WO2002022807A2 - Nouvelles proteines et molecules d'acide nucleique de dermatophagoides et leurs utilisations - Google Patents

Nouvelles proteines et molecules d'acide nucleique de dermatophagoides et leurs utilisations Download PDF

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Publication number
WO2002022807A2
WO2002022807A2 PCT/US2001/028730 US0128730W WO0222807A2 WO 2002022807 A2 WO2002022807 A2 WO 2002022807A2 US 0128730 W US0128730 W US 0128730W WO 0222807 A2 WO0222807 A2 WO 0222807A2
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Prior art keywords
seq
nucleic acid
protein
acid molecule
map
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PCT/US2001/028730
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English (en)
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WO2002022807A3 (fr
Inventor
Catherine A. Mccall
Shirley Wu Hunter
Eric R. Weber
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Heska Corporation
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Priority claimed from US09/662,293 external-priority patent/US7128921B1/en
Application filed by Heska Corporation filed Critical Heska Corporation
Priority to EP01973039A priority Critical patent/EP1328625A2/fr
Priority to AU2001292661A priority patent/AU2001292661A1/en
Priority to CA002420459A priority patent/CA2420459A1/fr
Priority to JP2002527249A priority patent/JP2004529605A/ja
Publication of WO2002022807A2 publication Critical patent/WO2002022807A2/fr
Publication of WO2002022807A3 publication Critical patent/WO2002022807A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43531—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from mites
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents

Definitions

  • the present invention also relates to reagents comprising non- proteinaceous epitopes that bind to IgE in mite-allergic dogs and/or cats as well as to antibodies raised against such epitopes.
  • the present invention also relates to therapeutic compositions or assay kits comprising such non-proteinaceous epitopes, as well as to methods to identify and/or desensitize an animal susceptible to an allergic response to a mite, comprising the use of non-proteinaceous epitopes of the present invention.
  • One embodiment of the present invention is at least one of the following isolated nucleic acid molecules: (a) a nucleic acid molecule comprising at least about 150 nucleotides, wherein such a nucleic acid molecule hybridizes, in a solution comprising IX SSC and 0% formamide, at a temperature of about 50°C, to a nucleic acid molecule comprising at least one of the following nucleic acid sequences: SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, and a nucleic acid sequence encoding a protein comprising the amino acid sequence of SEQ ID NO:33 and a complement thereof; and (b) a nucleic acid molecule comprising a fragment of any
  • Another embodiment of the present invention is an isolated protein encoded by at least one of the following nucleic acid molecules: (a) a nucleic acid molecule comprising at least about 150 nucleotides, wherein such a nucleic acid molecule hybridizes, in a solution comprising IX SSC and 0% formamide, at a temperature of about 50 °C, to a nucleic acid molecule comprising at least one of the following nucleic acid sequences: SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, and a complement of a nucleic acid sequence encoding a protein comprising the amino acid sequence SEQ ID NO: 33; and (b) a nucleic acid molecule comprising a fragment of any of the nucleic acid molecules of (a), wherein the fragment comprises at least about 15 nucleotides.
  • the present invention also includes an antibody that selectively binds to a protein of the present invention as well as methods to produce and use such proteins or antibodies.
  • the present invention also includes a therapeutic composition for treating an allergic response to a mite.
  • a therapeutic composition includes at least one of the following desensitizing compounds: (a) an isolated nucleic acid molecule of the present invention; (b) an isolated mite allergenic protein of the present invention; (c) a mimetope of such a mite allergenic protein; (d) a mutein of such a mite allergenic protein; (e) an antibody to such a mite allergic protein; and (f) an inhibitor of binding of such a mite allergic protein to IgE.
  • a method to desensitize a host animal to an allergic response to a mite includes the step of administering to the animal a therapeutic composition of the present invention.
  • One embodiment of the present invention is an assay kit for testing if an animal is susceptible to or has an allergic response to a mite.
  • a kit includes an isolated protein of the present invention and a means for determining if the animal is susceptible to or has that allergic response.
  • a means includes use of such a protein to identify animals susceptible to or having allergic responses to mites.
  • the present invention also includes a method to identify an animal susceptible to or having an allergic response to a mite. Such a method includes the steps of: (a) contacting an isolated protein of the present invention with antibodies of an animal; and (b) determining immunocomplex formation between the protein and the antibodies, wherein formation of the immunocomplex indicates that the animal is susceptible to or has such an allergic response.
  • the present invention includes a reagent that comprises a non-proteinaceous epitope having at least one of the following identifying characteristics: (a) the epitope is resistant to ⁇ -elimination of peptides; (b) the epitope is resistant to Proteinase-K digestion; and (c) the epitope is reactive to a test designed to detect glycosylated proteins.
  • a non-proteinaceous epitope having at least one of the following identifying characteristics: (a) the epitope is resistant to ⁇ -elimination of peptides; (b) the epitope is resistant to Proteinase-K digestion; and (c) the epitope is reactive to a test designed to detect glycosylated proteins.
  • Such an epitope binds to at least one of the following antibodies: canine IgE from dogs allergic to mites and feline IgE from cats allergic to mites.
  • an isolated antibody that selectively binds such a non-proteinaceous epitope
  • the present invention also relates to therapeutic compositions and assay kits comprising a non-proteinaceous epitope of the present invention, as well as methods to identify and/or desensitize an animal susceptible to an allergic response to a mite, comprising the use of a non-proteinaceous epitope of the present invention.
  • Fig. 1 illustrates high molecular weight Der proteins resolved by 12% Tris- Glycine SDS-PAGE.
  • Fig. 2 illustrates an about 60 kD er protein resolved by 14% Tris-Glycine SDS-PAGE.
  • One embodiment of the present invention is an isolated Dermatophagoides allergenic composition
  • a composition produced by a method comprising: (1) applying soluble proteins of a Dermatophagoides extract to a gel filtration column; (2) collecting excluded protein from the gel filtration column and applying the excluded protein to an anion exchange column; and (3) eluting proteins bound to the anion exchange column with about 0.3 M Tris-HCl, pH 8 to obtain the Dermatophagoides allergenic composition; and (b) a composition comprising a peptide of a protein produced in accordance with step (a), in which the allergenic composition is capable of a biological function including binding to IgE, stimulating a B lymphocyte response and stimulating a T lymphocyte response.
  • a or “an” entity refers to one or more of that entity; for example, a protein, a nucleic acid molecule, an antibody, an inhibitor, a compound or a therapeutic composition refers to "one or more” or “at least one” protein, nucleic acid molecule, antibody, inhibitor, compound or therapeutic composition respectively.
  • the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein.
  • the terms “comprising”, “including”, and “having” can be used interchangeably.
  • an isolated, or biologically pure, protein is a protein that has been removed from its natural milieu.
  • a Der HMW-map protein can be a full-length protein or any homolog of such a protein.
  • a protein can be a polypeptide or a peptide, as the terms are used by those of skill in the art.
  • a Der HMW-map protein comprises at least a portion of a Der HMW-map protein that comprises at least one epitope recognized by an IgE antibody (i.e., a protein of the present invention binds to an IgE antibody), an antibody on the surface of a B lymphocyte and/or a T cell receptor in the presence of a major histocompatability complex (MHC) molecule from an animal demonstrating IgE-mediated pathogenesis to a Der HMW-map protein.
  • IgE antibody i.e., a protein of the present invention binds to an IgE antibody
  • MHC major histocompatability complex
  • a peptide of the present invention includes a Der HMW-map protein of the present invention that is capable of binding to IgE, desensitizing an animal against mite allergen, stimulating a B lymphocyte response, and/or stimulating a T lymphocyte response.
  • a peptide of the present invention comprises a B lymphocyte epitope or a T lymphocyte epitope.
  • a peptide having a B lymphocyte epitope can bind to an antibody.
  • a peptide having a T lymphocyte epitope can bind to a MHC molecule in such a manner that the peptide can stimulate a T lymphocyte through a T cell receptor.
  • a Der HMW-map protein of the present invention can be identified in a straight-forward manner by the protein's ability to induce an allergic response to Der HMW-map protein.
  • Examples of Der HMW-map protein homologs include Der HMW-map protein in which amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of glycerophosphatidyl inositol) such that the homolog is capable of inducing an allergic response to a natural Der HMW-map protein.
  • nucleic acid sequence SEQ ID NO: 14 represents the deduced sequence of the coding strand of a cDNA (complementary DNA) denoted herein as Der HMW-map gene nucleic acid molecule nDerf98 1752 , the production of which is disclosed in the Examples.
  • Nucleic acid molecule nDerf98 1752 comprises an apparently full-length coding region.
  • the complement of SEQ ID NO: 14 (represented herein by SEQ ID NO: 16) refers to the nucleic acid sequence of the strand complementary to the strand having SEQ ID NO: 14, which can easily be determined by those skilled in the art.
  • a Der HMW-map gene or nucleic acid molecule can be an allelic variant that includes a similar but not identical sequence to SEQ ID NO: 14 or SEQ ID NO: 16, or any other Der HMW-map nucleic acid sequence cited herein.
  • an allelic variant of a Der HMW-map gene including SEQ ID NO: 14 or SEQ ID NO: 16 is a gene that occurs at essentially the same locus (or loci) in the genome as the gene including SEQ ID NO: 14 and SEQ ID NO: 16, but which, due to natural variations caused by, for example, mutation or recombination, has a similar but not identical sequence. Because natural selection typically selects against alterations that affect function, allelic variants (i.e.
  • the extent of homology required to form a stable hybrid under stringent conditions can vary depending on whether the homologous sequences are interspersed throughout a given nucleic acid molecule or are clustered (i.e., localized) in distinct regions on a given nucleic acid molecule.
  • the minimal size of a Der HMW-map protein homolog of the present invention is from about 4 to about 6 amino acids in length.
  • the maximal size of a nucleic acid molecule encoding a Der HMW-map protein of the present invention because a nucleic acid molecule of the present invention can include a portion of a gene, an entire gene, or multiple genes.
  • the preferred size of a protein encoded by a nucleic acid molecule of the present invention depends on whether a full-length, fusion, multivalent, or functional portion of such a protein is desired.
  • Stringent hybridization conditions are determined based on defined physical properties of the gene to which the nucleic acid molecule is being hybridized, and can be defined mathematically. Stringent hybridization conditions are those experimental parameters that allow an individual skilled in the art to identify significant similarities between heterologous nucleic acid molecules. These conditions are well known to those skilled in the art. See, for example, Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, and Meinkoth, et al, 1984, Anal. Biochem. 138, 267-284.
  • the determination of hybridization conditions involves the manipulation of a set of variables including the ionic strength (M, in moles/liter), the hybridization temperature (°C), the concentration of nucleic acid helix destabilizing agents (such as formamide), the average length of the shortest hybrid duplex (n), and the percent G + C composition of the fragment to which an unknown nucleic acid molecule is being hybridized.
  • M the ionic strength
  • °C the hybridization temperature
  • concentration of nucleic acid helix destabilizing agents such as formamide
  • n average length of the shortest hybrid duplex
  • percent G + C composition of the fragment to which an unknown nucleic acid molecule is being hybridized For nucleic acid molecules of at least about 150 nucleotides, these variables are inserted into a standard mathematical formula to calculate the melting temperature, or T m , of a given nucleic acid molecule.
  • T d dissociation temperature
  • T d 4(G + C) + 2(A + T).
  • a temperature of 5°C below T d is used to detect hybridization between perfectly matched molecules.
  • T m decreases about 1°C for each 1% of mismatched base-pairs for hybrids greater than about 150 bp
  • T d decreases about 5°C for each mismatched base-pair for hybrids below about 50 bp.
  • Hybridization reactions are often carried out by attaching the nucleic acid molecule to be hybridized to a solid support such as a membrane, and then hybridizing with a labeled nucleic acid molecule, typically referred to as a probe, suspended in a hybridization solution.
  • a labeled nucleic acid molecule typically referred to as a probe
  • Examples of common hybridization reaction techniques include, but are not limited to, the well-known Southern and northern blotting procedures.
  • the actual hybridization reaction is done under non-stringent conditions, i.e., at a lower temperature and/or a higher salt concentration, and then high stringency is achieved by washing the membrane in a solution with a higher temperature and/or lower salt concentration in order to achieve the desired stringency.
  • nucleic acid molecule that hybridizes under stringent hybridization conditions with a Dermatophagoides farinae and/or Dermatophagoides pteronyssius nucleic acid molecule of about 150 bp in length
  • the following conditions could preferably be used.
  • the average G + C content of Dermatophagoides farinae and Dermatophagoides pteronyssius DNA is about 39%.
  • the unknown nucleic acid molecules would be attached to a support membrane, and the 150 bp probe would be labeled, e.g. with a radioactive tag.
  • the hybridization reaction could be carried out in a solution comprising 2X SSC and 0% formamide, at a temperature of about 37°C (low stringency conditions).
  • Solutions of differing concentrations of SSC can be made by one of skill in the art by diluting a stock solution of 20X SSC (175.3 gram NaCl and about 88.2 gram sodium citrate in 1 liter of water, pH 7) to obtain the desired concentration of SSC.
  • the skilled artisan would calculate the washing conditions required to allow up to 30% base-pair mismatch. For example, in a wash solution comprising IX SSC and 0% formamide, the T m of perfect hybrids would be about 80°C:
  • hybridization washes would be carried out at a temperature of about 50°C. It is thus within the skill of one in the art to calculate additional hybridization temperatures based on the desired percentage base-pair mismatch, formulae and G/C content disclosed herein.
  • nucleic acid molecule to be tested for hybridization against nucleic acid molecules of the present invention having sequences specified herein becomes longer than 150 nucleotides, the T m for a hybridization reaction allowing up to 30% base-pair mismatch will not vary significantly from 50°C.
  • Der HMW-map proteins of the present invention include proteins that, when submitted to reducing 12% Tris gly cine SDS-PAGE, migrate as bands at a molecular weight of from about 98 kD to about 109 kD, as shown in Fig. 1.
  • the bands in Fig. 1 are obtained when proteins are collected from Dermataphagoides farinae mites using the method described in detail in Example 1.
  • Der HMW-map proteins of the present invention includes proteins having a molecular weight ranging from about 90 kD to about 120 kD, and more preferably from about 98 kD to about 109 kD.
  • Preferred Der HMW-map proteins of the present invention include map A and mapB, the identification of which is described in the Examples section.
  • Der HMW-map proteins of the present invention include proteins that, when submitted to reducing 14% Tris gly cine SDS-PAGE, migrate as a band at a molecular weight of about 60 kD, as shown in Fig. 2.
  • the band in Fig. 2 is obtained when proteins are collected from Dermataphagoides farinae mites using the method described in detail in Example 9.
  • Der HMW-map proteins of the present invention includes proteins having a molecular weight of about 60 kD.
  • Preferred Der HMW-map proteins of the present invention include mapD, the identification of which is described in the Examples section.
  • a preferred Der HMW-map protein includes a protein encoded by a nucleic acid molecule which is at least about 50 nucleotides, or about 150 nucleotides, and which hybridizes under conditions which preferably allow about 40% or less base pair mismatch, more preferably under conditions which allow about 35% or less base pair mismatch, more preferably under conditions which allow about 30% or less base pair mismatch, more preferably under conditions which allow about 25% or less base pair mismatch, more preferably under conditions which allow about 20% or less base pair mismatch, more preferably under conditions which allow about 15% or less base pair mismatch, more preferably under conditions which allow about 10% or less base pair mismatch and even more preferably under conditions which allow about 5% or less base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45 and a nucleic acid sequence en
  • Another embodiment of the present invention includes a Der HMW-map protein encoded by a nucleic acid molecule selected from the group consisting of: a nucleic acid molecule comprising at least about 150 nucleotides, wherein said nucleic acid molecule comprising at least about 150 nucleotides hybridizes, in a solution comprising IX SSC and 0% formamide, at a temperature of about 50°C, to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, and a complement of a nucleic acid sequence encoding a protein comprising the amino acid sequence SEQ ID NO: 33; and a nucleic acid molecule comprising a fragment of any of said nucleic acid molecules comprising at least about 15 nucleotides.
  • Yet another preferred Der HMW-map protein of the present invention includes a protein encoded by a nucleic acid molecule which is preferably at least about 60% identical, more preferably at least about 65% identical, more preferably at least about 70% identical, more preferably at least about 75% identical, more preferably at least about 80% identical, more preferably at least about 85% identical, more preferably at least about 90% identical and even more preferably at least about 95% identical to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, and/or a complement of a nucleic acid sequence encoding a protein comprising the amino acid sequence SEQ ID NO:33; also preferred are fragments of such proteins.
  • Additional preferred Der HMW-map proteins of the present invention include proteins having the amino acid sequence SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, and proteins comprising homologs of a protein having the amino acid sequence SEQ ID NO:l, SEQ ID NO:2,
  • a preferred isolated protein of the present invention is a protein encoded by at least one of the following nucleic acid molecules: nDerf98 1752 , nDerf98 1665 , nDerf98 1608 , nDerp98 ⁇ 621 , nDerp98 1527 , nDerp98 1470 , nDerf60 510 , or allelic variants of any of these nucleic acid molecules.
  • Another preferred isolated protein is encoded by a nucleic acid molecule having nucleic acid sequence SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43; or a protein encoded by an allelic variant of any of these listed nucleic acid molecule.
  • Translation of SEQ ID NO: 14, the coding strand of nDerf98 1752 yields a protein of about 555 amino acids, denoted herein as PDerf98 555 , the amino acid sequence of which is presented in SEQ ID NO: 15, assuming a first in-frame codon extending from nucleotide 1 to nucleotide 3 of SEQ ID NO: 14.
  • SEQ ID NO: 14 The complementary strand of SEQ ID NO: 14 is presented herein as SEQ ID NO: 16.
  • the amino acid sequence of PDerf98 555 is encoded by the nucleic acid molecule nDerf98 1665 , having a coding strand denoted SEQ ID NO: 17 and a complementary strand denoted SEQ ID NO: 19.
  • Analysis of SEQ ID NO: 15 suggests the presence of a signal peptide spanning from about amino acid 1 through about amino acid 19.
  • SEQ ID NO:43 the coding strand of nDerf60 5K
  • PDerf60 170 the amino acid sequence of which is presented as SEQ ID NO:44, assuming a first in-frame codon extending from nucleotide 1 to nucleotide 3 of SEQ ID NO:43.
  • SEQ ID NO:45 The complementary sequence to SEQ ID NO:43 is presented herein as SEQ ID NO:45.
  • Preferred Der HMW-map proteins of the present invention include proteins that are at least about 45%, preferably at least about 50%, more preferably at least about 55%, even more preferably at least about 60%, even more preferably at least about 65%, even more preferably at least about 70%, even more preferably at least about 75%, even more preferably at least about 80%, even more preferably at least about 85%, even more preferably at least about 90%, and even more preferably about 95% identical to PDerf98 555 .
  • Der HMW-map protein comprising PDerf98 555 , PDerf98 536 _ PDerp98 509 , PDerp98 490 , and/or PDerf60 170 ; and proteins encoded by allelic variants of nucleic acid molecules encoding proteins PDerf98 555 , PDerf98 536 ⁇ PDerp98 509 , PDerp98 490 , and/or PDerf60 170 .
  • Other preferred Der HMW-map proteins of the present invention include proteins having amino acid sequences that are at least about 45%, preferably at least about 50%, more preferably at least about 55%, even more preferably at least about 60%, even more preferably at least about 65%, even more preferably at least about 70%, even more preferably at least about 75%, even more preferably at least about 80%, even more preferably at least about 85%, even more preferably at least about 90%, and even more preferably about 95% identical to amino acid sequence SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO
  • Der HMW-map proteins comprising amino acid sequences SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, and/or SEQ ID NO:44; and Der HMW-map proteins encoded by allelic variants of nucleic acid molecules encoding Der HMW-map proteins having amino acid sequences SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID
  • Der HMW-map proteins comprise amino acid sequence SEQ ID NO: 15, SEQ ID NO:35, and/or SEQ ID NO:44 (including, but not limited to, the proteins consisting of amino acid sequence SEQ ID NO: 15, SEQ ID NO:35, and/or SEQ ID NO:44, fragments thereof, fusion proteins and multivalent proteins), and proteins encoded by allelic variants of nucleic acid molecules encoding proteins having amino acid sequence SEQ ID NO: 15, SEQ ID NO:35, and/or SEQ ID NO:44.
  • a Der HMW-map protein encoded by at least a portion nDerf98 1752 , nDerp98 1621 , and/or nDerf60 510 or by an allelic variant of these nucleic acid molecules.
  • a preferred Der HMW-map protein of the present invention is encoded by a nucleic acid molecule comprising an apparently full-length Der HMW-map coding region, i.e., a nucleic acid molecule encoding an apparently full-length Der HMW-map protein.
  • a Der HMW-map protein of the present invention is a fusion protein that includes a Der HMW-map protein-containing domain attached to one or more fusion segments.
  • Suitable fusion segments for use with the present invention include, but are not limited to, segments that can: enhance a protein's stability; act as an immunopotentiator to enhance an immune response against a er HMW-map protein, reduce an IgE response against a er HMW-map protein; and/or assist purification of a Der HMW-map protein (e.g., by affinity chromatography).
  • a suitable fusion segment can be a domain of any size that has the desired function (e.g., imparts increased stability, imparts increased immunogenicity to a protein, reduces an IgE response, and/or simplifies purification of a protein).
  • Such a multivalent desensitizing protein can be produced by culturing a cell transformed with a nucleic acid molecule comprising two or more nucleic acid domains joined together in such a manner that the resulting nucleic acid molecule is expressed as a multivalent desensitizing compound containing at least two desensitizing compounds capable of desensitizing an animal from allergens.
  • multivalent desensitizing compounds include, but are not limited to, a Der HMW-map protein of the present invention attached to one or more compounds that desensitize against allergies caused by one or more allergens, such as a plant allergen, an animal allergen, a parasite allergen or an ectoparasite allergen, including, but not limited to: pant allergens from grass, Meadow Fescue, Curly Dock, plantain, Mexican Firebush, Lamb's Quarters, pigweed, ragweed, sage, elm, cocklebur, Box Elder, walnut, cottonwood, ash, birch, cedar, oak, mulberry, cockroach, Dermatophagoides, Alternaria, Aspergillus, Cladosporium, Fusarium, Helminthosporium, Mucor, Penicillium, Pullularia, R izopus and/or Tricophyton; parasite allergens from helminths; or ectoparasite allergens from a
  • Mimetopes can be, but are not limited to: peptides that have been modified to decrease their susceptibility to degradation; anti-idiotypic and/or catalytic antibodies, or fragments thereof; non-proteinaceous immunogenic portions of an isolated protein (e.g., carbohydrate structures); synthetic or natural organic or inorganic molecules, including nucleic acids; and/or any other peptidomimetic compounds.
  • Mimetopes of the present invention can be designed using computer- generated structures of Der HMW-map protein of the present invention.
  • a mutein refers to a particular homolog of aDer HMW-map protein in which desired amino acid residues have been substituted or removed.
  • Preferred muteins of the present invention include Der HMW-map protein homologs in which amino acid residues have been changed to reduce an anaphylactic reaction by an animal when the mutein is administered to the animal in therapeutic doses.
  • More preferred muteins of the present invention include Der HMW-map protein homologs in which one or more cysteine residues of a Der HMW-map protein have been replaced or removed.
  • Methods to produce muteins are known to those of skill in the art and are disclosed herein.
  • a mutein is produced using recombinant techniques.
  • Another embodiment of the present invention is an isolated nucleic acid molecule comprising a Der HMW-map nucleic acid molecule.
  • the identifying characteristics of such nucleic acid molecules are heretofore described.
  • a nucleic acid molecule of the present invention can include an isolated natural Der HMW-map gene or a homolog thereof, the latter of which is described in more detail below.
  • a nucleic acid molecule of the present invention can include one or more regulatory regions, full-length or partial coding regions, or combinations thereof.
  • the minimal size of a nucleic acid molecule of the present invention is a size sufficient to allow the formation of a stable hybrid (i.e., hybridization under stringent hybridization conditions) with the complementary sequence of another nucleic acid molecule.
  • Isolated Der HMW-map nucleic acid molecules, and homologs thereof can include, for example, natural allelic variants and nucleic acid molecules modified by nucleotide insertions, deletions, substitutions, and/or inversions in a manner such that the modifications do not substantially interfere with the nucleic acid molecule's ability to encode aDer HMW-map protein of the present invention.
  • Nucleic acid molecule homologs can be selected by hybridization with a Der HMW-map nucleic acid molecule or by screening the function of a protein encoded by the nucleic acid molecule (e.g., ability to elicit an immune response against at least one epitope of a Der HMW-map protein or to effect Der HMW-map activity).
  • Allelic variants typically encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared. Allelic variants can also comprise alterations in the 5' or 3' untranslated regions of the gene (e.g., in regulatory control regions). Allelic variants are well known to those skilled in the art and would be expected to be found within a given dust mite since the genome is diploid and/or among a group of two or more dust mites.
  • the present invention also includes variants due to laboratory manipulation, such as, but not limited to, variants produced during polymerase chain reaction amplification.
  • An isolated nucleic acid molecule of the present invention can include a nucleic acid sequence that encodes at least one Der HMW-map protein of the present invention, examples of such proteins being disclosed herein.
  • nucleic acid molecule primarily refers to the physical nucleic acid molecule and the phrase “nucleic acid sequence” primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a Der HMW-map protein.
  • a preferred nucleic acid molecule of the present invention when administered to an animal, is capable of desensitizing that animal from allergic reactions caused by a Der HMW-map allergen.
  • a nucleic acid molecule can be, or encode, an antisense RNA, a molecule capable of triple helix formation, a ribozyme, or other nucleic acid-based drug compound.
  • One embodiment of the present invention is an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a Der HMW-map gene.
  • Stringent hybridization conditions refer to standard hybridization conditions described herein.
  • a preferred nucleic acid molecule of the present invention includes an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene encoding a protein comprising an amino acid sequence including SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID
  • a more preferred nucleic acid molecule of the present invention includes an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with the complement of a nucleic acid sequence that encodes a protein comprising an amino acid sequence including SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, and/or SEQ ID NO:44.
  • a more preferred nucleic acid molecule of the present invention includes an isolated nucleic acid molecule selected from the group consisting of: a nucleic acid molecule comprising at least about 150 nucleotides, wherein said nucleic acid molecule comprising at least about 150 nucleotides hybridizes, in a solution comprising IX SSC and 0% formamide, at a temperature of about 50°C, to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO: 45 and/or a nucleic acid sequence encoding a protein comprising the amino acid sequence SEQ ID NO:33 and a complement thereof.
  • the present invention also includes fragments of any nucleic acid molecule disclosed herein.
  • a fragment can include any nucleic acid molecule or nucleic acid sequence, the size of which can range between a length that is smaller than a sequence identified by a SEQ ID NO of the present invention and the minimum size of an oligonucleotide as defined herein.
  • the size of a fragment of the present invention can be any size that is less than about 1752 nucleotides and greater than 11 nucleotides in length.
  • a preferred Der HMW-map nucleic acid molecule includes an isolated nucleic acid molecule which is at least about 50 nucleotides, or at least about 150 nucleotides, and which hybridizes under conditions which preferably allow about 40% or less base pair mismatch, more preferably under conditions which allow about 35% or less base pair mismatch, more preferably under conditions which allow about 30% or less base pair mismatch, more preferably under conditions which allow about 25% or less base pair mismatch, more preferably under conditions which allow about 20% or less base pair mismatch, more preferably under conditions which allow about 15% or less base pair mismatch, more preferably under conditions which allow about 10% or less base pair mismatch and even more preferably under conditions which allow about 5% or less base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:34, SEQ ID NO:36
  • nucleic acid molecule comprising at least about 150 base-pairs, wherein the nucleic acid molecule hybridizes, in a solution comprising IX SSC and 0% formamide, at a temperature of about 50°C, to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, and/or a nucleic acid sequence encoding a protein comprising the amino acid sequence SEQ ID NO: 33 and a complement thereof.
  • Additional preferred nucleic acid molecules of the present invention include fragments of an isolated nucleic acid molecule comprising at least about 150 base-pairs, wherein said nucleic acid molecule hybridizes, in a solution comprising IX SSC and 0% formamide, at a temperature of about 50°C, to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45 and a nucleic acid sequence encoding a protein comprising the amino acid sequence SEQ ID NO:33 and complement thereof.
  • Additional preferred Der HMW-map nucleic acid molecules of the present invention include an isolated nucleic acid molecule which is at least about 50 nucleotides, or at least about 150 nucleotides, comprising a nucleic acid sequence that is preferably at least about 60% identical, more preferably at least about 65% identical, more preferably at least about 70% identical, more preferably at least about 75% identical, more preferably at least about 80% identical, more preferably at least about 85% identical, more preferably at least about 90% identical and even more preferably at least about 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, and a nucleic acid sequence en
  • One embodiment of the present invention is a nucleic acid molecule comprising all or part of nucleic acid molecules nDerf98 1752 , nDerf98 1665 and nDerf98 1608 , nDerp98 1621 , nDerp98 1527 , nDerp98 1470 , and/or nDerf60 510 , or allelic variants of these nucleic acid molecules.
  • nucleic acid molecule of the present invention includes at least a portion of nucleic acid sequence SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45 and/or a nucleic acid sequence encoding a protein comprising the amino acid sequence SEQ ID NO: 33, as well as allelic variants of nucleic acid molecules having these nucleic acid sequences and homologs of nucleic acid molecules having these nucleic acid sequences; preferably such a homolog encodes or is complementary to a nucleic acid molecule that encodes at least one epitope that elicits and an immune response against a protein having an amino acid sequence SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ
  • nucleic acid molecules can include nucleotides in addition to those included in the SEQ ID NOs, such as, but not limited to, a full-length gene, a full-length coding region, a nucleic acid molecule encoding a fusion protein, or a nucleic acid molecule encoding a multivalent protective compound.
  • a Der ITMW-map nucleic acid molecule of the present invention encodes a protein that is at least about 45%, preferably at least about 50%, more preferably at least about 55%, even more preferably at least about 60%, even more preferably at least about 65%, even more preferably at least about 70%, even more preferably at least about 75%, even more preferably at least about 80%, even more preferably at least about 85%, even more preferably at least about 90%, and even more preferably about 95% identical to PDerf98 555 , PDerp98 509 , and/or PDerf60 ⁇ 70 .
  • nucleic acid molecule encoding PDerf98 555 , PDerf98 536 , PDerp98 509 , PDerp98 490 , and/or PDerf60 170 , and/or an allelic variant of such nucleic acid molecules.
  • the present invention also includes a er HMW-map nucleic acid molecule encoding a protein having at least a portion of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 18 , SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:41, and/or SEQ ID NO:44, as well as allelic variants of aDer HMW-map nucleic acid molecule encoding a protein having these sequences, including
  • a preferred Der HMW-map nucleic acid molecule encodes a er HMW-map protein comprising at least about at least about 35 amino acids in length, preferably at least about 50 amino acids in length, more preferably at least about 100 amino acids in length, more preferably at least about 200 amino acids in length, even more preferably at least about 250 amino acids in length.
  • the present invention also includes nucleic acid molecules that are oligonucleotides capable of hybridizing, under stringent hybridization conditions, with complementary regions of other, preferably longer, nucleic acid molecules of the present invention such as those comprising Der HMW-map nucleic acid molecules or other Der HMW-map nucleic acid molecules.
  • Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either. The minimum size of such oligonucleotides is the size required for formation of a stable hybrid between an oligonucleotide and a complementary sequence on a nucleic acid molecule of the present invention.
  • One embodiment of the present invention includes a recombinant vector, which includes at least one isolated nucleic acid molecule of the present invention, inserted into any vector capable of delivering the nucleic acid molecule into a host cell.
  • a vector contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to nucleic acid molecules of the present invention and that preferably are derived from a species other than the species from which the nucleic acid molecule(s) are derived.
  • the vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid.
  • Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulation of Der HMW- map nucleic acid molecules of the present invention.
  • a recombinant molecule comprises a nucleic acid molecule of the present invention operatively linked to an expression vector.
  • the phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell.
  • an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified nucleic acid molecule.
  • the expression vector is also capable of replicating within the host cell.
  • Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids.
  • Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, endoparasite, insect, other animal, and plant cells.
  • Preferred expression vectors of the present invention can direct gene expression in bacterial, yeast, insect and mammalian cells and more preferably in the cell types disclosed herein.
  • expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present invention.
  • recombinant molecules of the present invention include transcription control sequences.
  • Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art.
  • Mycobacteria T ⁇ choplusia, BHK (baby hamster kidney) cells, MDCK cells (normal dog kidney cell line for canine herpesvirus cultivation), CRFK cells (normal cat kidney cell line for feline herpesvirus cultivation), CV-1 cells (African monkey kidney cell line used, for example, to culture raccoon poxvirus), COS (e.g., COS-7) cells, and Vero cells.
  • Particularly preferred host cells are Escherichia coli, including E.
  • a recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules of the present invention operatively linked to an expression vector containing one or more transcription control sequences.
  • the phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell.
  • an expressed recombinant protein of the present invention may be improved by fragmenting, modifying, or derivatizing nucleic acid molecules encoding such a protein.
  • Isolated Der HMW-map proteins of the present invention can be produced in a variety of ways, including production and recovery of natural proteins, production and recovery of recombinant proteins, and chemical synthesis of the proteins.
  • an isolated protein of the present invention is produced by culturing a cell capable of expressing the protein under conditions effective to produce the protein, and recovering the protein.
  • a preferred cell to culture is a recombinant cell of the present invention. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production.
  • Proteins of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization. Proteins of the present invention are preferably retrieved in "substantially pure” form. As used herein, “substantially pure” refers to a purity that allows for the effective use of the protein as a therapeutic composition or diagnostic. A therapeutic composition for animals, for example, should exhibit no substantial toxicity and preferably should be capable of desensitizing a treated animal.
  • the present invention also includes isolated (i.e., removed from their natural milieu) antibodies that selectively bind to a Der HMW-map protein of the present invention or a mimetope thereof (i.e., anti-Der HMW-map protein antibodies).
  • a Der HMW-map protein of the present invention or a mimetope thereof (i.e., anti-Der HMW-map protein antibodies).
  • the term "selectively binds to" a er HMW-map protein refers to the ability of antibodies of the present invention to preferentially bind to specified proteins and mimetopes thereof of the present invention. Binding can be measured using a variety of methods standard in the art including enzyme immunoassays (e.g., ELISA), immunoblot assays, etc.; see, for example, Sambrook et al., ibid.
  • An anti-Der HMW- map protein antibody preferably selectively binds to a portion of a Der HMW-map protein that induces an immune
  • Isolated antibodies of the present invention can include antibodies in a bodily fluid (such as, but not limited to, serum), or antibodies that have been purified to varying degrees.
  • Antibodies of the present invention can be polyclonal or monoclonal. Functional equivalents of such antibodies, such as antibody fragments and genetically- engineered antibodies (including single chain antibodies or chimeric antibodies that can bind to more than one epitope) are also included in the present invention.
  • a preferred method to produce antibodies of the present invention includes (a) administering to an animal an effective amount of a protein, peptide or mimetope thereof of the present invention to produce the antibodies and (b) recovering the antibodies.
  • antibodies of the present invention are produced recombinantly using techniques as heretofore disclosed to produce Der HMW-map proteins of the present invention.
  • Antibodies raised against defined proteins or mimetopes can be advantageous because such antibodies are not substantially contaminated with antibodies against other substances that might otherwise cause interference in a diagnostic assay or side effects if used in a therapeutic composition.
  • Antibodies of the present invention have a variety of potential uses that are within the scope of the present invention.
  • such antibodies can be used (a) as tools to detect mite allergen, in particular Der HMW-map protein; (b) as tools to screen expression libraries; and/or (c) to recover desired proteins of the present invention from a mixture of proteins and other contaminants.
  • Antibodies of the present invention can also be used, for example, to inhibit binding of Der HMW-map protein to IgE that binds specifically to Der HMW-map protein, to prevent immunocomplex formation, thereby reducing hypersensitivity responses to mite allergens.
  • a Der HMW-map protein of the present invention can be included in a chimeric molecule comprising at least a portion of a Der HMW-map protein that induces an immune response in an animal and a second molecule that enables the chimeric molecule to be bound to a substrate in such a manner that the Der HMW-map protein portion can bind to IgE in essentially the same manner as a Der HMW-map protein that is not bound to a substrate.
  • An example of a suitable second molecule includes a portion of an immunoglobulin molecule or another ligand that has a suitable binding partner that can be immobilized on a substrate, e.g., biotin and avidin, or a metal-binding protein and a metal (e.g., His), or a sugar-binding protein and a sugar (e.g., maltose).
  • a substrate e.g., biotin and avidin
  • a metal-binding protein and a metal e.g., His
  • sugar-binding protein and a sugar e.g., maltose
  • a Der HMW-map protein of the present invention can be contained in a formulation, herein referred to as a Der HMW-map protein formulation.
  • a Der HMW-map protein can be combined with a buffer in which the Der HMW-map protein is solubilized, and/or with a carrier. Suitable buffers and carriers are known to those skilled in the art.
  • Carriers can be mixed with Der HMW-map protein or conjugated (i.e., attached) to Der HMW-map protein in such a manner as to not substantially interfere with the ability of the Der HMW-map protein to selectively bind to an antibody that specifically binds to Der HMW-map protein.
  • a Der HMW-map protein of the present invention can be produced by a cell comprising the Der HMW-map protein.
  • a preferred Der HMW-map protein-bearing cell includes a recombinant cell comprising a nucleic acid molecule encoding a er ITMW-map protein of the present invention.
  • a Der HMW-map protein formulation of the present invention can include not only a Der HMW-map protein but also one or more additional antigens or antibodies useful in desensitizing an animal against allergy, or preventing or treating mite allergen pathogenesis.
  • an antigen refers to any molecule capable of being selectively bound by an antibody.
  • an allergen refers to any antigen that is capable of stimulating production of antibodies involved in an allergic response in an animal.
  • selective binding of a first molecule to a second molecule refers to the ability of the first molecule to preferentially bind (e.g., having higher affinity higher avidity) to the second molecule when compared to the ability of a first molecule to bind to a third molecule.
  • the first molecule need not necessarily be the natural ligand of the second molecule.
  • Allergens of the present invention are preferably derived from mites, and mite-related allergens including, but not limited to, other insect allergens and plant allergens.
  • One embodiment of the present invention is a reagent comprising a non- proteinaceous epitope that is capable of binding to IgE of an animal that is allergic to mites, of desensitizing an animal against mite allergen, of stimulating a B lymphocyte response, and/or of stimulating a T lymphocyte response.
  • a Der NP epitope can exist as part of a Der HMW-map protein of the present invention or can be isolated therefrom.
  • Such an epitope exists, for example, on a protein contained in the D. farinae HMW-map composition produced in accordance with Example 1.
  • a Der NP epitope of the present invention can be isolated from its natural source or produced synthetically.
  • Such a method includes the steps of contacting a reagent comprising a Der NP epitope with antibodies of an animal and determining immunocomplex formation between the reagent and the antibodies, wherein formation of the immunocomplex indicates that the animal is susceptible to or has said allergic response.
  • Another embodiment of the present invention is a method to desensitize a host animal to an allergic response to a mite.
  • Such a method includes the step of administering to the animal a therapeutic composition that includes a reagent comprising a Der NP epitope as a desensitizing compound.
  • Another embodiment of the present invention is a Der HMW-map protein lacking Der NP epitopes. Without being bound by theory, it is believed that such a protein would be a better desensitizing compound since such a protein is expected to have a reduced ability to bind to IgE.
  • Such a protein can be produced by, for example, removing Der NP epitopes from a native Der HMW-map protein or by producing the protein recombinantly, for example in E. coli.
  • Preferred amounts of Der HMW-map protein for use in a skin test of the present invention range from about 1 x 10 "8 micrograms ( ⁇ g) to about 100 ⁇ g, more preferably from about 1 x 10 "7 ⁇ g to about 10 ⁇ g, and even more preferably from about 1 x 10 "6 ⁇ g to about 1 ⁇ g of Der HMW-map protein. It is to be appreciated by those of skill in the art that such amounts will vary depending upon the allergenicity of the protein being administered.
  • phenolated phosphate buffered saline available from Greer Laboratories, Inc., Lenoir, NC.
  • Hypersensitivity of an animal to one or more formulations of the present invention can be evaluated by measuring reactions (e.g., wheal size, induration or hardness; using techniques known to those skilled in the art) resulting from administration of one or more experimental sample(s) and control sample(s) into an animal and comparing the reactions to the experimental sample(s) with reactions resulting from administration of one or more control solution.
  • Preferred devices for intradermal injections include individual syringes.
  • Preferred devices for scratching include devices that permit the administration of a number of samples at one time.
  • the hypersensitivity of an animal can be evaluated by determining if the reaction resulting from administration of a formulation of the present invention is larger than the reaction resulting from administration of a negative control, and/or by determining if the reaction resulting from administration of the formulation is at least about the same size as the reaction resulting from administration of a positive control solution.
  • an experimental sample produces a reaction greater than or equal to the size of a wheal produced by administration of a positive control sample to an animal, then that animal is hypersensitive to the experimental sample.
  • an experimental sample produces a reaction similar to the reaction produced by administration of a negative control sample to an animal, then that animal is not hypersensitive to the experimental sample.
  • the ability or inability of an animal to exhibit an immediate hypersensitive response to a formulation of the present invention is determined by measuring wheal sizes from about 2 minutes to about 30 minutes after administration of a sample, more preferably from about 10 minutes to about 25 minutes after administration of a sample, and even more preferably about 15 minutes after administration of a sample.
  • the ability or inability of an animal to exhibit a delayed hypersensitive response to a formulation of the present invention is determined by measuring induration and/or erythema from about 18 hours to about 30 hours after administration of a sample, more preferably from about 20 hours to about 28 hours after administration of a sample, and even more preferably at about 24 hours after administration of a sample.
  • a delayed hypersensitivity response can also be measured using other techniques such as by determining, using techniques known to those of skill in the art, the extent of cell infiltrate at the site of administration during the time periods defined directly above.
  • a skin test of the present invention comprises intradermally injecting into an animal at a given site an effective amount of a formulation that includes Der HMW-map protein, and intradermally injecting an effective amount of a control solution into the same animal at a different site. It is within the scope of one of skill in the art to use devices capable of delivering multiple samples simultaneously at a number of sites, preferably enabling concurrent evaluation of numerous formulations.
  • a preferred Der HMW-map protein for use with a skin test includes full-length protein.
  • a preferred positive control sample can be a sample comprising histamine.
  • a preferred negative control sample can be a sample comprising diluent.
  • Animals suitable and preferred to test for hypersensitivity to Der HMW-map protein using a skin test of the present invention are disclosed herein.
  • Particularly preferred animals to test with a skin test of the present invention include humans, canines, felines and equines, with human, canines and felines being even more preferred.
  • canine refers to any member of the dog family, including domestic dogs, wild dogs and zoo dogs. Examples of dogs include, but are not limited to, domestic dogs, wild dogs, foxes, wolves, jackals and coyotes.
  • feline refers to any member of the cat family, including domestic cats, wild cats and zoo cats.
  • cats include, but are not limited to, domestic cats, lions, tigers, leopards, panthers, cougars, bobcats, lynx, jaguars, cheetahs and servals.
  • equine refers to any member of the horse family, including horses, donkeys, mules and zebras.
  • One embodiment of the present invention is a method to detect antibodies in vitro that bind to Der HMW-map protein (referred to herein as anti-Der HMW-map antibody) which includes the steps of: (a) contacting an isolated Der HMW-map protein with a putative anti-Der HMW-map antibody-containing composition under conditions suitable for formation of aDer HMW-map protein: antibody complex; and (b) detecting the presence of the antibody by detecting the Der HMW-map protein: antibody complex. Presence of such a er HMW-map protein: antibody complex indicates that the animal is producing antibody to a mite allergen.
  • Preferred anti-Der HMW-map antibody to detect include antibodies having an IgE or IgG isotype.
  • Preferred anti-Der HMW-map antibody to detect include feline antibody, canine antibody, equine antibody and human antibody, with feline, canine and human antibody being particularly preferred.
  • Formation of a complex between a Der HMW-map protein and an antibody refers to the ability of the Der HMW-map protein to selectively bind to the antibody in order to form a stable complex that can be measured (i.e., detected).
  • the term selectively binds to an antibody refers to the ability of a Der HMW-map protein of the present invention to preferentially bind to an antibody, without being able to substantially bind to other antibodies that do not specifically bind to Der HMW-map protein.
  • Binding between a Der HMW-map protein and an antibody is effected under conditions suitable to form a complex; such conditions (e.g., appropriate concentrations, buffers, temperatures, reaction times) as well as methods to optimize such conditions are known to those skilled in the art, and examples are disclosed herein. Examples of complex formation conditions are also disclosed in, for example, in Sambrook et al., ibid.
  • detecting complex formation refers to determining if any complex is formed, i.e., assaying for the presence (i.e., existence) of a complex. If complexes are formed, the amount of complexes formed can, but need not be, determined.
  • Complex formation, or selective binding, between Der HMW-map protein and an antibody in the composition can be measured (i.e., detected, determined) using a variety of methods standard in the art (see, for example, Sambrook et al. ibid.), examples of which are disclosed herein.
  • a putative antibody-containing composition of the present method includes a biological sample from an animal.
  • a suitable biological sample includes, but is not limited to, a bodily fluid composition or a cellular composition.
  • a bodily fluid refers to any fluid that can be collected (i.e., obtained) from an animal, examples of which include, but are not limited to, blood, serum, plasma, urine, tears, aqueous humor, cerebrospinal fluid (CSF), saliva, lymph, nasal secretions, milk and feces.
  • CSF cerebrospinal fluid
  • Such a composition of the present method can, but need not be, pretreated to remove at least some of the non-IgE or non-IgG isotypes of immunoglobulin and/or other proteins, such as albumin, present in the fluid.
  • Such removal can include, but is not limited to, contacting the bodily fluid with a material, such as the lectin jacalin or an antibody that specifically binds to the constant region of an IgA immunoglobulin (i.e., anti-IgA isotype antibody), to remove IgA antibodies and/or affinity purifying IgE or IgG antibodies from other components of the body fluid by exposing the fluid to, for example, Concanavalin A or protein G, respectively.
  • a composition includes collected bodily fluid that is pretreated to concentrate immunoglobulin contained in the fluid.
  • immunoglobulin contained in a bodily fluid can be precipitated from other proteins using ammonium sulfate.
  • a preferred composition of the present method is serum.
  • an antibody-containing composition of the present method includes a cell that produces IgE or IgG.
  • a cell can have IgE or IgG bound to the surface of the cell and/or can secrete IgE or IgG.
  • An example of such a cell includes myeloma cells. IgE or IgG can be bound to the surface of a cell either directly to the membrane of the cell or bound to a molecule (e.g., an antigen) bound to the surface of the cell.
  • a complex can be detected in a variety of ways including, but not limited to use of one or more of the following assays: an enzyme-linked immunoassay, a radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a lateral flow assay, an agglutination assay, a particulate-based assay (e.g., using particulates such as, but not limited to, magnetic particles or plastic polymers, such as latex or polystyrene beads), an immunoprecipitation assay, a BioCoreTM assay (e.g., using colloidal gold) and an immunoblotting assay (e.g., a western blot).
  • an enzyme-linked immunoassay e.g., a radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a lateral flow assay, an agglutination assay, a particulate
  • assays are well known to those skilled in the art. Assays can be used to give qualitative or quantitative results depending on how they are used. Some assays, such as agglutination, particulate separation, and immunoprecipitation, can be observed visually (e.g., either by eye or by a machines, such as a densitometer or spectrophotometer) without the need for a detectable marker.
  • conjugation i.e., attachment
  • a detectable marker to the Der HMW-map protein, to antibody bound to the Der HMW-map protein, or to a reagent that selectively binds to the Der HMW-map protein or to the antibody bound to the Der HMW-map protein (described in more detail below) aids in detecting complex formation.
  • detectable markers include, but are not limited to, a radioactive label, an enzyme, a fluorescent label, a chemiluminescent label, a chromophoric label or a ligand.
  • a ligand refers to a molecule that binds selectively to another molecule.
  • Preferred detectable markers include, but are not limited to, fluorescein, a radioisotope, a phosphatase (e.g., alkaline phosphatase), biotin, avidin, a peroxidase (e.g., horseradish peroxidase) and biotin-related compounds or avidin- related compounds (e.g., streptavidin or ImmunoPure® NeutrAvidin available from Pierce, Rockford, EL).
  • a complex is detected by contacting a putative antibody- containing composition with a Der HMW-map protein that is conjugated to a detectable marker.
  • a suitable detectable marker to conjugate to a er HMW-map protein includes, but is not limited to, a radioactive label, a fluorescent label, an enzyme label, a chemiluminescent label, a chromophoric label or a ligand.
  • a detectable marker is conjugated to a Der HMW-map protein in such a manner as not to block the ability of the Der HMW-map protein to bind to the antibody being detected.
  • a er HMW-map protein: antibody complex is detected by contacting the complex with an indicator molecule that selectively binds to an IgE antibody (referred to herein as an anti-IgE reagent) or an IgG antibody (referred to herein as an anti-IgG reagent.
  • an indicator molecule that selectively binds to an IgE antibody (referred to herein as an anti-IgE reagent) or an IgG antibody (referred to herein as an anti-IgG reagent.
  • Suitable shapes for substrate material include, but are not limited to, a well (e.g., microtiter dish well), a plate, a dipstick, a bead, a lateral flow apparatus, a membrane, a filter, a tube, a dish, a celluloid-type matrix, a magnetic particle, and other particulates.
  • a particularly preferred substrate comprises an ELISA plate, a dipstick, a radioimmunoassay plate, agarose beads, plastic beads, latex beads, immunoblot membranes and immunoblot papers.
  • a substrate, such as a particulate can include a detectable marker.
  • a preferred method to detect antibody that binds to Der HMW-map protein is an immunoabsorbent assay.
  • An immunoabsorbent assay of the present invention comprises a capture molecule and an indicator molecule.
  • a capture molecule of the present invention binds to an IgE or an IgG in such a manner that the IgE or IgG is immobilized to a substrate.
  • a capture molecule is preferably immobilized to a substrate of the present invention prior to exposure of the capture molecule to a putative IgE-containing composition or a putative IgG-containing composition.
  • An indicator molecule of the present invention detects the presence of an IgE or an IgG bound to a capture molecule.
  • Excess non-bound material, if any, is removed from the substrate under conditions that retain anti-IgE antibody:IgE complex or anti- IgG antibody:IgG complex binding to the substrate.
  • Der HMW-map protein is added to the substrate and incubated to allow formation of a complex between the Der HMW-map protein and the anti-IgE antibody: IgE complex or anti-IgG antibody: IgG complex.
  • the Der HMW-map protein is conjugated to a detectable marker (preferably to biotin, an enzyme label or a fluorescent label).
  • Excess Der HMW-map protein is removed, a developing agent is added if required, and the substrate is submitted to a detection device for analysis.
  • an immunoabsorbent assay of the present invention does not utilize a capture molecule.
  • a biological sample collected from an animal is applied to a substrate, such as a microtiter dish well or a dipstick, and incubated under conditions suitable to allow for IgE or IgG binding to the substrate. Any IgE or IgG present in the bodily fluid is immobilized on the substrate. Excess non-bound material, if any, is removed from the substrate under conditions that retain IgE or IgG binding to the substrate.
  • Der HMW-map protein is added to the substrate and incubated to allow formation of a complex between the Der HMW-map protein and the IgE or IgG.
  • the Der HMW-map protein is conjugated to a detectable marker (preferably to biotin, an enzyme label or a fluorescent label). Excess Der HMW-map protein is removed, a developing agent is added if required, and the substrate is submitted to a detection device for analysis.
  • a detectable marker preferably to biotin, an enzyme label or a fluorescent label.
  • Another preferred method to detect IgE or IgG is a lateral flow assay, examples of which are disclosed in U.S. Patent No. 5,424,193, issued June 13, 1995, by Pronovost et al.; U.S. Patent No.
  • the capture zone receives labeling reagent from the labeling zone which is directed downstream by the flow path.
  • the capture zone contains the capture reagent, in this case an anti-IgE or anti-IgG antibody, or both, as disclosed above, that immobilizes the IgE and/or IgG complexed to the Der HMW-map protein in the capture zone.
  • the capture reagent is preferably fixed to the support structure by drying or lyophilizing.
  • the labeling reagent accumulates in the capture zone and the accumulation is assessed visually or by an optical detection device.
  • the delayed hypersensitivity of the animal to Der HMW-map protein can be tested using an skin test of the present invention.
  • Immunosuppression refers to inhibiting an immune response by, for example, killing particular cells involved in the immune response.
  • Immunotolerization refers to inhibiting an immune response by anergizing (i.e., diminishing reactivity of a T cell to an antigen) particular cells involved in the immune response.
  • a membrane-bound desensitizing compound of the present invention is useful for: (1) inhibiting a Type II hypersensitivity reaction by blocking IgG: antigen complex formation on the surface of cells leading to complement destruction of cells; (2) inhibiting a Type II hypersensitivity reaction by blocking IgG regulated signal transduction in immune cells; and (3) inhibiting a Type IV hypersensitivity reaction by blocking T cytotoxic cell killing of antigen-bearing cells.
  • desensitizing compounds include, but are not limited to, muteins, mimetopes and antibodies of the present invention, as well as other inhibitors of the present invention that inhibit binding between a protein of the present invention and IgE.
  • a preferred Der HMW-map mutein comprises at least a portion of Der HMW-map protein, in which a suitable number of cysteine residues have been removed or replaced with a non-cysteine residue such that the altered Der HMW-map protein is not toxic to an animal (e.g., does not cause anaphylaxis).
  • a therapeutic composition of the present invention includes a nucleic acid molecule encoding a Der HMW-map protein that can be administered to an animal in a fashion to enable expression of that nucleic acid molecule into a Der HMW-map protein in the animal.
  • Nucleic acid molecules can be delivered to an animal in a variety of methods including, but not limited to, (a) administering a naked (i.e., not packaged in a viral coat or cellular membrane) nucleic acid molecule (e.g., as naked DNA or RNA molecules, such as is taught, for example in Wolff et al., 1990, Science 247, 1465-1468) or (b) administering a nucleic acid molecule packaged as a recombinant virus or as a recombinant cell (i.e., the nucleic acid molecule is delivered by a viral or cellular vehicle).
  • a naked nucleic acid molecule e.g., as naked DNA or RNA molecules, such as is taught, for example in Wolff et al., 1990, Science 247, 1465-1468
  • a nucleic acid molecule packaged as a recombinant virus or as a recombinant cell i.e., the nucleic acid molecule is delivered by a
  • a recombinant vims of the present invention When administered to an animal, infects cells within the recipient animal and directs the production of a protein or RNA nucleic acid molecule that is capable of reducing Der lTMW-map protein-mediated biological responses in the animal.
  • a recombinant virus comprising a Der HMW-map nucleic acid molecule of the present invention is administered according to a protocol that results in the animal producing an amount of protein or RNA sufficient to reduce Der HMW-map protein-mediated biological responses.
  • a preferred single dose of a recombinant virus of the present invention is from about 1 x 10 4 to about 1 x 10 7 virus plaque forming units (pfu) per kilogram body weight of the animal.
  • Administration protocols are similar to those described herein for protein- based compositions, with subcutaneous, intramuscular, intranasal and oral administration routes being preferred.
  • a recombinant cell vaccine of the present invention includes recombinant cells of the present invention that express at least one protein of the present invention.
  • Preferred nucleic acid molecules to use with a therapeutic composition of the present invention include any Der HMW-map nucleic acid molecule disclosed herein, in particular SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45 and/or a nucleic acid sequence encoding a protein comprising the amino acid sequence SEQ ID NO:33 and a complement thereof.
  • a recombinant cell useful in a therapeutic composition of the present invention includes recombinant cells of the present invention that comprises Der HMW-map protein of the present invention.
  • Preferred recombinant cells for this embodiment include Salmonella, E. coli, Listeria, Mycobacterium, S. frugiperda, yeast, (including Saccharomyces cerevisiae), BHK, CV-1, myoblast G8, COS (e.g., COS-7), Vero, MDCK and CRFK recombinant cells.
  • a recombinant cell of the present invention can be administered in a variety of ways but have the advantage that they can be administered orally, preferably at doses ranging from about 10 s to about 10 12 cells per kilogram body weight. Administration protocols are similar to those described herein for protein compositions.
  • Recombinant cells can comprise whole cells, cells stripped of cell walls or cell lysates.
  • the efficacy of a therapeutic composition of the present invention to effect an allergic response to Der HMW-map protein can be tested using standard methods for detecting Der HMW-map protein-mediated immunity including, but not limited to, immediate hypersensitivity, delayed hypersensitivity, antibody-dependent cellular cytotoxicity (ADCC), immune complex activity, mitogenic activity, histamine release assays and other methods such as those described in Janeway et al., ibid.
  • the present invention also includes a therapeutic composition comprising one or more therapeutic compounds of the present invention. Examples of such therapeutic compounds include, for example, other allergens disclosed herein.
  • compositions of the present invention can be formulated in an excipient that the animal to be treated can tolerate.
  • excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions.
  • Nonaqueous vehicles such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used.
  • Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran.
  • Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability.
  • Protein adjuvants of the present invention can be delivered in the form of the protein themselves or of nucleic acid molecules encoding such proteins using the methods described herein.
  • a preferred controlled release formulation of the present invention is capable of effecting a treatment preferably for at least about 1 month, more preferably for at least about 3 months, even more preferably for at least about 6 months, even more preferably for at least about 9 months, and even more preferably for at least about 12 months.
  • Therapeutic compositions of the present invention can be sterilized by conventional methods which do not result in protein degradation (e.g., filtration) and/or lyophilized.
  • a therapeutic composition of the present invention can be administered to any animal susceptible to mite allergy as herein described.
  • Acceptable protocols by which to administer therapeutic compositions of the present invention in an effective manner can vary according to individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art.
  • An effective dose refers to a dose capable of treating an animal against hypersensitivity to mite allergens. Effective doses can vary depending upon, for example, the therapeutic composition used and the size and type of the recipient animal.
  • Effective doses to immunomodulate an animal against mite allergens include doses administered over time that are capable of alleviating a hypersensitive response by an animal to mite allergens.
  • a first tolerizing dose can comprise an amount of a therapeutic composition of the present invention that causes a minimal hypersensitive response when administered to a hypersensitive animal.
  • a second tolerizing dose can comprise a greater amount of the same therapeutic composition than the first dose.
  • Effective tolerizing doses can comprise increasing concentrations of the therapeutic composition necessary to tolerize an animal such that the animal does not have a hypersensitive response to exposure to mite allergens.
  • An effective dose to desensitize an animal can comprise a concentration of a therapeutic composition of the present invention sufficient to block an animal from having a hypersensitive response to exposure to a mite allergen present in the environment of the animal.
  • Effective desensitizing doses can include repeated doses having concentrations of a therapeutic composition that cause a minimal hypersensitive response when administered to a hypersensitive animal.
  • a suitable single dose is a dose that is capable of treating an animal against hypersensitivity to mite allergens when administered one or more times over a suitable time period.
  • a preferred single dose of a mite allergen, or mimetope therapeutic composition is from about 0.5 ng to about 1 g of the therapeutic composition per kilogram body weight of the animal.
  • Further treatments with the therapeutic composition can be administered from about 1 day to 1 year after the original administration. Further treatments with the therapeutic composition preferably are administered when the animal is no longer protected from hypersensitive responses to mite allergens.
  • Particular administration doses and schedules can be developed by one of skill in the art based upon the parameters discussed above. Modes of administration can include, but are not limited to, subcutaneous, intradermal, intravenous, nasal, oral, transdermal and intramuscular routes.
  • a therapeutic composition of the present invention can be used in conjunction with other compounds capable of modifying an animal's hypersensitivity to mite allergens.
  • an animal can be treated with compounds capable of modifying the function of a cell involved in a hypersensitive response, compounds that reduce allergic reactions, such as by systemic agents or anti-inflammatory agents (e.g., anti-histamines, anti-steroid reagents, anti-inflammatory reagents and reagents that drive immunoglobulin heavy chain class switching from IgE to IgG).
  • systemic agents or anti-inflammatory agents e.g., anti-histamines, anti-steroid reagents, anti-inflammatory reagents and reagents that drive immunoglobulin heavy chain class switching from IgE to IgG.
  • Suitable compounds useful for modifying the function of a cell involved in a hypersensitive response include, but are not limited to, antihistamines, cromolyn sodium, theophylline, cyclosporin A, adrenalin, cortisone, compounds capable of regulating cellular signal transduction, compounds capable of regulating adenosine 3 ',5 '-cyclic phosphate (cAMP) activity, and compounds that block IgE activity, such as peptides from IgE or IgE specific Fc receptors, antibodies specific for peptides from IgE or IgE- specific Fc receptors, or antibodies capable of blocking binding of IgE to Fc receptors.
  • compositions of the present invention can be administered to any animal having or susceptible to mite allergen hypersensitivity.
  • Preferred animals to treat include mammals and birds, with felines, canines, equines, humans and other pets, work and/or economic food animals.
  • Particularly preferred animals to protect are felines and canines.
  • a preferred method for prescribing treatment for mite allergen hypersensitivity comprises: (1) intradermally injecting into an animal at one site an effective amount of a formulation containing a mite allergen of the present invention, or a mimetope thereof (suitable and preferred formulations are disclosed herein); (2) intradermally injecting into the animal at a second site an effective amount of a control solution; (3) evaluating if the animal has mite allergen hypersensitivity by measuring and comparing the wheal size resulting from injection of the formulation with the wheal size resulting from injection of the control solution; and (4) prescribing a treatment for the mite allergen hypersensitivity.
  • An alternative preferred method for prescribing treatment for mite allergen hypersensitivity comprises: (1) contacting a first portion of a sample of bodily fluid obtained from an animal to be tested with an effective amount of a formulation containing mite allergen, or a mimetope thereof (suitable and preferred formulations are disclosed herein) to form a first immunocomplex solution; (2) contacting a positive control antibody to form a second immunocomplex solution; (3) evaluating if the animal has mite allergen hypersensitivity by measuring and comparing the amount of immunocomplex formation in the first and second immunocomplex solutions; and (4) prescribing a treatment for the mite allergen hypersensitivity. It is to be noted that similar methods can be used to prescribe treatment for allergies using mite allergen formulations as disclosed herein.
  • Another aspect of the present invention includes a method for monitoring animals susceptible to or having mite allergen hypersensitivity, using a formulation of the present invention.
  • In yivo and in vitro tests of the present invention can be used to test animals for mite allergen hypersensitivity prior to and following any treatment for mite allergen hypersensitivity.
  • the present invention also includes antibodies capable of selectively binding to mite allergen, or mimetope thereof.
  • an antibody is herein referred to as an anti- mite allergen antibody.
  • selectively binds to refers to the ability of such an antibody to preferentially bind to mite allergens and mimetopes thereof.
  • the present invention includes antibodies capable of selectively binding to Der HMW-map protein.
  • Binding can be measured using a variety of methods known to those skilled in the art including immunoblot assays, immunoprecipitation assays, enzyme immunoassays (e.g., ELISA), radioimmunoassays, immunofluorescent antibody assays and immunoelectron microscopy; see, for example, Sambrook et al., ibid.
  • the blot was then incubated with donkey anti-mouse IgG antibody conjugated to horseradish peroxidase (1:1000 dilution; available from Jackson Labs, Maine). The presence of HRP-conjugated antibody bound to the blot was detected using standard techniques. An about 70-kD protein was identified in the 0.2 M Tris-HCl, pH 8 fraction, an about 98-kD protein and an about 109-kD protein were identified in the 0.3 M Tris-HCl, pH 8 fraction.
  • the fraction described above that was eluted using 0.3 M Tris-HCl, pH 8 was concentrated in a Centriprep 30 concentrator and then diluted in 20 mM Na-Ac, pH 5.6. The diluted fraction was then applied to a PolyCat A HPLC cation exchange column (available from PolyLC, Columbia, MD). The column was eluted with about 10 ml of 20 mM Na-Ac, pH 5.6, and then with about 45 ml of a linear gradient from 0 to 0.5 M NaCl in the 20 mM Na-Ac, pH 5.6 buffer at a flow rate of about lml/min.
  • the N- terminal partial amino acid sequence of map(7) was determined to be Asp Met Ala Gin Asn Tyr Lys Tyr Arg Gin Gin Phe He Gin Ser Val Leu Asn Asn Gly Ala Thr Arg Gin, also denoted SEQ ID NO:8.
  • the N-terminal partial amino acid sequence of map(8) was determined to be Asp Glu Xaa Asn Val Met Xaa Tyr Val Leu Tyr Thr Met His Tyr Tyr Leu Asn Asn Gly Ala Thr Arg, also denoted SEQ ID NO:9, in which Xaa represents any amino acid.
  • This example describes the purification of a 70-kD protein that binds to IgE from dogs known to be allergic to mite allergens.
  • This example describes the binding of the D. farinae HMW-map composition (i.e., containing mapA and mapB) to canine IgE in dog sera isolated from dogs known to be allergic to mite allergens.
  • Multiple wells of an Immulon ⁇ microtiter plate were coated with about 100 nanograms per well (ng/well) of a D. farinae HMW-map composition isolated according to the method described above in Example 1, diluted in CBC buffer. The plate was incubated overnight at 4°C. Following incubation, the D. farinae HMW-map composition-containing solution was removed from the plate, and the plate was blotted dry.
  • a negative control group of sera was also added to the plate comprising a combination of sera from six dogs that were raised in a barrier facility (available from Harlan Bioproducts, Indianapolis, IN). Some wells did not receive dog sera so that background binding levels could be determined.
  • the plate was incubated for about 1 hour at room temperature and then washed four times with PBST.
  • This example describes the binding of mapA, mapB or mapC proteins to feline IgE in cat sera isolated from cats shown by in vitro testing to be hypersensitive to mite allergens.
  • Multiple wells of an Immulon E microtiter plate were coated with about 100 ng/well of a D. farinae HMW-map composition (isolated according to the method described above in Example 1) and 70-kD D. farinae protein (isolated according to the method described above in Example 3).
  • Other wells of the plate were coated with 400 ng/well of whole Dermatophagoides pteronyssius extract (available from Greer Laboratories, Inc., Lenoir, NC; concentrated 8-fold prior to use) or whole D.
  • HDM refers to cats that are sensitive to house dust mite allergens (by serological test, i.e. an ELISA to whole D. farinae extract). Table 1.
  • HMW-map composition of the present invention Der HMW-map composition nucleic acid molecules were identified and isolated as follows.
  • SEQ ED NO: 34 Translation of SEQ ED NO: 34 yields a protein of about 509 amino acids, denoted PDerp98 509 , the amino acid sequence of which is presented as SEQ ED NO: 35.
  • the nucleic acid molecule consisting of the coding region encoding PDerp98 509 is referred to herein as nDerp98 1527 , the nucleic acid sequence of which is represented as SEQ ED NO:37 (the coding strand), and SEQ ED NO:39 (the complementary strand).
  • the amino acid sequence of PDerp98 509 also represented herein as SEQ ED NO:38, has an estimated molecular weight of about 58.9 kD and an estimated pi of about 5.61.
  • RAST radio-allergo-absorbent test
  • a standard curve was derived by performing RAST with several dilutions of a well-characterized chimeric human/mouse IgE monoclonal antibody against Derp2, (human IgE/monoclonal anti-Derp2, following the procedure of Schuurman, et al. (1997) J Allergy Clin Immunol. 99: pp 545-550). Briefly, 50 ⁇ g of the HMW-map composition, purified as described in
  • Example 4 The remainder of the residue from each sample was subjected to ELISA analysis as described in Example 4. Briefly, 100 ng of either the ⁇ -eliminated sample or of non- ⁇ -eliminated sample of the HMW-map composition was coated onto the Immulon plates, and ELISAs were carried out as described in Example 4 with aD. farinae sensitive dog sera pool, a D. farinae sensitive cat sera pool, and various individual dog sera that are either D. farinae sensitive or not sensitive (as measured by
  • This example describes the isolation and sequencing of a nucleic acid molecule encoding the full length Dermatophagoides farinae 60 kD allergen.
  • farinae 60 kD allergen was converted into a double stranded recombinant molecule, herein denoted as nDerf60 1455 , using the ExAssistTM helper phage and SOLRTM E. coli according to the in vivo excision protocol described in the ZAP-cDNA Synthesis Kit (available from Stratagene).
  • Double-stranded plasmid DNA was prepared using an alkaline lysis protocol, such as that described in Sambrook et al., ibid.
  • This example describes the sequencing of a D. farinae nucleic acid molecule of the present invention.
  • SEQ ED NO: 51 suggests the presence of a signal peptide encoded by a stretch of amino acids spanning from amino acid 1 through amino acid 25.
  • the proposed mature protein denoted herein as PDerf60 437 , contains about 437 amino acids which is represented herein as SEQ ED NO: 56.
  • the amino acid sequence of PDerf60 437 i.e., SEQ ED NO: 56
  • predicts that PDerf60 462 has an estimated molecular weight of about 50.0 kD, an estimated pi of about 5.61. and one predicted asparagine- linked glycosylation site extending from amino acids 313 through 315.
  • Nucleic acid molecule nDerf98 1752 of Example 8 was inserted into appropriate expression vectors and expressed in E. coli and P. pastoris.
  • PDerf98 555 was expressed in E. coli or P. pastoris
  • sensitized dog sera produced as described in Example 4
  • failed to recognize the recombinant protein This is in contrast to the positive results obtained when the native D. farinae HMW-map composition of Example 1 (also referred to as native Der f 15) was used; see Example 4.
  • the non-reactivity of the protein expressed in E. coli is consistent with the results shown in Example 16, in which it was shown that the native HMW allergens retain their character as allergens, even after the amino acids are removed.
  • the native Der f 15 antigen was analyzed for carbohydrate content. A substantial amount of carbohydrate was found, about 30% by weight. Specifically, mannose constituted approximately 2.8% by weight of the antigen; galactose approximately 23.2%; glucose approximately 4.3% (the presence of glucosyl residue must be considered tentative as glucose often contaminates glycoprotein samples); and HexNAc at detectable levels; further investigation revealed that the HexNAc were GlcNAc and GalNAc.
  • the native Der f 15 protein was treated with base in the presence of NaBKt, . and analyzed by a P-4 sizing chromatography. O-linked oligosaccharides present in Der f 15 were found to void the column.

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Abstract

L'invention concerne des protéines de Dermatophagoïdes de poids moléculaire élevée, des molécules d'acide nucléique codant ces protéines, ainsi que des réactifs thérapeutiques et diagnostiques obtenus à partir de ces protéines.
PCT/US2001/028730 2000-09-14 2001-09-14 Nouvelles proteines et molecules d'acide nucleique de dermatophagoides et leurs utilisations WO2002022807A2 (fr)

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AU2001292661A AU2001292661A1 (en) 2000-09-14 2001-09-14 Novel dermatophagoides nucleic acid molecules, proteins and uses thereof
CA002420459A CA2420459A1 (fr) 2000-09-14 2001-09-14 Nouvelles proteines et molecules d'acide nucleique de dermatophagoides et leurs utilisations
JP2002527249A JP2004529605A (ja) 2000-09-14 2001-09-14 Ｄｅｒｍａｔｏｐｈａｇｏｉｄｅｓ核酸分子、タンパク質およびそれらの使用

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CN106146640A (zh) * 2016-05-31 2016-11-23 深圳大学 尘螨变应原及其应用
WO2019025388A1 (fr) * 2017-07-31 2019-02-07 Laboratorios Leti S.L. Unipersonal Produit vétérinaire

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CN106146640A (zh) * 2016-05-31 2016-11-23 深圳大学 尘螨变应原及其应用
WO2019025388A1 (fr) * 2017-07-31 2019-02-07 Laboratorios Leti S.L. Unipersonal Produit vétérinaire
CN111132734A (zh) * 2017-07-31 2020-05-08 莱蒂个人实验室 兽用产品
IL272348B (en) * 2017-07-31 2022-09-01 Leti Pharma S L Veterinary product

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