDTLGEESYKDSTLIMQLLRDNLTLWTSDLTEEG GAEDGKEASKGEGAEGQ
MAKLAEQAERYEEMVEYMEKVAKTVDVEELTVEERNLLSVAYKNVIGARRASWRIVS SIEQKEEGRGNEEHVTLIKEYRGKIEAELSKICDGILKLLDSHLVPMSTAAESKVFYLK
675 86 LoLp MKGDYHRYLAEFKASAERKEAAESTMVAYKAAQDIALAELAPTHPIRLGLALNFSVFY
YEILNSPDKACNLAKQAFDEAISELDTLGEESYKDSTLIMQLLRDNLTLWTSDITDDA GDEIKEASKPETGEGHQ
PTRRHCHAGPAPSAPGDLLKSPPLLLRLPHKRVHLSPPSPDPLAHPSLFATMSPAEPTR
EESVYMAKLAEQAERYEEMVEFMERVAKATGGAGPGEELSVEERNLLSVAYKNVIGA
RRASWRIISSIEQKEEGRGNDAHAATIRSYRTQIEAELAKICEGILALLDSHLVPSAGA
676 86 Poa_p
AESKVFYLKMKGDYHRYLAEFKSGAERKEAAESTMNAYKAAQDIALADLAPTHPIRLG
LALNFSVFYYEILNSPDRACNLAKQAFDEAISELDSLGEESYKDSTLIMQLLRDNLTLW
TSDTNEEGGDDIKEAPAPKESGDGQ
QTRGKMSTAEATREENVYMAKLAEQAERYEEMVEFMEKVAKTADVGELTVEERNLLS VAYKNVIGARRASWRIISSIEQKEESRGNEAYVASIKEYRTRIETELSKICDGILKLLDS
677 86 Poa_p H LVPSATAAES KVFYLKM KG DYH RYLAEFKAGAERKEAAE NTLVAYKSAQD IALADLP
TTHPIRLGLALNFSVFYYEILNSPDRACNLAKQAFDEAIAELDSLGEESYKDSTLIMQLL RDNLTLWTSDN ADEGGDEIKEASKPEGEGH
RTRGKMSTAEATREENVYMAKLAEQAERYEEMVEFMEKVAKTADVGELTVEERNLLS VAYKNVIGARRASWRIISSIEQKEESRGNEAYVASIKEYRTRIETELSKICDGILKLLDS
678 86 Poa_p H LVPSATAAES KVFYLKM KG DYH RYLAEFKAGAERKEAAE NTLVAYKSAQD IALADLP
TTHPIRLGLALNFSVFYYEILNSPDRACNLAKQAFDEAIAELDSLGEESYKDSTLIMQLL RDNLTLWTSDN ADEGGDEIKEASKPEGEGH
MSPSEPTREESVYMAKLAEQAERYEEMVEFMERVARSAGGAGGGEELSVEERNLLSV
AYKNVIGARRASWRIISSIEQKEEGRGNEAHAASIRAYRSKIEAELARICDGILALLDS
86/5
679 Cyn_d HLVPSAGAAESKVFYLKMKGDYHRYLAEFKSGTERKEAAESTMNAYKAAQDIALADL 1
APTHPIRLGLALNFSVFYYEILNSPDRACNLAKQAFDEAISELDSLGEESYKDSTLIMQ
LLRDNLTLWTSDTNEDGGDEIKEAAAPKESGD
MAAKVYIVYYSTYGHVGKLAEEIKKGASSVEGVEAKLWQVPETLSEEVLGKMGAPPK PDVPVITPQELAEADGILFGFPTRFGMMAAQMKAFFDATGGLWREQSLAGKPAGIFF
680 87 Cyn_d
STGTQGGGQETTPLTAITQLTHHGMVFVPVGYTFGAKLFGMDQVQGGSPYGAGTFA ADGSRWPSEVELEHAFHQGKYFAGIAKKLKGSA MAVKVYVVFYSTYGHVAKLAEEIKKGAASVEGVEVKLWQVPETLSEEVLGKMGAPPK
681 87 Cyn_d TDVPVITPQELAESDGSLFGFPTRFGMMAAQMKAFFDATGGLWREQSLAGKPAGIFF
STGTQGGGQE
QGGGQETTPLTAVTQLTHHGMVFVPVGYTFGAKMFDMESVHGGSPYGAGTFAGDG
682 87 Cyn_d
SRWPTEVELEHAFHQGKYFAGI
KRIVKLVNDVGAVVNNDPDVNKYLKVVFIPNYNVSVAEVLIPGSELSQHISTAGMEAS
683 89 Ant_o
GTSNMKFSLNGCVIIGTLDG
SFPKIVRLAQFLGRAIAVPSRPLQKAPTGSHLSPSPIRCPNSEALSPPPPHARRLRIPHH
SAMSAADKVKPAANPAAEDAKAIAGNISYHAQYSPHFSPLAFGPEPAYFATAESVRDH
LLQRWNDTYLHFHKTDPKQTYYLSMEYLQGRALTNAVGNLNITGAYAEAVKKFGYEL
EALAGQERDMALGNGGLGRLAACFLDSMATLNLPAWGYGLRYRYGLFKQRITKEGQ
EEVAEDWLEKFSPWEIVRHDVVYPVRFFGHVEISPDGSRKVAGGEVLNALAYDVPIP
GYKTKNAISLRLWDAKASAEDFNLFQFNDGQYESSAQLHSRAQQICAVLYPGDATEE
GKLLRLKQQFFLCSASLQDIIFRFKERKSDRVSGKWSEFPSKVAVQMNDTHPTLAIPE
LMRLLMDEEGLGWDEAWEVTNKTVAYTNHTVLPEALEKWSQAVMRKLLPRQMEIIEE
684 89 Ant_o
IDKRFREMVISTRKDMEGKLDLMSVLDNSPQKPVVRMANLCVVSAHTVNGVAELHS
NILKEELFADYVSIWPNKFQNKTNGITPRRWLRFCNPELSEIVTKWLKTDKWTSNLDL
LTGLRKFADDEKLHTEWAAAKLASKKRLAKHVLDVTGVTIDPNSLFDIQIKRIHEYKR
QLMNILGAVYRYKKLKEMSAEEKQKVTPRTVMVGGKAFATYTNAKRIVKLVTDVGAV
VNNDPDVNKYLKVVFIPNYNVSVAEVLIPGSELSQHISTAGMEASGTSNMKFSLNGC
VIIGTLDGANVEIREEVGEDNFFLFGAKADQVAGLRKDRENGLFKPDPRFEEAKNYIR
SGTFGTYDYTPLLDSLEGNSGFGRGDYFLVGYDFPSYIDAQARVDEAYKN KKRWIKM
SILNTAGSGKFSSDRTIAQYAKEIWGITASPVP
SRPRPVYRIRRPPHVSPARLLEKPLPGSQTSSHSRSSIPRSWSVLVRRESPRLLDAIPQ
CREPAMPESKCGAAEKVAPAATPAAEKPADIAGNISYHATYSPHFAPLNFGPEQAFYA
TAESVRDHLIQRWNETYLHFHKTDPKQTYYLSMEYLQGRALTNAVGNLGITGAYAEA
VKKFGYELEALAAEEKDAALGNGGLGRLASCFLDSMATLNLPAWGYGLRYRYGLFKQ
RISKEGQEEIAEDWLDKFSPWEIPRHDVVFPVRFFGHVEILPNGTRKWVGGEVMKAL
AYDVPIPGYKTKNAISLRLWEAKATAEDFNLFQFNDGQYESSAQLHSRAQQICAVLYP
GDATEEGKLLRLKQQFFLCSASLQDMIARFKERNPDRASGKWAEFPTKVAVQLNDTH
PTLAIPELMRLLMDEEGLGWDEAWDITYRTVSYTNHTVLPEALEKWSQIVMRKLLPRH
685 89 Cyn_d
MEIIEEIDKRFREMVISSHKEMEGKIDSMKVLDSSNPQKPVVRMANLCVVSSHTVNG
VAELHSNILKQELFADYVSIWPSKFQNKTNGITPRRWLRFCNPELSELVTKWLKTDD
WTSNLDLLTGLRKFADDEKLHAEWASAKLASKKRLAKYVLDVTGVEIDPTSLFDIQIK
RIHEYKRQLLNILGVVYRYKKLKEMSAEERQKVTPRTVMLGGKAFATYTNAKRIVKLV
NDVGAVVNNDPDVNKYLKVVFIPNYNVSVAEVLIPGSELSQHISTAGMEASGTSNMK
FSLNGCVIIGTLDGANVEIREEVGEENFFLFGAKADQIAGLRKDRENGLFKPDPRFEEA
KQLIRSGAFGSYDYEPLLDSLEGNSGFGRGDYFLVGYDFPSYIDAQNLVDKAYKDKK
KWITMSILNTAGSGKFSSDRTIAQYAKEIWDIKASPVA
RCANSEALSPPPPHALAQRIPHHTAMSAADKVKPAASPAAEDPAAIAGNISFHAQYSP
HFSPLTFGPEPAYFATAESVRDHLLQRWNDTYLHFHKTDPKQTYYLSMEYLQGRALTN
AVGNLNITGAYAEAVKKFGYELEALAGQERDMALGNGGLGRLAACFLDSMATLNLPA
WGYGLRYRYGLFKQRITKEGQEEVAEDWLEKFSPWEIVRHDVVYPVRFFGHVEISPD
GRRKAVGGEVLNALAYDVPIPGYKTKNAISLRLWDAKASAEDFNLFQFNDGQYESAA
QLHSRAQQICAVLYPGDATEEGKLLRLKQQFFLCSASLQDIIFRFKERKPDRASGKWS
EFPSKVAVQMNDTHPTLAIPELMRILMDEEGLGWDEAWDVTNKTVAYTNHTVLPEAL
686 89 LoLp EKWSQAVMRKLLPRQMEIIEEIDKRFRELVISTRKDMEGKLDSMSVLDNSPQKPVVR
MANLCVVAAHTVNGVAELHSNILKEELFADYLSIWPNKFQNKTNGITPRRWLRFCNP
ELSEIVTKWLKTDQWTSNLDLLTGLRKFADDEKLHAEWAAAKLASKKRLAKHVLDVT
GVTIDPNSLFDIQIKRIHEYKRQLMNILGAVYRYKKLKEMSAEEKQKVTPRTVMVGGK
AFATYTNAKRIVKLVTDVGAVVNNDPDVNKYLKVVFIPNYNVSVAEVLIPGSELSQHI
STAGMEASGTSNMKFSLNGCVIIGTLDGANVEIREEVGQDNFFLFGAKADQVAGLRK
DRENGLFKPDPRFEEAKQFVRSGAFGTYDYTPLLDSLEGNSGFGRGDYFLVGYDFPSY
IDAQARVDEAYKDKKRWIKMSILNTAGSGKFSSDRTIAQYAKEIWGITASPVP
GVLPVPPFGAPRLITSPATHAHRERSTQFPTAMSAADKVKPAASPAAEDPAAIAANISY
HAQYSPHFSPLAFGPEPAYFATAQSVRDHLLQRWNDTYLHFHKTDPKQTYYLSMEYL
QGRALTNAVGNLDITGAYAEAVKKFGYELEALAGQERDMALGNGGLGRLAACFLDS
687 89 Poa_p
MATLNLPAWGYGLRYRYGLFKQRIAKEGQEEIAEDWLEKFSPWEIVRHDVVYPVRFF
GHVEISPDGTRKSAGGEVLKALAYDVPIPGYKTKNAISLRLWDAKASAEDFNLFQFN
DGQYESAAQLHSRAQQICAVLYPGDATEEGKLLRLKQQFFLCSASLQDIIFRFKERKS
Figure imgf000078_0002
Table 4
Table 4 shows NTGA's ranked according to the number of PG peptides contained in the NTGA. The table also shows the number of peptides (Th+) recognized by T cells of a grass allergic donor population (n=20) .
Figure imgf000078_0001
Figure imgf000079_0001
PG: refers to a peptides with a mismatch of less than 3 to a corresponding peptide in at least one other grass pollen species Table 5
Table 5 shows NTGA's or homologs thereof, which release from Phi p pollen as well as Cyn d pollen is initiated within 30 minutes after hydration of pollen. Table 5 also shows NTGA's which release from Phi p and Cyn d pollen is not initiated within 30 minutes after hydration of pollen.
Figure imgf000080_0001
Table 6
Table 6 lists pollen species of the grass plant families Poaceae. Pollen species used for the present conservation analysis are highlighted in grey colour.
Table 6 - List of Grass Pollen species
Major
Common Latin name of
ID Genus Family Order Taxonomic
Name species
group
Sweet vernal Anthoxanthum Anthoxanthu
Ant o Poaceae Poales Liliopsida
grass odoratum m
Bermuda
Cyn d Cynodon dactylon Cynodon Poaceae Poales Liliopsida
grass
Orchard Dactylis glomerata
Dactylis Poaceae Poales Liliopsida
Grass L.
Meadow
Festuca pratensis Festuca Poaceae Poales Liliopsida fescue
Velvet Grass Holcus lanatus Holcus Poaceae Poales Liliopsida
Barley Hordeum vulgare Hordeum Poaceae Poales Liliopsida
Lol p Rye grass Lollium Perenne Lollium Poaceae Poales Liliopsida
Rice Oryza sativa Oryza Poaceae Poales Liliopsida
Bahia grass Paspalum notatum Paspalum Poaceae Poales Liliopsida
Canary Grass Phalaris aquatica Phalaris Poaceae Poales Liliopsida
Timothy
Phi p Phleum Pratense Phleum Poaceae Poales Liliopsida
grass
Kentucky
Poa P Poa pratensis Poa Poaceae Poales Liliopsida
blue grass
Rye Secale Cereale Secale Poaceae Poales Liliopsida
Johnson
Sorghum halepense Sorghum Poaceae Poales Liliopsida grass
Wheat Triticum aestivum Triticum Poaceae Poales Liliopsida
Maize Zea mays Zea Poaceae Poales Liliopsida Table 7
Table 7 shows a Panel of 25 MHC II molecules (alleles) for which peptide binding affinities were predicted .
Locus HLA DP Locus HLA DQ Locus HLA DR
DPA1*01-DPB1*0401 DQA1*0101-DQB1*0501 DRB1*0101 DRB1*0802
DPA1*0103- DQA1*0102-DQB1*0602 DRB1*0301 DRB1*0901
DPA1*0201- DQA1*0301-DQB1*0302 DRB1*0401 DRB1*1101
DPA1*0201- DQA1*0401-DQB1*0402 DRB1*0404 DRB1*1302
DPA1*0301- DQA1*0501-DQB1*0201 DRB1*0405 DRB1*1501
DQA1*0501-DQB1*0301 DRB1*0701 DRB3*0101
DRB4*0101
DRB5*0101
Table 8
Table 8 shows individual peptide data for the cross reactivity experiments. Each peptide was used to stimulate cells and cross reactivity was tested for extracts from each pollen species. The number of mismatches (# of mm) for each peptide compared to the pollen species and the reactivity of the extracts as a percentage of the reactivity compared to the peptide are shown. Peptides are SEQ ID NO's 321, 303, 414, 689, 690, 28, 691-713 in order of appearance, , e.g. peptide NGSQFFLCTAKTAWL of NTGA 91 has SEQ ID NO: 1110
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
EXAMPLES
Example 1
This example includes a description of transcriptomic analysis of various pollen species and conservation analysis.
A set of 93 proteins from Timothy grass (TG) pollen and the assembly of 822 peptides (15 mers) predicted to promiscuously bind HLA class II molecules shown in Table 7 and the immune reactivity in allergic donors have been reported in PCT application WO
2013/119863. Promiscuous binders were determined by predicting the binding affinity to a panel of 25 HLA class II molecules using a consensus prediction approach (Wang P, et a/. (2008) and Wang P, et a/. (2010) . Peptides with predicted binding scores in the top 20% for a given allele were considered potential binders. Peptides predicted to bind 13 or more of the HLA molecules in Table 7 at this threshold were considered promiscuous binders, and selected for synthesis (after eliminating peptides overlapping by more than 9 contiguous residues) . If less than 5 peptides from a given protein met this threshold, the top 5 peptides were chosen, and up to 4 peptides in proteins where length was prohibitive. In total, this resulted in the selection of 822 TG peptides from a total of 21,506 distinct 15-mers encoded in 620 ORFs derived from the transcriptomic analysis. Immune reactivity was determined by the production of IL-5 or IFNg from cultured PBMCs of the allergic donors in response to stimulation with a peptide and IL-5 and IFNg were measured by ELISPOT as described in Oseroff C et al, 2010. In short, T cell immune reactivity was determined using PBMCs isolated from study participants and stored in liquid nitrogen until further use. For experimental testing, PBMCs were thawed and expanded in vitro with TG pollen extract (50 μg/mL) or the peptide pool (5 μg/mL). The TG extract and peptide pools had each been previously titrated to determine optimal stimulation concentrations.
Cytokine production by cultured PBMCs in response to antigen stimulation was measured by ELISPOT. Cells (1 x 105 cells/well) were plated and incubated with peptide (10 μg/mL), the peptide pool (5 μg/mL), or the TG extract (50 μg/mL). Phytohaemagglutinin (10 μg/mL) and medium alone were used as positive and negative controls, respectively. Samples were considered to produce a cytokine if 100 spot-forming cells (SFCs)/105 PBMCs were detected, with P≤ .05 and a stimulation index of 2 or more. Criteria for individual peptides were the same except that a minimum of 20 SFCs were required for a sample to be counted as positive.
To study the conservation of the 822 TG peptides in other grass pollen species, RNA- sequencing was performed on pollen samples of four additional grass pollen species
(Kentucky blue grass (Poa pratensis, Poa p), Sweet vernal grass (Anthoxanthum odoratum, Ant o), Rye grass (Lollium Perenne, Lol p), Bermuda grass (Cynodon dactylon, Cyn d)). RNA-seq was run at UCSD, using an Illumina HiSeq 2000. RNA-seq was run at UCSD, using an Illumina HiSeq 2000. The table below shows the number of reads assembled for each of the different pollens (top), with over 500 million reads over two replicate runs per allergen. Sequences were assembled into transcripts using Trinity (bottom), resulting in over 50 thousand transcripts per allergen with minimum lengths of 200 nucleotides. The transcripts include related variants, such as isoforms, and homologs.
Sequencing was performed on an Illumina Genome Analyzerllx (GAIIx). Briefly, adaptor- ligated cDNA was loaded into an Illumina flow cell. DNA was then bridge-amplified within the flow cell to generate millions of DNA clusters by using specific reagents and enzymes (Illumina Paired-End Cluster Generation Kit). The flow cell was loaded onto the GAIIx equipped with a paired-end module, and 72 sequencing cycles were performed to generate sequence in both directions by using Illumina Sequencing Kit v4. Replicate samples were run in seven of the eight lanes on the flow cell, producing 280 million raw sequence reads of 72 bp in length. Reads went through several preprocessing steps using the FastX toolkit (2) before they were assembled into contigs: (i) the 3 ' terminal base was removed; (ii) low- complexity reads were removed; (iii) portions of reads downstream of a low-quality score were removed; and (iv) portions of reads corresponding to adapter sequencers were removed. The remaining reads were assembled into contigs by using Velvet (Version 1.0.15) (3). Because of the excessive memory requirements inherent to de novo sequence assembly, the reads for each lane were considered separately and were each run with five different values for the word size parameter (k=21, 23, 25, 27, 29). We and others (4) have observed that different sets of contigs are obtained for each value for k. The contigs were further merged with Oases (Version 0.18.1; D. R. Zerbino, European Bio-informatics Institute, Hinxton, United Kingdom) into putative transcripts.
Table showing pollen RNA-seq reads for various grass pollen species
Figure imgf000087_0001
Example 2
This example includes a description of how to identify which of the TG peptides that are conserved across a Phi p grass pollen and other selected grass pollen species (across Cyn d and Lol p as well as across Cyn d, Lol p, Ant o and Poa p).
The degree of conservation of the known 15-mer peptides deriving from TG pollen proteins was determined across the different pollens. For the purpose of this analysis, peptides that have a homologous hit with 0, 1 or 2 mismatches are considered as being conserved. Any substitution of an amino acid sequence within the 15mer TG peptide is considered to constitute a mismatch. All 15mer peptides (overlapping by 10 aa) of the
representative/construct sequence were created in silico and compared against the protein sequences of non-TG species. All peptides with 2 or less mismatches to the TG construct peptides were run through the IEDB MHC class II peptide binding predictor for 20 common class II alleles. In total 519 of the 822 TG peptides have a mismatch of less than 3 (0, 1, or 2 mismatces) to a homologous peptide in another grass pollen species, thus resulting in 519 PG peptides.
Table 1 list the 517 PG peptides and indicates for each of the PG peptides (SEQ ID NOs 1- 519) in which other grass pollen species (Cyn d and Lol p as well as Cyn d, Lol p, Ant o and Poa p) a matching peptide with either less than 3, less than 2 or zero mismatches are found. The number of TG grass allergic donors (n=20) with an in vitro T cell response to the TG peptide sequence is also shown. The immune reactivity of the TG peptide was assessed as the number of TG grass allergic donors (n=20) having in vitro T cell response against the TG peptide. Example 3
This example includes a description of how the full length sequence of a selection of NTGA's and their homologs in other grass pollen species are identified.
Full length sequence of NTGA's were assembled using multiple sequence alignments of transcripts from the different pollens, thereby identifying with more confidence the full length sequence of selected antigens of interest based on conserved start- and stop-codons. For example this made it possible to distinguish between multiple variants of TG transcripts identified in the initial assembly, and then pick high confidence candidate sequences that are starting points for protein synthesis.
In order to identify the correct coding region of each transcript, there was identified the closest homologous sequence in the rice (Oryza sativa japonica) proteome (via Blast). Rice was chosen since it is a species closely related to Timothy grass with a completely sequenced and annotated genome. Homologous rice sequences were identified for 180 Timothy grass sequences. Subsequently, homologous sequences were identified (via Blast) in the translated transcriptomes of the grass pollen species of all identified sequences, the one(s) sharing the largest number of conserved peptides with the Phi p sequence was selected as homolog. In addition, there was found evidence of the presence of the NTGA's upon extracting pollen in a buffered aqueous solution for at least 2 hours and detecting the NTGA's by mass spectrometry analysis of the trypsin-treated extract and comparing mass signals to protein databases. Table 2 shows Phi p amino acid sequences of the identified NTGA's in grass pollen and Table 3 shows amino acid sequences of proteins with high identity and similarity to the Phi p sequence.
During the work with assembling the full length sequences it was found that PG peptides of NTGA's 5 and 64 derives from the same full length sequence, thus hereinafter named NTGA 5/64. Likewise, PG peptides of NTGA's 86 and 51 derive from the same full length sequence, and the full length protein is hereinafter named 86/51. PG peptides of NTGA's 49 and 54 derive from the same full length sequence, thus hereinafter named NTGA 49/54. PG peptides of NTGA's 39 and 59 derive from the same full length sequence, thus hereinafter named NTGA 39/59.
Example 4
This example includes a description of how to identify PG peptides with high correlation between conservation across grass pollen species and immune reactivity.
Some PG peptides are conserved among several grass pollen species and produce a T cell response in a higher fraction of the donors. Those PG peptides are derived from the proteins numbered 2, 6, 7, 9, 22, 24, 27, 49/54, 52, 89, 90 and 91 and PG peptides are identified by the SEQ ID NOs 19, 20, 24, 25, 26, 28, 32, 37, 69, 71, 72, 73, 74, 78, 79, 80, 81, 82, 84, 86, 87, 88, 89, 90, 91, 93, 95, 99, 107, 166, 169, 173, 174, 176, 180, 193, 303, 313, 319, 321, 492, 505, 506, 510, 511, 513, 514, 518, 519 (those with > than 2 responders).
Example 5
This example includes a description of NTGA's with high number of conserved peptides among grasses.
Table 4 shows the ranking of NTGA's according to the number of pan-grass peptides (PG) it incorporates. The top number NTGA's containing most PG peptides are NTGA 89, 6, 30, 2, 72, 1, 73, 83, 4, 13, 86/51, 77, 24, 29, 7, 39/59, 34, 20, 76, 84 and 91, respectively. A fraction of those NTGA's (NTGA's 2, 6, 7, 49/54, 89, 53 and 91) contains highly T cell reactive sequences (SEQ ID Nos: 19, 20, 24, 25, 26, 32, 37, 69, 71, 72, 73, 74, 78, 79, 80, 81, 82, 84, 86, 87, 88, 89, 90, 91, 93, 95, 99, 319, 321, 506, 510 set out in Table 1)
This example includes a description of how to identify PG peptides having high correlation between immune reactivity and conservation across grass and non-grass pollen species.
Example 6
This example includes a description of how to examine release patterns of proteins from pollen. (Screening for co-release of NTGA's with major allergens from various pollen species) and detecting polypeptides of the invention by Mass Spectrometry. Raw pollen or defatted pollen of various pollen sources, Glass bottles (100 ml) for extraction, PD-10 columns with PE bed support combined with 10 ml syringe with silicone tubing, PBS buffer, pH 7.2 containing the following salts:
Salt Mw (g/mol) Cone. g/L Cone. mM
Sodium chloride NaCI 58.44 8.0 137
Potassium chloride KCI 74.55 0.2 2.7
Na-phosphate Na2HP04, 2H20 175.98 1.44 8.2
K-phosphate KH2P04 136.09 0.2 1.5
Phosphate cone : 8.2 + 1.5 = 9.7 mM phosphate
NaCI : μ = Vi * (137 * 12 + 137 * 12) = 137 mM
KCI : μ = /2 * (2.7 * 12 + 2.7 * 12) = 2.7 mM
Na2HP04: μ = Vi * ((8.2 * 2 * 12) + (8.2 * 22)) = 24.6 mM
KH2P04: μ = Vi * ((1.5 * 12) + (1.5 * 12)) = 1.5 mM
Total ionic strength : μ = 165.8 mM « 0.17 M
Extraction Procedure (at room temperature, 21-24°C) :
5.0 g of pollen are weighed into a glass bottle and 50 ml of PBS is added and the bottle is immediately rotated, first 5 minutes by hand and thereafter rotated in a sample rotator during the entire extraction.
5 ml of slurry is taken out after 20 sec, transferred to a column with a bed filter and dragged through the filter with a syringe. The syringe is immediately transferred to a filter unit and the extract is pushed through the combined filters into a labelled test tube. The tube is stored in an ice bath until the sample is pipetted in aliquots for further analysis and frozen. About 5 ml of the suspension is taken out at various time points.
Samples are analysed for NTGA and major allergens by MS (Mass Spectrometry) : Buffers/solutions for reduction, alkylation and digestion of the sample:
Sample buffer: 8 M urea in 0.4 M NH4HC03
DTT (45 mM) : Make it fresh from the frozen stock 1.0 M : 45 μΙ 1 M DTT + 955 μΙ water Iodoacetamide (IAA) : Make fresh solution, Iodoacetamide 100 mM,
Trypsin : Sigma T6567, Dissolve one vial in 20 μΙ of 1 mM HCI . This results in a solution containing 1 μ9/μΙ trypsin. After reconstitution in 1 mM HCI frozen aliquots can be stored for up to 4 weeks.
Enzymatic digestion with trypsin in solution for mass spectrometry: Dilute the dried sample in 5 μΙ of water, add 15 μΙ of sample buffer (8 M Urea in 0.4 M NH4HC03), add 5 μΙ 45 mM DTT, incubate at 56°C for 15 min, cool it to room temperature, add 5 μΙ of 100 mM Iodoacetamide, incubate in the dark in room temperature for 15 min, add 90 μΙ of water to lower the concentration of urea < l-2 M, add 1 μς trypsin, incubate at 37DC over night.
Chromatography: Reverse phase chromatography (Ultimate 3000 HPLC, Dionex) was performed using a C18 pre- and analytical column. The eluting peptides were sprayed directly into an ESI-QTOF mass spectrometer (MaXis, Bruker). After washing the trap column with 0.05% v/v formic acid for 5 min with a flow rate of 30 μΙ/min, the peptides were eluted with an acetonitrile gradient at a flow rate of 2 μΙ/min using solvent A: 0.05% v/v formic acid and solvent B: 80% v/v acetonitrile/0.04% v/v formic acid and the gradient: 4-50% B in 200 minutes; 50-80% B inlO minutes; 100% B in 10 min, 4% B in 5 min. Spectra were acquired in the mass range 50-2599 m/z and a spectra rate of 1.5 Hz. The instrument was tuned and calibrated using ESI-L Low concentration Tunning Mix from Agilent Technology.
Data acquisition and instrument control were carried out with Bruker Compass HyStar 3.2. Data processing was performed using DataAnalysis 4.0 (Bruker). Protein identification was performed using the program Biotools3.2 (Bruker) and two different data bases, i.e. Swiss prot and NCBInr. The MS/MS data sets for the tryptic digest were analysed using the following parameters; peptide tolerance 10 ppm and fragment tolerance 0.05 Da.
Procedure: The extraction samples were all evaporated (50 μΙ) and re-suspended in 5 μΙ of water. The sample is then reduced, alkylated and digested with trypsin. Resulting peptides are separated and identified by reversed phase chromatography followed by MS/MS.
Results: The release of major allergens from the various pollen species investigated is initiated almost instantly after hydration with buffer and the release continues with high rate within a time range of at least 30 to 60 minutes. Table 5 shows which NTGA's and the Cyn d homolog thereof that starts to release from pollen within a period overlapping with the release of major allergens from Phi p grass pollen (Phi p) and Cyn d grass pollen (Cyn d).
It is found that at least the NTGA's 1, 3, 4, 6, 5/64, 20, 24, 26, 30, 39/59, 47, 62, 76, 86/51, 89 and 91 (SEQ ID Nos: 520, 522, 523, 524, 525, 533, 535, 536, 539, 542, 546, 555, 558, 561, 563, 564) releases from Phi p grass pollen and the corresponding Cyn d homolog releases from Cyn d grass pollen. Those, NTGA's contain PG peptides with SEQ ID NOs: 1-18, 38-40, 41-55, 56-62, 63-89, 148-156, 169-182, 183-190, 211-232, 270-277, 338-348, 292-296, 354-356, 409-419, 467-481, 485-513 and 519, respectively Example 7
This example describes how to determine that T cells responding to a particular PG peptide (Phi p sequence) also recognizes a sequence of a corresponding peptide identified in another grass pollen species. PBMCs from Phi p reactive donors were expanded with individual PG peptides as well as peptides derived from major allergens of Phi p for 14 days (peptides shown in Table 8). For each peptide, the mismatch to a corresponding sequence in a non-grass pollen species or a pollen species other than Phi p were determined. Cytokine IL-5 responses were measured in response to the peptide itself, Phi p extract and extracts of the other pollen species.
Reponses to extracts and peptide pools were expressed as the relative fraction of the response to the peptide itself and plotted as a function of conservation of the peptide in the different extracts (Fig 1). The data points for each peptide are contained in Table 8. A clear hierarchy of responses was observed, with non-Phi p extracts in which the peptide is completely conserved (zero mismatches) showing the highest response, followed by non-Phi p extracts with 1-2 mismatches, and lowest responses with non-Phi p extracts with 3 or more mismatches. The exact same hierarchy was observed when analyzing peptides from the major allergens and the NTGA-derived peptides separately. Thus, Phi p epitopes conserved in other pollen species including pollen of Cyn d 1, Poa P, Ant o and Lol p were indeed able to induce cross-reactive T cell immune responses.
Example 8
This example describes how to determine the ability of a NTGA or a corresponding sequence found in another grass pollen species to relieve an allergic immune response in mice.
Initially, the sensitization pattern of an immunogen of the invention (NTGA 86/51) was investigated in BALB/c mice sensitized to Phi p extract (Figure 2). For the purposes of these studies, the immunogen were expressed in E. Coli using standard expression protocols.
Mice were sensitized by one intraperitoneal injection with Phi p extract adsorbed to aluminium hydroxide. Eleven days later the mice were euthanized and splenocytes were stimulated in vitro with Phi p extract, Phi p 1, Phi p 5, NTGA 86/51. The cells were incubated for 6 days at 37°C under 5% C02 and incorporated 3H-thymidine was counted and used as a measure for T cell proliferation.
The results show that the in vitro T-cell response towards NTGA 86/51 is much weaker compared to the response to Phi p 5. This correlates well with the human situation, where Phi p 5 is considered to be a major T-cell allergen. In line with this, the results also show that the response towards NTGA 86/51 is much weaker compared to the response towards the Phi p extract that was used for intraperitoneal sensitization.
Then the tolerance induction of NTGA 86/51 was investigated in a prophylactic mice model using sublingual administration (figure 3) The ability of NTGA 86/51 and NTGA 6 to induce prophylactic tolerance was investigated by SLIT treating naive BALB/c mice with NTGA 86/51 or NTGA 6 for two weeks (Monday-Friday) followed by one Phi p extract sensitization or sensitization to the immunogen itself (NTGA 86/51 or NTGA 6) as described above. Eleven days after the sensitization, splenocytes were harvested and stimulated in vitro with NTGA 86/51 as well as Phi p extract. The result is presented in Figures 3A-C and show that prophylactic SLIT treatment with
NTGA 86/51 is capable of inducing tolerance towards itself (3A) as well as towards the Phi p extract (3B), as shown by the reduced proliferation in splenocytes from the NTGA 86/51- treated mice compared to Buffer (sham) treated mice. In addition, it was shown that NTGA 6 is capable of inducing tolerance towards itself (3C) Bystander tolerance induction by prophylactic SLIT treatment with A0086 (Figure 4). The ability of NTGA 86/51 to induce bystander tolerance, i.e. to induce tolerance against a non- related protein was investigated by SLIT treating the mice for two weeks with NTGA 86/51 followed by an IP sensitization with NTGA 86/51 together with the unrelated protein ovalbumin (OVA). Following this splenocytes were stimulated in vitro either with NTGA 86/51to confirm the ability of this protein to induce tolerance towards itself, or with OVA to investigate if NTGA 86/51 can induce bystander tolerance towards an unrelated protein.
As shown in Figure 4A, prophylactic SLIT treatment with NTGA 86/51 is capable of inducing direct tolerance (towards NTGA 86/51 itself), as demonstrated by reduced proliferation of splenocytes from NTGA 86/51 treated mice compared to buffer treated mice. Furthermore, Figure 4B shows that SLIT treatment with OVA is also able to downregulate the NTGA 86/51 specific in vitro response, demonstrating bystander tolerance induction by OVA SLIT.
Likewise, SLIT treatment with NTGA 86/51 is also able to induce bystander tolerance, as measured by the decreased OVA specific in vitro proliferation of splenocytes from A NTGA 86/51-SLIT treated mice compared to buffer treated mice. The mechanism behind tolerance induction towards major allergens using proteins that are not themselves major allergens is believed to be induction of regulatory T-cells specific for the proteins used for SLIT treatment. At challenge it is therefore important that these proteins are present in the pollen grains in sufficient amounts to re-activate the regulatory T-cells, in order for the tolerance to spill over to the major allergens. When targeting multiple pollen allergies by one immunogen, it is crucial that this immunogen or one highly conserved thereto is present in all the pollen species of interest in sufficient amounts (pan- pollen immunogen). Furthermore, it may be important that the epitopes recognized by the regulatory T-cells induced during SLIT treatment is sufficiently conserved across the immunogens - otherwise the regulatory T-cells will not be re-activated and tolerance will not occur (illustrated in figure 5).
Whether an immunogen of the invention can relieve an immune response triggered by a pollen allergen in mice that are sensitized to the pollen allergen when starting SLIT treatment can be investigated in a therapeutic mice model. For example, BALB/cJ mice or HLA-transgenic mice may be IP sensitized with model allergen adsorbed to aluminium hydroxide (e.g. an extract of a grass pollen species, e.g. cyn d, Poa p, Phi p or a model allergen like OVA). Subsequently, the mice might be treated by sublingual
immunotherapy (SLIT) with an immunogen of the invention for a period of about 4 weeks, followed by about 2 weeks of intranasal challenge with model allergen together with the immunogen or model allergen alone to induce an allergic immune response in the airways. Mice are then sacrificed one day after the last challenge and blood, bronchoalveolar fluid (BAL), spleen and cervical lymph nodes may be collected for analysis. Clinically relevant readouts, such as sneezes, airway hyper-reactivity and presence of eosinophils, might be obtained on the last day of intranasal challenge. For example, sneezed may be observed in an 8 min-period after intranasal administration of model allergen and the numbers of sneezes be counted during this period. Airway hyper-reactivity may be determined using a whole body pletysmograph, airflow obstruction might be induced by increasing concentrations of aerosolized metacholine. Pulmonary airflow obstruction may be measured by enhanced pause (penh) in a period of 6 minutes after administration of metacholine. Differential counting of bronchial fluid (BAL) is performed after centrifugation of BAL fluid and removal of supernatant. The pellet was re-suspended in PBS and the fraction of eosinophils might be determined by an automated cell counter (Sysmex).
The results may show that an immunogen of the invention is able to reduce the number of sneezes, number of eosinophils, airway obstruction, T cell proliferation of spleen cells or cervical lymph nodes and may be shown to depend on the co-exposure of model allergen and immunogen at the target organ (airways). Whether SLIT treatment with pan pollen immunogens is capable of inducing tolerance that can be re-activated by a non-identical, but highly conserved immunogen from a different pollen source can be addressed in several different in vivo models, as outlined below.
Experiment 1 : 1 . SLIT treatment with immunogen A
2. IP Sensitization with immunogen B (contains conserved regions overlapping with A)
3. in vitro stimulation with immunogen B
Where results verify that the specific in vitro proliferation to immunogen B is down- regulated in mice SLIT-treated with immunogen A, then cross-species tolerance induction has been demonstrated for this immunogen, since the two immunogens are sufficiently similar in order for cross-species tolerance induction to occur.
Experiment 2:
1 . SLIT treatment with immunogen A
2. IP Sensitization with extract of pollen source containing immunogen B (pollen extract containing the homologous immunogen B)
Where results verify that the specific in vitro proliferation to immunogen B extract is down- regulated in mice SLIT-treated with immunogen A, then cross-species tolerance induction has been demonstrated for this immunogen. Furthermore, it has been demonstrated that pollen source B contains sufficient amounts of immunogen B to re-activate the tolerance induced by SLIT treatment with immunogen A.
In the above-mentioned mice model, Balb/cJ mice have been suggested . However, in vivo studies may instead be carried out in humanized mice models using transgenic mice, e.g. "HLA-DRB1*0401 transgenic mice" that may be obtained from Taconic. Also, in the above- mentioned mice models, the immune response against an allergen of a grass pollen (phi p grass extract) have been investigated, but other models may investigate the immune reponse against non-grass pollen allegens, e.g. allergens of weed or tree pollen, or there may be used model allergens like OVA protein.
Furthermore, the T cell responses in mice or humans may be evaluated by in-vitro T cell proliferation assays or ELISPOT assays. The production of IL-5 and IFN-γ from cultured PBMCs (Peripheral blood monocytes) obtained from mice or human in response to stimulation with an immunogen disclosed herein. Such assays are well known in the art. The assays may be able to analyze various different cytokines or cellular mediators associated with the immune response, e.g the cytokines IL-2, IL-4, IL-5, IL-9, IL-10, IL-12, IL-13, IL-17, IL-22, IL-31 and IFN-gamma.

Claims

Claims
1. A method for relieving an allergic immune response against a grass pollen allergen, wherein the grass pollen allergen is not a grass pollen allergen of the genus Phleum, in a subject in need thereof, comprising administering an effective amount of an immunogenic molecule, wherein said molecule comprises or consists of a) a polypeptide, which includes at least one amino acid sequence with 0, 1 or 2
mismatches compared to a sequence selected from any one of SEQ ID NOs: 63-89, 1-62 and 90-519; or b) a polypeptide comprising an amino acid sequence having at least 65% sequence similarity or identity to a sequence selected from any one of SEQ ID NOs: 525, 520-524 and 526-565.
2. The method according to any one of preceding claims, wherein the polypeptide of option b) comprises an amino acid sequence having at least 85% similarity or identity to a sequence selected from any one of SEQ ID NOs: 525, 520-524 and 526-565.
3. The method according to any one of preceding claims, wherein the subject is sensitized to a pollen allergen of a plant genus selected from any of Anthoxanthum
Cynodon, Lollium and Poa.
4. The method according to any one of preceding claims, wherein the allergic immune response at least is against a grass pollen allergen of a plant genus selected from any of Anthoxanthum, Cynodon, Lollium and Poa.
5. The method according to any one of preceding claims, wherein the subject is sensitized to grass pollen allergens of two or more of the genera Anthoxanthum, Cynodon, Lollium and Poa 6. The method according to any one of preceding claims, wherein the allergic immune response is against grass pollen allergens of two or more of the genera
Anthoxanthum, Cynodon, Lollium and Poa.
1. The method according to any one of preceding claims, wherein the polypeptide of option a) comprises a T cell epitope and includes at least one amino acid sequence with 0, 1 or 2 mismatches to a sequence selected from any one of SEQ ID NOs: 44, 8, 9, 10, 14, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 45, 47, 50, 51, 63, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 9798, 99, 102, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 135, 136, 137, 154, 155, 162, 163, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 187, 191, 192, 193, 194, 195, 197, 199, 200, 203, 220, 236, 237, 239, 240, 244, 250, 251, 252, 253, 254, 273, 274, 278, 279, 285, 293, 294, 301, 303, 305, 308, 309, 310, 312, 313, 314, 317,
318, 319, 320, 321, 322, 327, 341, 343, 345, 346, 347, 348, 354, 356, 357, 359, 360, 361, 362, 379, 385, 398, 404, 409, 413, 414, 416, 417, 419, 426, 427, 432, 433, 435, 436, 437, 439, 440, 441, 443, 444, 445, 446, 447, 448, 451, 452, 453, 454, 455, 456, 457, 460, 461, 463, 481, 485, 487, 489, 490, 492, 502, 504, 505, 506, 507, 508, 509, 510, 511, 513, 514, 515, 517, 518 and 519.
8. The method according to any one of preceding claims, wherein the polypeptide of option a) includes at least one amino acid sequence with 0 or 1 mismatches compared to a sequence selected from any one of SEQ ID NOs: 4, 8, 9, 10, 14, 19, 20, 21, 22, 24, 25, 26, 29, 30, 32, 33, 34, 35, 37, 38, 41, 42, 45, 65, 66, 67, 69, 71, 72, 73, 74, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 98, 99, 102, 106,
107, 109, 110, 116, 117, 135, 136154, 155, 162, 163, 166, 167, 168, 169, 170, 171, 172, 174, 175, 177, 178, 179, 180, 182, 187, 192, 193, 194, 199, 220, 236, 237, 239, 240, 244, 252, 254, 273, 274, 278, 279, 285, 305, 309, 310, 312, 319, 320, 321, 327, 345, 346, 347, 348, 354, 357, 359, 360, 361, 379, 385, 398, 409, 413, 416, 417, 419, 426, 427, 432, 433, 436, 440, 441, 443, 444, 445, 446, 447, 448, 453, 454, 455, 456, 457, 460, 461, 463, 481, 485, 487, 489, 502, 504, 506, 507, 508, 509, 510, 515 and 517.
9. The method according to any one of preceding claims, wherein the polypeptide of option a) comprises a T cell epitope and includes at least one amino acid sequence with 0, 1 or 2 mismatches to a sequence selected from any one of SEQ ID NOs: 19, 20, 24, 25, 26, 28, 32, 37, 69, 71, 72, 73, 74, 78, 79, 80, 81, 82, 84, 86, 87, 88, 89, 90, 91, 93, 95, 99, 107, 166, 169, 173, 174, 176, 180, 193, 303, 313, 319, 321, 492, 505, 506, 510, 511, 513, 514, 518 and 519.
10. The method according to any one of preceding claims, wherein the polypeptide of option a) comprises a T cell epitope and includes at least one amino acid sequence with 0, 1 or 2 mismatches to a sequence selected from any one of SEQ ID NOs: 19, 20, 24, 25, 26, 32, 37, 69, 71, 72, 73, 74, 78, 79, 80, 81, 82, 84, 86, 87, 88, 89, 90, 91, 93, 95, 99,
319, 321, 506 and 510.
1 1 . The method according to any one of preceding claims, wherein the polypeptide of option b) comprises a T cell epitope and comprises an amino acid sequence with at least 65% similarity or identity to a sequence selected from any of SEQ ID NOs: 520, 521, 523, 525, 526, 531, 533, 535, 538, 539, 541, 542, 557, 558, 559, 561, 563 and 564.
12. The method according to any one of the preceding claims, wherein a polypeptide of option a) and option b) is derived from a full length protein that co-releases/co-elutes with a major allergen of grass pollen of the genera Cynodon and Phleum, and optionally of a grass pollen selected from any of the plant genera Anthoxanthum, Dactylis, Festuca, Lollium, Paspalum, Phalaris, Poa and Sorghum,
13. The method according to any one of the preceding claims, wherein a polypeptide of option a) and option b) is derived from a full length protein that co-releases/co-elutes with a major allergen of grass pollen of the genera Phleum, Anthoxanthum, Cynodon, Lollium and Poa.
14. The method according to claim 12, wherein the polypeptide of option b) comprises a T cell epitope and comprises an amino acid sequence with at least 65% similarity or identity to a sequence selected from any of SEQ ID NOs: 520, 522, 523, 524, 525, 533, 535, 536, 539, 542, 546, 555, 558, 561, 563 and 564.
15. The method according to claim 12, wherein the polypeptide of option a) comprises a T cell epitope and includes at least one amino acid sequence with 0, 1 or 2 mismatches to a sequence selected from any one of SEQ ID NOs 1-18, 38-40, 41-55, 56- 62, 63-89, 148-156, 169-182, 183-190, 211-232, 270-277, 338-348, 292-296, 354-356, 409-419, 467-481, 485-513 and 519.
16. The method according to any one of claims claim 20 or 21, wherein the wild type amino acid sequence is a protein present in, based upon or derived from a pollen of a plant family of Poaceae, optionally the genera of any of Phleum, Anthoxanthum, Cynodon, Lollium and Poa.
17. The method according to any of the preceding claims, wherein a polypeptide of option a) and b) is present in, based upon or derived from a pollen protein of a plant genera selected from any of Anthoxanthum, Cynodon, Lollium and Poa.
18. The method according to claim 17, wherein a polypeptide b) comprises an amino acid sequence having at least 65% identity to any of SEQ ID Nos: 566-688.
19. The method according to any one of preceding claims, wherein the polypeptide of option a) includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 485-513; the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 563 (NTGA 89) .
20. The method according to any one of preceding claims, wherein the polypeptide of option a) includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 63-89; the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 525.
21 . The method according to any one of preceding claims, wherein the polypeptide of option a) includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 169- 182; the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 535.
22. The method according to any one of preceding claims, wherein the polypeptide of option a) includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 198-210; the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 538.
23. The method according to any one of preceding claims, wherein the polypeptide of option a) includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 519; the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 564.
24. The method according to any one of preceding claims, wherein the polypeptide of option a) includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 270-277, 338-348; the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 542.
25. The method according to any one of preceding claims, wherein the polypeptide of option a) includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 467-481 ; the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 561.
26. The method according to any one of preceding claims, wherein the polypeptide of option a) includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 301-305, 319-322; the polypeptide of option b) comprises an amino acid sequence having at least 65% sequence similarity or identity to SEQ ID NOs: 548.
27. The method according to any one of preceding claims, wherein the subject additional is sensitized to a grass pollen allergen of the plant genus Phleum.
28. The method according to any of the preceding claims, wherein the allergic immune response is atopic dermatitis, allergic conjunctivitis, allergic rhinitis, or allergic asthma.
29. The method or uses according to any one of preceding claims, wherein the subject has exhibited a symptom of, or suffers from, an allergic immune response.
30. The method according to any one of the preceding claims, wherein the method relieves one or more symptoms of an allergic immune response or delays the onset of symptoms, slows the progression of symptoms, or induce disease modification.
31 . The method according to claim 30, wherein the symptom(s) of an allergic reaction is selected from any of nasal symptoms in the form of itchy nose, sneezing, runny nose, blocked nose; conjunctival symptoms in the form of itchy eyes, red eyes, watery eyes; and respiratory symptoms in the form of decreased lung function.
32. The method according to any one of the preceding claims, wherein relieving in an allergic response is observed by the patient's need for less concomitant treatment with corticosteroids or HI antihistamines to suppress the symptoms.
33. The method according to any one of preceding claims, wherein the treatment comprises immunotherapy.
34. A molecule for use in relieving an allergic immune response against a pollen allergen other than a grass pollen allergen in a subject, wherein said molecule comprises or consists of a) a polypeptide, which includes at least one amino acid sequence with 0, 1 or 2 mismatches compared to a sequence selected from any one of SEQ ID NOs: 63-89, 1-62 and 90-519; or b) a polypeptide comprising an amino acid sequence having at least 65% sequence similarity or identity to a sequence selected from any one of SEQ ID NOs: 525, 520-524 and 526-565.
35. Use of a molecule for preparation of a medicament for relieving an allergic immune response against a pollen allergen other than a grass pollen allergen in a subject, wherein said molecule comprises or consists of a) a polypeptide, which includes at least one amino acid sequence with 0, 1 or 2
mismatches compared to a sequence selected from any one of SEQ ID NOs: 63-89, 1-62 and 90-519; or b) a polypeptide comprising an amino acid sequence having at least 65% sequence similarity or identity to a sequence selected from any one of SEQ ID NOs: 525, 520-524 and 526-565.
36. A molecule comprising or consisting of a) a polypeptide, which includes at least one amino acid sequence with 0, 1 or 2
mismatches compared to a sequence selected from any one of SEQ ID NOs: 63-89, 1-62 and 90-519; or b) a polypeptide comprising an amino acid sequence having at least 65% sequence similarity or identity to a sequence selected from any one of SEQ ID NOs: 525, 520-524 and 526-565.
37. The polypeptide according to claim 36, wherein the amino acid sequence of option b) has at least 85% similarity or identity to a sequence selected from any one of SEQ ID NOs: 525, 520-524 and 526-565.
38. The polypeptide according to any one of claims 36 and 37, wherein the protein is isolated or purified.
39. A composition comprising a polypeptide according to any one of claims 36 to 38 and a pharmaceutically acceptable ingredient or carrier.
40. The composition according to claim 39 that is lyophilized.
41 . The composition according to any one of claims 39 and 40 that is sterile.
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